Infertility Manual - Kamini Rao
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SECTION 3: Male Factor Infertility
15. Semen Analysis....................................................................................................................................................... 125
Veronica Irene Yuel
16. Evaluation of Sperm Damage................................................................................................................................ 132
Contents
Chandrika Kulkarni
17. Current Trends in the Management of Azoospermia and Oligoasthenospermia............................................. 140
Soumya Davuluri
18. Sexual Dysfunctions in Male Infertility................................................................................................................ 147
BV Srinivas
SECTION 1: A General Overview 19. Surgical Sperm Retrieval....................................................................................................................................... 152
Madhu Lakshmikantha, Kamini A Rao
1. Oogenesis and Spermatogenesis..............................................................................................................................3
Mandeep Kaur
2. The Human Sperm and Egg: Key Players of Fertilization.........................................................................................9 SECTION 4: Female Factor Infertility
P Jyothishmathi Sharma, Kamini A Rao 20. Female Age and Fertility Preservation................................................................................................................. 159
3. Molecular Mechanisms: Endometrial Receptivity and Implantation....................................................................16 Nupur Garg
Nameeta Mokashi - Bhalerao 21. Congenital Uterine Malformations and Reproduction....................................................................................... 166
4. Infertile Couple—Initial Investigations...................................................................................................................26 Santosh Kumar Jena, Kamini A Rao
Chaitra Nayak 22. Tubal Factor Infertility and Ectopic Pregnancy—Diagnosis and Management................................................ 173
5. Counseling in Infertility............................................................................................................................................30 Richa Sharma
Shreedevi Kumar 23. The Prediction and Management of Poor Responder......................................................................................... 181
6. Thyroid Disorders and Infertility.............................................................................................................................36 R Karthigayeni, V Swetha, Divyashree PS, Kamini A Rao
GB Ashwini 24. Endometriosis, Adenomyosis, and Infertility...................................................................................................... 195
7. Ultrasound in Infertility............................................................................................................................................43 Anjali Gahlan
Arunima Halder, Divyashree PS 25. Role of Fibroids in Infertility.................................................................................................................................. 203
8. Unexplained Infertility.............................................................................................................................................56 Amit J Upadhyay, Kamini A Rao
Navya Vavuluru 26A. Reproductive Tract Infections............................................................................................................................... 210
Vyshnavi A Rao
SECTION 2: Endocrinological Disorders of Infertility 26B. Genital TB and Infertility....................................................................................................................................... 217
Vyshnavi A Rao
9. Menstrual Cycle........................................................................................................................................................ 65
J Sneha
10. Normal and Abnormal Puberty................................................................................................................................71 SECTION 5 : Ovarian Stimulation
Meghana Nyapathy
27. Drugs Used in Ovarian Stimulation...................................................................................................................... 229
11. Polycystic Ovarian Syndrome...................................................................................................................................80 Arveen Vohra
Deepika Krishna
28. Ovulation Induction and Superovulation............................................................................................................ 249
12. Hyperprolactinemia and Infertility.........................................................................................................................95 Mekhala Dwarakanath
Priya Khamatkar
29. Controlled Ovarian Stimulation and Individualization of Stimulation (iCOS).................................................. 258
13. Luteal Phase Defect................................................................................................................................................ 106 Mamta Dighe
Pratibha Malik, Kamini A Rao
30. Monitoring of Ovarian Stimulation...................................................................................................................... 267
14. Obesity.................................................................................................................................................................... 112 Sandhya Krishnan
Rajitha
Contents xv
31. Ovarian Hyperstimulation Syndrome.................................................................................................................. 275
Divya Sardana
32. Ovulatory Triggers................................................................................................................................................. 283
SE C T I O N
1
Vinay Kumar, Ratnakar Majalekar
33. Luteal Phase Support in Assisted Reproduction................................................................................................. 289
Harpreet Kaur
SECTION 6 : Assisted Reproduction Techniques
34. Intrauterine Insemination..................................................................................................................................... 297
R Karthigayeni, Divyashree PS
35. Oocyte Retrieval and Embryo Transfer................................................................................................................. 308
Shivani Singh, Shalu Gupta
36. Prognosticating and Optimizing In Vitro Fertilization Outcome........................................................................ 328
G Ashwini Sidhmalswamy
A General Overview
37. Third-party Reproduction..................................................................................................................................... 339
Nivedita Shetty
38. Adoption: Review and Current Status.................................................................................................................. 347
Suvarna A Rathor
SECTION 7: Laboratory Management
39. Quality Control in the ART Laboratory................................................................................................................. 355
Hemalatha Ravikumar
40. Follicular Fluid Screening...................................................................................................................................... 364
Hemantha Kumar
41. IVF, ICSI, IMSI, IVM.................................................................................................................................................. 379
Ratnakar Majalekar, Vinay Kumar, Kamini A Rao
42. Assessment of Fertilization................................................................................................................................... 386
Arunima Halder, Dileep Kumar, Divyashree PS
43. Culture Systems...................................................................................................................................................... 396
Anuja Kamath
44. Cryopreservation................................................................................................................................................... 402
Mohammed Ashraf
45. PolScope, Embryoscope and Assisted Hatching.................................................................................................. 411
Mir Jaffar, Waseem Andrabi
46. Embryology Instruments....................................................................................................................................... 418
Snehal Dhobale-Kohale
47. Recent Advances in Infertility............................................................................................................................... 453
Sachin A Narvekar
48. Critical Analysis of the Current ART Guidelines................................................................................................... 461
P Jyothishmathi Sharma, Manishi Mittal, Divyashree PS, Kamini A Rao
Index ........................................................................................................................................................................................................................... 469
C HA PTE R
Oogenesis and Spermatogenesis 1 Mandeep Kaur
Chapter Outline
• Indifferent Stage of the Gonad • Role of Anti-Müllerian Hormone
• Differentiation of the Gonad (6–9 Weeks) • Spermatogenesis
• Stage of Oogonial Multiplication and Oocyte Formation • Endocrine Control
• Follicle Formation • Sperm Transport
• Neonatal, Childhood, and Adult Ovary
INTRODUCTION • Primordial germ cells first appear as a group of about
100 cells in dorsal yolk sac endoderm between 3 and
The human ovary has two main physiologic responsibili- 4 weeks of gestation.1
ties—monthly release of egg and steroid hormone pro- • Indifferent stage lasts for about 7–10 days.
duction. The ovary is made up of cortex, medulla, and the • By 6 weeks of gestation, the germ cells increase in
hilum. Oocytes are covered with layers of cells called the number by mitosis to about 10,000.
follicles, and they are enclosed in the inner part of cortex. • The germ cells migrate along the dorsal mesentery to
During fetal life, the human ovary passes through four the gonadal ridge between 4 and 6 weeks of gestation.2
stages: Gonadal ridge provides a place where these germ cells
1. Indifferent stage of the gonad; can survive.
2. Differentiation of the gonad; • Fibronectin and laminin are the factors that seem to be
3. Stage of oogonial multiplication and oocyte involved in the migration.
formation;
4. Follicle formation.
DIFFERENTIATION OF
THE GONAD (6–9 WEEKS)
INDIFFERENT STAGE OF THE GONAD
• Differentiation to testis or ovary will occur accord-
• Ovaries are formed at 5 weeks of gestation from the
ing to the genetic constitution and receptivity of the
gonadal ridges (Fig. 1.1).
tissues.
• Gonadal ridges are coelomic prominences overlying
• If the indifferent gonad has to become testis, differen-
the mesonephros.
tiation will occur; in absence of testicular differentia-
tion, formation of ovary occurs.
• Both testis and ovarian development require domi-
nantly acting genes. SRY (sex-determining region on
Y chromosome) via upregulation of SOX-9 (SRY box 9
region) causes testis development and W NT-4 (mem-
ber of wingless family of genes) and RSPO1 (R-spon-
din-1) by suppression of SOX-9 cause development of
the ovary (Fig. 1.2).3
Fig. 1.1: Series of events in development of gonads. Fig. 1.2: Development of the gonad.
4 The Infertility Manual Oogenesis and Spermatogenesis 5
STAGE OF OOGONIAL MULTIPLICATION Table 1.2. Germ cell number in relation to age. egg, carry genetic material for zygote formation, and carry
centrosome to enable cell division.
AND OOCYTE FORMATION Age Number of follicles
3 wk 100
• WNT4 and RSPO1 are the genes responsible for ovar-
6 wk 10,000
Development
ian development and activate beta-catenin signaling
pathway, which results in loss of adhesion between 8 wk 600,000 • The testis begins its differentiation at 6–7 weeks by
cells and that is required for entry into meiosis.4 20 wk 6–8 million appearance of Sertoli cells and spermatogonia from
• Germ cells multiply by mitosis and reach 6–7 million Birth 1–2 million
the primordial germ cells.
in number at 16–20 weeks, and this is the maximum • Development of male phenotype depends on produc-
Puberty 300,000
number. After this point, the number keeps decreasing. tion of AMH and testosterone.
35 y 25,000
• Germ cells give rise to oogonia by week 7. • Sertoli cell differentiation begins at 7 weeks and is a site
Menopause 1,000 for production of androgen-binding protein (ABP) to
• Oogonia forms oocyte at first meiotic division (week 8),
which gets arrested in the prophase. maintain high local androgen environment and inhibin.
• Retinoic acid is considered to be a meiotic inducing • Leydig cells appear at 8 weeks from the interstitial
• Each ovary contains similar total number of follicles.
factor.5 Fig. 1.3: Diagrammatic representation of the series of events. component.
• Levels of gonadotropins are higher in female fetus
• Meiosis progresses to diplotene stage during the preg- • Leydig cells produce testosterone and the secre-
compared to male as there is no negative feedback by
nancy and gets completed by birth. tion increases with increase in number and peaks at
• Primary to preovulatory follicle may take up to 85 days. sex steroids.
• Ovum is formed by two meiotic divisions of the oocyte, 15–18 weeks.
• Preantral follicle is formed by more complete granu- • Most common abdominal masses in female fetuses
one just before ovulation and one at the time of sperm • Male primordial germ cells unlike in the female do not
losa cell proliferation by sixth month of gestation. and newborn are ovarian cyst as a result of gonadotro-
penetration (Table 1.1). start with the meiotic division before the onset of puberty.
• Formation of antrum and theca cell formation from pin stimulation.
• The testosterone production in the local tissues causes
the surrounding mesenchyme occurs and forms the • Ovary increases in size during childhood by almost
the differentiation of the Wolffian system.
FOLLICLE FORMATION antral follicle and is seen by the end of pregnancy. 10-fold due to active synthesis of proteins.11
• Testis is an oval-shaped organ that is located in the
• Each follicle that ovulates, about 1,000, undergoes
• Follicle formation starts at 18–20 weeks, and as vas- • Anti-Müllerian hormone (AMH) is a dimeric glycopro- scrotum and has a volume of about 15 mL (measured
tein produced by the granulosa cells of growing pre- atresia in that process.
cularity increases, perivascular cells become pre- with Prader orchidometer), length of about 5 cm and
antral and small antral follicles.8 • And as women age, cycles become shorter due to early
granulosa cells.7 rubbery in consistency.
recruitment in the initial years and later on the cycles
• Primordial follicle consists of oocyte which is arrested • Primordial follicles grow and pass through various • Testis needs a temperature of 2–3°C lower than the
stages of development and reach the antral follicle become longer due to more anovulatory cycles.
in prophase of meiotic division which is covered by a body temperature for normal functioning.
stage and this process is known as initial recruitment • Rising follicle-stimulating hormone (FSH) reflects
layer of spindle-shaped granulosa cells. • Each testis contains 200–300 seminiferous tubules
(gonadotropin independent) as factors other than reduced quality and quantity of follicles in ageing
• Primordial to preovulatory follicle may take up to 1 year. which increase the surface area to about 1–2 m, and
gonadotropins are responsible for this process. ovary.
• Primary follicle is formed by change of pregranulosa this is required for the process of spermatogenesis.
cells from spindle-shaped to cuboidal-shaped cells • A set of antral follicles are recruited for further growth, • The seminiferous tubules are the site for spermato-
dominance and ovulation by cyclic stimulation of gon- ROLE OF ANTI-MÜLLERIAN HORMONE
and formation of multiple layers of granulosa cells is genesis.
called secondary follicle. adotropins, and this is known as cyclic recruitment • Anti-Müllerian hormone (AMH) is first detected at • The seminiferous tubules are lined by stratified germi-
(gonadotropin dependent). 36 weeks of fetal life in the granulosa cells of develop- nal epithelium and contain germ cells and supporting
• The primordial follicles start growing into primary fol- ing preantral follicles. The levels peak at puberty and cells/Sertoli cells.
Table 1.1. Series of events in follicle formation licles during fetal life and this continues after birth reduce to undetectable levels at menopause.12 • Five percent of the total testicular tissue consists of the
and maturation. until the ovarian reserve is depleted, and only 1,000 • AMH is an important marker of ovarian reserve. Leydig cells, and they are responsible for testosterone
Oogonial mitosis 5–28 wk primordial follicles are left in the ovary at the time of • AMH has the least inter- and intracycle variability and production.
Oogonial atresia 8–28 wk menopause (Fig. 1.3 and Table 1.2).9 as a result a good test for evaluation in random blood • A cross-section of the seminiferous tubule contains
Oocyte formation (entry into meiosis) 8–28 wk samples. four to five generations of germ cells, younger one
Follicle formation 16 wk–6 mo6 NEONATAL, CHILDHOOD, AND • AMH levels correlate well with the number of antral toward the basement membrane and mature ones
Follicle atresia 24 wk–menopause ADULT OVARY follicles in the ovary and also the number of oocytes toward lumen (Fig. 1.4).
Completion to diplotene stage of Completed by birth retrieved.13
• Germ cell content in the neonatal ovary is 500,000– Spermatogenesis is completed in 64 days in human
prophase of meiosis males and can be divided into three parts:
2 million at birth, and 80% of the germ cells are lost
Completion of first meiotic division Just before ovulation SPERMATOGENESIS 1. Proliferation
before the female newborn enters life.
Completion of second meiotic At time of sperm • Right ovary is larger, heavier, and more in protein as Sperm is a unique cell having a specialized ability to 2. Meiosis
division penetration well as DNA content than the ovary on the left side.10 migrate through the female genital tract and fertilize the 3. Differentiation
6 The Infertility Manual Oogenesis and Spermatogenesis 7
Table 1.3. Contribution of each of the structures to the ejaculate.
Source of secretion Percentage Important components
Testes 5 Testosterone, inhibin, carnitine, LDH, glycerophosphorylcholine
Seminal vesicles 46–80 Fructose, prostaglandins, and substrate for semen clotting
Prostrate 13–33 Acid-phosphatase, citrate, protease, peptidase, hyaluronidase,
vesiculose, spermine, Zn, Ca, Mg
Bulbourethral and urethral glands 2–5 Mucoproteins and IgG
Abbreviations: Ca, calcium; IgG, immunoglobulin G; LDH, lactic dehydrogenase; Mg, magnesium; Zn, zinc.
• Semen is a gray opalescent mixture of secretions of SPERM TRANSPORT
testis, epididymis, prostrate, seminal vesicles, and bul-
bourethral glands. • Cervical crypts act as a reservoir of sperms providing
Fig. 1.4: Cross-section of seminiferous tubule.
• The propulsion of secretions into the prostatic urethra supply for up to 72 hours.
Fig. 1.5: The process of spermatogenesis. • Process of capacitation starts in the cervix and is com-
is called emission and to the exterior from the penile
pleted in the fallopian tube.
Proliferation urethra is called ejaculation (Table 1.3).
• Capacitation is the cellular change that makes the
• Formation of spermatogonia and begins at puberty. • There are three major modifications: chromatin spermatozoa fertilizable.
condensation, formation of acrosome, and formation ENDOCRINE CONTROL
• Proliferation occurs by mitosis. • Capacitation is the ability to undergo acrosomal reac-
• Multiplication occurs not only to replenish base cells of the tail. • The hypothalamic-pituitary-gonadal (HPO) axis pro- tion, ability to attach to the zona pellucida, and acqui-
but also to form committed cells. • Deoxyribonucleic acid (DNA) condensation is packag- vides feedback loops for spermatogenesis and devel- sition of hypermotility.
• Type Ad (dark) spermatogonium is the precursor and ing with protamines instead of histones and adjusts in opment of secondary sexual characters. • Acrosome reaction is the fusion of the sperm mem-
divides into type Ad and Ap (pale) types of spermato- 40 times smaller volume compared to a somatic cell. • Hypothalamic hormones stimulate the pituitary to brane and outer acrosomal membrane allowing
gonium. • The advantages are that the sperm becomes lighter produce FSH and luteinizing hormone (LH). release of enzymes and changes in the inner acroso-
• Type Ap spermatogonium finally forms type B sper- during travel through the genital tract, keeps the • FSH stimulates Sertoli cells to produce ABP and inhibin, mal membrane necessary for fusion with the oocyte
matogonium, which subsequently forms primary genome inactive, and protects the genetic material and LH stimulates Leydig cells to produce testosterone. cell membrane.
from external insults. • Sertoli cells form a site for spermatogenesis and Leydig
spermatocyte. • Sperms typically undergo capacitation in female
• Spermiation is the extrusion of elongated spermatids cells produce testosterone for its maintenance.
• Estimated number of spermatid formed from a single reproductive tract but can also acquire ability to ferti-
from Sertoli cells to tubular lumen (Fig. 1.5). • There is inverse relationship between FSH and inhibin
spermatogonium is 516. lize in defined media.
and measurement of inhibin is considered to be a best
Epididymal Maturation marker of spermatogenesis (Flowchart 1.1).
Meiosis PROBABLE QUESTIONS
• Epididymis lies along the posterolateral border of tes-
• Primary spermatocytes are arrested in the prophase of tis and consists of three parts—caput (head), corpus Flowchart 1.1: Endocrine control of spermatogenesis. 1. Write a short note on gonadal development.
first meiotic division until puberty. (body), and cauda (tail). 2. Write a short note on folliculogenesis.
• At puberty, primary spermatocytes develop by meiosis • Sperms pass across the epididymis in 7–10 days and 3. Draw and explain about different stages of sperm
into haploid secondary spermatocytes. attain maturity, motility, and fertilization capacity development found in seminiferous tubule of a male.
• Secondary spermatocytes have the shortest life span in it. 4. Write a short note on oogenesis.
and form four haploid spermatids. • Cauda is the site for storage of sperms before ejacula- 5. Explain about the endocrine control of spermatogen-
• Development requires the presence of Sertoli cells and tion, and the sperms reach the cauda 72 days after the esis and oogenesis.
so in vitro development has not been achieved. beginning of the process of spermatogenesis.
• In the epididymis, sperm acquires a sperm-coating
protein beta-defensin 126 and a high negative charge
REFERENCES
Differentiation that is critical for movement through the cervical 1. Mc KD, Hertig AT, Adams EC. Danziger S. Histochemical
• Round spermatids are differentiated into elongated mucus. observations on the germ cells of human embryos. Anat
Rec. 1953;2:201-19.
cells with a condensed nucleus and a flagellum and • The sperms then pass into the vas deferens (25 cm in
2. Pereda M, Zorn T, Soto-Suazo M. Migration of human
a shape specific to each species, and this process is length) and are joined by the secretions of the seminal and mouse primordial germ cells and colonization of the
called spermiogenesis. vesicles (5 cm in length) and pass through the pros- developing ovary: an ultrastructural and cytochemical
• Spermiogenesis takes about 2 weeks to complete. trate via the ejaculatory ducts into the urethra. study. Microscopy Res Technique. 2006;69:386.
C HA PTE R
8 The Infertility Manual
3. Cool J, Capel B. Mixed signals: development of the testis.
Seminars. Reprod Med. 2009;27:5.
4. Chassot AA, Ranc F, Gregoire EP, Roepers-Gajadien
HL, Taketo MM, Camerino G, et al. Activation of beta-
catenin signaling by Rspo1 controls differentiation of the
mammalian ovary. Hum Mol Genet. 2008;17:1264.
9. Oktem O, Oktay K. The ovary: anatomy and function
throughout human life. Ann NY Acad Sci. 2008;1127:1-9.
10. Mittwoch U, Mahadevaiah S. Comparison of development
of human fetal gonads and kidneys. J Reprod Fertil.
1980;58:463.
11. Bridges NA, Cooke A, Healy MJ, Hindmarsh PC, Brook CG.
The Human Sperm and Egg:
Key Players of Fertilization
2
P Jyothishmathi Sharma, Kamini A Rao
5. Wilhelm D, Palmer S, Koopman P. Sex determination and Standards for ovarian volume in childhood and puberty.
gonadal development in mammals. Physiol Rev. 2007;87:1. Fertil Steril. 1993;60:456.
Chapter Outline
6. Maheshwari A, Fowler PA. Primordial follicular assembly 12. de Vet A, Laven JS, de Jong FH, Themmen AP, Fauser BC.
• History • Structure of the Oocyte
in humans—revisited. Zygote. 2008;4:285-96. Antimullerian hormone serum levels: a putative marker for
• Structure of Sperm Cell • Maturation and Activation of Spermatozoa
7. Ammini AC, Pandey J, Vijyaraghavan M, Sabherwal U. ovarian aging. Fertil Steril. 2002;2:357-62.
Human female phenotypic development: role of fetal 13. Ebner T, Sommergruber M, Moser M, Shebl O, Schreier-
ovaries. J Clin Endocrinol Metab. 1994;79:604. Lechner E, Tews G. Basal level of anti-Mullerian hormone
8. Visser JA, Themmen AP. Anti-Mullerian hormone and is associated with oocyte quality in stimulated cycles. Hum INTRODUCTION • Oscar Hertwig (1876)—First discovered the fusion
folliculogenesis. Mol Cell Endocrinol. 2005;1–2:81-6. Reprod. 2006;8:2022-2026. of sperm and egg nuclei during fertilization in sea
Structure of sperm cell—Contains head, acrosomal cap,
urchins.1
neck, midpiece, and tail. Acrosome contains proteolytic
enzymes.
Structure of oocyte—One of the largest cells in human
STRUCTURE OF SPERM CELL
body. Covered by zona pellucida(ZP). ZP has specific • Paddle shaped, round head 4–5 µm long, 2–3 µm wide,
receptors for sperm binding and prevents polyspermy. and tail of about 50 µm (Figs. 2.1A to C).
Maturation and Activation of spermatozoa—Four • Consist of head, neck, midpiece, and tail.
stages have been described: • Head is covered by acrosomal cap. Acrosome contains
proteolytic enzymes and spans the region between
1. Storage in the epididymis-Maturation
the nuclear membrane inside and plasma membrane
2. Ejaculation-Activation of motility
outside.
3. Ascension to the tubes-capacitation • Neck: Carries the centrosome that carries a proximally
4. Near the oocyte-Acrosome reaction placed, functional centriole.2
Oocyte maturation—Includes both nuclear and cyto- • The mid piece contains mitochondria arranged in
plasmic maturation. Nuclear maturation is evident by spiral fashion.
extrusion of first polar body. • The plasma membrane covers almost the end of the
Fertilization—Oocyte activation occurs following inva- tail.
• Tail consists of 11 microtubules, nine of which are
sion by spermatozoa. Polyspermy is prevented by tran-
arranged peripherally and remaining two are present
sient block due to membrane depolarization followed by
in the center. Each microtubule is arranged as a pair of
permanent block due to cortical reaction. Fertilization is
two units joined by dyenin arms.
recognized by the appearance of two pronuclei (male and • Consists of several membrane proteins like SPAM—
female) with the extrusion of the second polar body. helps to bind to ZP and disperses the cumulus,
Impact of ICSI on fertilization—Nuclear decondensa- Fertilin—helps in fusion with oocyte.
tion may be slower due to presence of acrosomal materi-
als, but male pronuclear formation is not hindered.
STRUCTURE OF THE OOCYTE
Fertilization is a fascinating process that leads to the
re-creation of an entire being from the fusion of a single • Between 100–200 µm in diameter, one of the largest
sperm and egg. cells in human body (Fig. 2.2).
• Surrounded by a gelatinous glycoprotein shell, the
HISTORY zona pellucida (ZP), and several layers of follicular
cells, the cumulus oophorus.
• Leewenhoek (1677)—First saw sperm under micro- • Contains basic cellular organelles with cortical gran-
scope and called them “little animals of semen.” ules. Ribosomes are usually not seen.
• Von Baer (early 19th century)—First saw mammalian • MII spindle is located beneath the polar body I. It is
egg under microscope. anastral and arranged at right angles to surface.
10 The Infertility Manual The Human Sperm and Egg: Key Players of Fertilization 11
istinctly from the stage of secondary spermatocytes
d • β-Defensin aids movement through cervical mucus
and round spermatids. The sperm head becomes and coats the spermatozoa.4
smaller and more compact. • Uterine contractions play their part and push the
• In order not to use up their stored energy, the motility sperms upward. It takes 5 minutes for the sperm to
of spermatozoa is inhibited by an additional coating reach the fallopian tube post insemination.
of a glycoprotein layer and other molecules. These
motility inhibitors are then removed stepwise in the Ascension to the Ovary—Capacitation
course of later activation steps.
• Definition includes molecular changes in the sperm
• The structure of plasma membrane differs in mature
plasma membrane and in the components of signal
and immature spermatozoa. During the process of transduction that enable the sperm to undergo hyper-
maturation, sterols in the plasma membrane are activated motility and the acrosome reaction.
removed and phospholipid content increases—helps • Takes 5–6 hours. Calcium ions play a major role in
in increased motility. the process of capacitation by activating ATPase. This
• Also excess cytoplasm is removed—seen as small vesicles is followed by glycoprotein, glycolipid, and mannose
in the neck region—helps in assessing mature sperm. receptor redistribution on the sperm surface. Changes
in membrane cholesterol/phospholipid ratio further
Ejaculation—Activation of Motility enhance calcium ion influx.
• Motile capabilities of the spermatozoa are abruptly • Hyperactivated motility ensues. It involves increased
activated after ejaculation. Mechanisms are as follows: side to side movement of the head, increased fre-
quency, and wider displacement of the tail.
(1) mechanical stimulation during the ejaculation, and
• Maturation mechanisms occur in small groups over a
(2) admixture of the glandular secretions.
A period of time because of unpredictability of the tim-
B C • Following intercourse, semen has a gel-like consist-
ing of ovulation.
ency. Liquefaction occurs due to prostatic enzymes
Figs. 2.1A to C: (A) Sperm ultrastructure. (B) Whole intact sperm bound by its plasma membrane to the surface of the zona 15,000×. (C) Sperm
within 20–30 minutes.
head showing surface membranes 70,000×.
• The sperms move rapidly and reach the cervical Near the Oocyte—Acrosome Reaction
mucus within 90 seconds of ejaculation.3 Hence- • It is disruption and fusion of outer acrosomal mem-
forth, only the active spermatozoa progress further brane and plasma membrane (Figs. 2.3A to D).
• Zona pellucida consist of two major functions: in their path as they have to make thier way through • These two membranes fuse intermittently appear-
a. Species-specific ligands the pores, smaller than the sperm head size seen in ing like vesicles (vesiculation) around the anterior
b. Zona reaction to prevent polyspermy the cervical mucus. two-thirds of the sperm head releasing enzymes
• Composed of various glycoproteins ZP1, ZP2, ZP3, and
ZP4. Among these, the most important is ZP3. It is pre-
sent in large quantities compared to others. Sperm binds
via ZP3 and ZP4. ZP2 helps to prevent polyspermy.
MATURATION AND ACTIVATION
OF SPERMATOZOA
Four stages have been described:
1. Storage in the epididymis—maturation
2. Ejaculation—activation of motility a b c
3. Ascension to the ovary—capacitation A B C D
4. Near the oocyte—acrosome reaction Figs. 2.3A to D: Sperm acrosome reaction. (A) Sperm acrosome reaction: two stages. Note vesiculation of surface membranes: fused plasma
membrane and outer acrosome membranes. (a) Acrosome intact, (b) acrosome cap reacted, (c) acrosome reacted. (B) Acrosome-intact sperm
Storage in the Epididymis—Maturation attached to zona(z) by its plasma membrane 30,600×. (C) Acrosome-reacting sperm (cap region) at the surface of the zona. Note connection
Fig. 2.2: Human oocyte fine structure. This illustration incorporates
between plasma membrane and outer acrosome, where the acrosome has still not reacted (arrowhead) 35,700×. (D) Acrosome reacted sperm
cellular organelles of immature and mature oocytes, as well as two fol- • Histones are replaced by protamines. Acrosomal cap penetrating the zona. Vesiculation is complete and myriads of vesicles are still attached to the inner acrosome membrane 25,000×.
licle cells that play an important role in oocyte maturation. is formed from golgi complex. The midpiece is seen AC, acrosomal cap; ES, equatorial segment; PA, postacrosomal region.
12 The Infertility Manual The Human Sperm and Egg: Key Players of Fertilization 13
(hyaluronidase and acrosin) for digesting a pathway
for penetration through the cumulus and ZP.5,6
• Calcium influx occurs causing changes in the pH. This
is followed by the release of acrosomal contents and
exposure of zona-binding receptors.
• Acrosomal contents include hyaluronidase, neurami-
nidase-like factor, cumulus-dispersing enzyme, and
acrosin. A prerequisite for the success of the acrosome
reaction is the previous binding of the spermatozoon
to the pellucid zone. The same reaction can be induced
in vitro by human follicular fluid.
• Capacitation and the acrosome reaction last for about
2 hours, and sperm traverses the zona over another
hour. A B
Figs. 2.5A and B: Sperm–egg fusion and incorporation. (A) Fusion of the oocyte and sperm membranes: the midsegment of the acrosome
reacted sperm has fused with the oolemma (between arrows). The egg has extended a phagocytic process (P) that engulfs the head of the sperm.
Oocyte Maturation M, mitochondira, MP, midpiece, S, smooth endoplasmic reticulum, 30,600×. (B) Normal sperm incorporation into the ooplasm (O): the c hromatin
Oocyte maturation includes both nuclear and cytoplasmic has decondensed and the sperm head has expanded to form an MPN. Note centriole (arrow) in the neck region. CH, chromatin, M, m itochondira,
NE, nuclear envelope 13,200×.
maturation.
Nuclear maturation: The oocyte resumes meiosis and Fig. 2.4: Major events at fertilization. (A) The sperm fuses with the
the nuclear maturation is evident by the extrusion of the oocyte at the midsegment, followed by phagocytosis and formation of
male pronucleus (MPN). (B) Completion of second meiotic m aturation
first polar body.
(telophase II). The second polar body (PB2) is released and the female
Cytoplasmic maturation inclues the following: pronucleus (FPN) is developing. PB1, first polar body. (C) Cortical
reaction (CR): exocytosis of cortical granules (C). (D) Zona reaction (ZR):
1. Organelle redistribution: Endoplasmic reticulum and dispersed contents of cortical granules interacting with the zone (Z).
cortical granules are redistributed to increased concen (E) Associationn of MPN and FPN occurs eventually (arrows).
trations near the cortex. They are required for calcium
release and cortical reaction, respectively. Lipid drop-
lets, which are source of energy, accumulate around fashion and integrated into zygote membrane to form
the spindle. Mitochondria that were predominantly a mosaic membrane.
seen in the cortex become dispersed in the cytoplasm. • All structural and biochemical changes invoked in
2. Cytoskeleton Changes: The microfilaments scattered oocytes by the fertilizing spermatozoa are collectively
in the cytoplasm in GV stage migrate toward the called oocyte activation.
cortex. • The earliest visible indications of oocyte activation are A B
3. Protein Synthesis also occurs as a part of cytoplasmic cortical granule extrusion (CGE).
• Transient block to other sperm is due to depolariza- Figs. 2.6A and B: Sperm incorporation soon after fusion (diagram and TEM). This sperm (incorporated 3 hours post insemination) shows how a
maturation.
pathway opens for molecular interactions between sperm and ooplasm via the fusogenic domain. The sperm factor that activates the egg could
tion of the membrane, and permanent block is due
originate from the fusogenic, midsegment of the sperm head where sperm–egg membrane fusion occured (receptor or cytosolic mediated)
to cortical reaction. Cortical reaction involves protein or the perinuclear theca between the IAM and nuclear envelope (cytosolic mediated). C, centriole, I, inner acrosome membrane, MP, midpiece,
Sperm–Oocyte Fusion: Fertilization cross-linking resulting in inaccessibility and inactiva- O, ooplasm, T, phagocytic process of egg.
• Spermatozoa enter perivitelline space at an angle. tion of ligands for sperm receptors.
Inner acrosomal membrane (IAM) remains intact • Activation of metaphase II is indicated by the emer-
gence of pronuclei in the oocyte along with extrusion 1. The fusion theory—It is believed that the sperm head facilitate sperm entry post fertilization. It is believed
when the sperm reaches the perivitelline space. contains certain elements that bring about calcium to be triggered by phospholipase C zeta inositol-3-
Oocyte is sphere shaped while sperm head flat shaped of second polar body.
release upon fusion with oocyte. phosphate and mediated by IP3 receptor system.
(Fig. 2.4). 2. The receptor theory—This theory proposes that the
• First site of contact is equatorial region, i.e. distal por- Nature of Activating Stimulus oocyte harbours receptors that cause signal transduc-
tion of acrosome (Figs. 2.5A and B).7
Formation of Pronuclei
It is unclear how exactly the spermatozoa bring about tion that further causes influx of calcium.
• Whole of sperm plasma membrane posterior to acro- oocyte activation, and three theories have been proposed 3. The ‘‘calcium bomb’’ theory8—The source of c alcium • The second polar body is extruded after oocyte
some is internalized into the oolemma in a zipper-like (Fig. 2.6A and B): could be the sperm stores or channels in the sperm that activation.
14 The Infertility Manual The Human Sperm and Egg: Key Players of Fertilization 15
Impact of Intracytoplasmic Sperm 4. Tollner TL, Yudin AL, Treece CA, Overstreet JW, Cherr human oocytes following intracytoplasmic injection. Hum
GN. Macaque sperm coating protein DEFB126 facilitates Reprod. 1995;10(4):896-902.
Injection on Fertilization sperm penetration of cervical mucus. Hum Reprod. 9. Sathananthan AH, Ratnam SS, Ng SC, Tarin JJ, Gianaroli L,
2008;23:2523.
• In intracytoplasmic sperm injection (ICSI)—entire Trounson A. The sperm centriole: its inheritance, replication
5. Sathanathan AH, Ng S-C, Edirisinghe R, Wong PC. Human
spermatozoa + a small amount extracellular medium sperm egg interaction in vitro. Gamete Res. 1986b;15:317-26. and perpetuation in early human embryos. Hum Reprod.
6. Chen C, Sathananthan H. Sperm oocyte membrane fusion I 1996;11:345-56.
are deposited in an oocyte, which otherwise does not
the human during monospermic fertilization. Gamete Res. 10. Z P Nagy, Janssenswillen C, Janssens R, De Vos A,
happen in natural conception. 1986;15:177-86. Staessen C, Van de Velde H, et al. Timing of oocyte
• Following ICSI, the time taken for various critical steps 7. Sathananthan H, Ng S-C, Bongso A, Trouson A, Ratnam activation, pronucleus formation and cleavage in humans
is as follows: S. Visual atlas of early human development for assisted
reproductive technology. National University of Singapore, after intracytoplasmic sperm injection (ICSI) with testicular
Beginning of sperm nucleus decondensation— 1993. p. 209. spermatozoa and after ICSI or in-vitro fertilization on
30 minutes 8. Gearon CM, Taylor AS, Forman RG. Fertilization and early sibling oocytes with ejaculated spermatozoa. Hum Reprod
Oocyte activation (cortical granule exocytosis)— embryology: factors affecting activation and fertilization of 1998;13(6):1606-12.
60 and 90 minutes
Both pronuclei seen distinctly—about 4 hours
Fig. 2.7: The fertilized oocyte with zygote formation. • Pronuclear formation time does not significantly dif-
fer between ejaculated and testicular spermatozoa.10
However, in ICSI, nuclear decondensation may be
• Sperm head swells progressively due to chromatin slower due to the presence of acrosomal materials. But
decondensation. The IAM is intermittently discarded the male pronuclear development is not hindered.
and new nuclear envelope is organized by intercala- • In comparision to other species such as hamsters
tion of flattened element of egg smooth endoplasmic and guinea pigs, human spermatozoa do not possess
reticulum to accommodate the expanding male pro- huge acrosomes or plasma membranes with increased
nucleus. stability. This could be a reason why ICSI in humans is
• Simultaneously FPN forms from maternal chromatin so successful. Also oocyte tolerance to lytic acrosomal
decondensation. enzymes could be an additional factor.
• DNA duplication occurs following chromatin decon-
densation and it takes roughly 12–18 hours. Ultrastructural details of sperm and oocyte morphology
• Meanwhile, the two pronuclei also get closer to each and their contributions to fertilization and embryogenesis
other with the help of microtubules. Paternal centro- have been made possible by the use of electron micros-
some is the site of origin of microtubules, it lies beside copy, transition electron microscopy (TEM), and freeze
the forming paternal pronucleus. The microtubular pro- fracture models of study.
teins themselves arise from the cytoplasm of the oocyte.
PROBABLE QUESTIONS
Zygote Formation
1. Describe the process of fertilization.
• No merging of pronuclei happens. Dissolution of 2. Write in detail about sperm and oocyte maturation.
nuclear membrane occurs. Chromosomes of both align 3. Describe the morphology of mature oocyte and sperm.
themselves on the spindle apparatus at the e quator. 4. What is oocyte activation?
• The zygote, with new genome is formed when the mater-
nal and paternal chromosomes are aligned together on
a common spindle apparatus (Fig. 2.7). REFERENCES
• Mitotic spindle is formed about 22 hours post 1. Hertwig O. Beitrage zur Kenntniss der Bildung, Befruchtung
fertilization. und Tehilung des thierischen Eies. Morphol Jahrb.
• Approximately 3 hours post insemination, meiosis is 1876;1:347-434.
completed. First cell division plane is determined by the 2. Sathanathan AH. Visual Atlas of Human Sperm Structure
and Function for Assisted Reproductive Technology.
centriole of sperm.9 Gamma tubulin of the oocytes helps Singapore: National University; 1996. p. 279.
to activate sperm centriole to form aster, while α-tubulin 3. Sobrero AJ, MacLeod J. The immediate postcoital test.
aids to assemble aster and form first mitotic spindle. Fertil Steril. 1962;13:184.
C HA PT E R
Molecular Mechanisms: Endometrial
Receptivity and Implantation
3 Molecular Mechanisms: Endometrial Receptivity and Implantation 17
Nameeta Mokashi - Bhalerao
Chapter Outline
• Definition of Implantation • Impact of Ovarian Stimulation on Endometrial Receptivity
• Stages of Secretory Phase • The Science of Omics
• Steps of Implantation • Endometrial Receptivity Array
• Summary of Embryo Implantation • Future Directions
• Endometrial Receptivity (ER)
INTRODUCTION attachment to endometrial epithelium, i.e. 9–12 days post-
ovulation and then rises exponentially.7-9
In vitro fertilization (IVF) is considered to be one of the The human endometrium is a dynamic tissue; it under-
major medical breakthroughs of the 20th century. How- goes changes at multiple levels during the menstrual cycle Fig. 3.3: Stages of secretory endometrium. CAM, cellular adhesion
ever, despite consistent improvements and progress in in response to ovarian hormones and paracrine s ecretions molecule. Fig. 3.4: Preimplantation embryo development.11-14
assisted reproductive techniques (ARTs), implantation is (Fig. 3.2).6
still considered the “black box” or “bottle neck” of ART
outcomes.1 The majority of women undergoing IVF and
embryo transfer procedures achieve oocyte retrieval,
fertilization, and embryo development, but a relatively
small percentage of the transferred embryos actually
implant and develop into a viable pregnancy. In inter- Fig. 3.5: Cell types of a blastocyst.
national reports, pregnancy rates (PRs) range between
19 and 39% in IVF.2 In the best case scenario for patients
with a good prognosis and embryo selection, PRs still STAGES OF SECRETORY PHASE STEPS OF IMPLANTATION (FIG. 3.6)
only reach 61–70%.3,4 These results suggest that the uterus The following are the stages within the secretory Implantation has three steps,16 which are as follows:
Fig. 3.1: Contributors of implantation.
itself might play a more important role than previously endometrium (Fig. 3.3).10
considered.1 Molecular Mediators of Implantation and
Preimplantation, Embryo Development, Endometrial Receptivity
DEFINITION OF IMPLANTATION and Genomic Activation (Fig. 3.4) The endometrium produces proteins, cytokines, and other
Implantation is considered to occur when a blastocyst molecules in a cycle-dependent manner. Many of these
Blastocyst has two cell types: show increased expression in a temporal relation to the
breaches the luminal endometrial epithelium (Fig. 3.1).5
WOI and disappear thereafter. These are seen as chemical
It is a highly complex and multifactorial process involving
Hatching messengers recognized by the embryo and facilitate
spatiotemporally regulated endocrine, paracrine, auto- embryonic growth and differentiation. The use of such
crine, and juxtacrine modulators that span cell–cell and • Zona pellucida (ZP) keeps blastocyst inert and nonad-
proteins as biomarkers might help to define the period of
cell–matrix interactions. hesive to tube until window of implantation (WOI). receptivity, understand the implantation process, and may
Successful implantation at the very least requires the • As fluid accumulates in central blastocoelic cavity → identify women with implantation failure.17
presence of a healthy embryo, a receptive endometrium, a blastocyst expands → ZP thins out.
1. Mucins:
synchronized and successful molecular dialogue between • Trophectoderm cells produce proteolytic enzymes to
• High-molecular-weight glycoprotein interfering
the two and immune protection from the host.6 digest and escape out from ZP (Fig. 3.5).14 with cell–cell and cell–matrix adhesion.
The only established clinical marker of implantation is • Occurs at 72 hours after entry into the uterus. • Promotes implantation by repelling and guiding
hCG, produced by trophectoderm cells of blastocyst.5 It is • In humans: Invasive/interstitial/eccentric: Embed- blastocyst to find a spot devoid of glycocalyx for
Fig. 3.2: Dynamics of human endometrium. ding of blastocyst within subepithelial stroma.15
detected in maternal circulation within 3 days of embryo attachment.18
18 The Infertility Manual Molecular Mechanisms: Endometrial Receptivity and Implantation 19
8. Growth factors:31
• EGF, transforming growth factor (TGF), fibro-
blast growth factor (FGF), IGH-f, PDGF, heparin-
binding EGF-like growth factor (HB-EGF)
• Glycoproteins or peptides
• Regulated by paracrine, autocrine, and endocrine
signaling
9. Prostaglandins:19,32
• Prostaglandin D2 (PGD2), prostaglandin E2
(PGE2), prostaglandin F2 α (PGF2α), prostacyclin
Fig. 3.6: Steps of implantation. BM, basement membrane, CAM, cellular adhesion molecule. Fig. 3.8: Types of cellular adhesion molecules. (PGI2)
• PGE2, PGF2α, and PGI2 → ↑ vascular permeabil-
ity and edema at implantation site→ promotes
• Local loss of MUC1 at the precise site of attach- • IL-8 (α family): Potent chemo-attractant and acti- implantation
ment helps embryo apposition.20,21 vator of neutrophils and T lymphocytes 10. Glycodelin A or placental protein 14:
• Expression increased in luteal and implantation • Monocyte chemoattractant protein-1 (MCP-1) • Suppresses NK cells → embryo protection from
phase.19 (β family): Activator of monocytes, macrophages, maternal immune system33
• MUC1 and 6: Important markers of endometrial T cells, basophils, mast cells, and natural killer • The most abundantly secreted and consistently
receptivity (Fig. 3.7). (NK) cells:25,26 upregulated glycoprotein in late secretory endo-
• Mucin midsecretory expression reduced in recur- 1. LIF: Stimulators—IL-1α, TNF, platelet- metrium34
rent implantation failure (RIF). contained growth factor (PDGF), epidermal • A very promising marker:
growth factor (EGF), hCG, insulin-like growth Easily obtainable (serum/uterine flushing)
2. Cellular adhesion molecules (Fig. 3.8): factor (IGF)-1, IGF-2; inhibitors—interferon Good discriminative qualities17
• Serve as receptors for extracellular matrix or (IFN)27 11. HOXA (Homeobox) genes:
cell-surface ligands. 2. Colony-stimulating factor-1/macrophage CSF • Transcription factors and regulators of embryonic
• Act as modulators of endometrial and embryonic (CSF-1/M-CSF) induces proliferation and dif- morphogenesis and differentiation35
function. ferentiation of cells belonging to the mononu- • HOXA 10 and HOXA 11 upregulation in window
• Integrins proposed as markers for endome- clear phagocytic lineage.28 of implantation (WOI)
trial receptivity. Integrin αvβ3 is stimulated by 3. Interleukins29,30 (Table 3.1) • Reduced expression in hydrosalpinx, endome-
interleukin-α (IL-α), interleukin-β (IL-β), and triosis, polycystic ovary syndrome (PCOS)—
tumor necrosis factor-α (TNF-α) (Fig. 3.7).22,23 reduced implantation36
Table 3.1: Interleukin and its role in implantation.
3. Cadherins:
• Group of glycoproteins responsible for calcium-
Interleukin (IL) Role in implantation Additional Factors Influencing
IL-1 Induction of integrin expression, stimula- Implantation
dependent cell-to-cell adhesion24
tion of matrix metalloproteinase (MMP)
• Especially E-cadherin
• Dual function: IL-6 Immunomodulation, activation of MMP, 1. Inhibins A and B, activin, follistatin:
trophoblast growth • Members of TGF-β superfamily
1. Initial—Upregulated for adhesiveness
IL-10 Inhibits proliferation of Th1 leukocytes • Regulate angiogenesis, decidualization, immu-
2. Later—Downregulated to enable epithelial
IL-12 Vascular remodeling, uterine natural killer
nomodulation, and embryo implantation37
cells dissociation and blastocyst invasion
(uNK) cells activation 2. Relaxin:
4. Immunoglobulins: • Ovarian peptide hormone of IGF family
IL-15 Growth factor and activation of uNK cells
• Intercellular adhesion molecule-1 (ICAM-1) and • Increases glycodelin and vascular endothelial
CD-54 IL-17 Angiogenesis and immunoregulation growth factor (VEGF)
• Essential for transendothelial migration of LKs IL-18 Vascular remodeling, activation of uNK 3. MMPs and tissue inhibitors of metalloproteinase
and for various immunological functions cells (TIMP):38
Fig. 3.7: Profiles across the menstrual cycle of factors of importance to
5. Chemoattractant cytokines/chemokines: the interaction between human blastocyst and endometrium.17 CSF-1, • MMP 2, 9, and TIMP are key mediators for matrix
• Family of small polypeptides (70–80 amino acids) colony-stimulating factor-1; IL, interleukin; LH, luteinizing hormone; 6. Plasminogen activator digests intercellular matrix degradation during implantation and deciduali-
• α and β subtypes LIF, leukemia inhibitory factor. 7. Collagenases zation
20 The Infertility Manual Molecular Mechanisms: Endometrial Receptivity and Implantation 21
Fig. 3.11: Dynamics of steroids E and P in regulating estrogen receptor (ER).10
and induce changes in the stroma, which results in the 2. Emergence of microvilli on the luminal epithelium
process of implantation.42 This short period during which 3. Pinopodes:
the endometrium is receptive to embryo implantation is • Smooth mushroom or balloon—like projections
Fig. 3.10: Role of Th1–Th2 interplay of mutual homeostasis in
implantation.46 Abbreviations: APC, antigen-presenting cell; GM-CSF, referred to as the “Window of Implantation.” that arise from the apical surface of the luminal
granulocyte macrophage colony-stimulating factor; IL, interleukin; epithelium of the endometrium during WOI.
INFγ, interferon γ; TNF-α, tumor necrosis factor-α. Window of Implantation • Literally means “drinking foot,” signifying their func-
The endometrium is normally non-receptive for the tion of pinocytosis and endocytosis of uterine fluid
Fig. 3.9: Role of hCG in embryo–maternal signaling.40 CSF-1, colony- Mechanisms:41 embryo, except during WOI when the uterine environment and macromolecules, thereby facilitating adhesion
stimulating factor-1; GnRH, gonadotropin-releasing hormone; hCG, is conducive to blastocyst acceptance and subsequent of the blastocyst to the luminal epithelium.
human chorionic gonadotropin; LIH, leukemia inhibitory factor; LH, lu- 1. Placenta lacks MHC antigens40 implantation.
teinizing hormone; M-CSF, macrophage CSF; MMP, metalloproteinase; 2. Placenta secretes placenta-induced blocking factor
VEGF, vascular endothelial growth factor. (PIBF) and facilitating/enhancing antibodies
Salient features of WOI43 are as follows: Clinical Value of Pinopodes as a
3. Trophoblast cells express unique HLA-G, HLA-E, • Restricted time frame in the mid-luteal phase Marker of Endometrial Receptivity44
classical class I MHC molecule—HLA-C41 • Endometrium acquires anatomical, morphological,
• Balance between MMP and TIMP is crucial for molecular, and functional changes leading to a coordi- • Not useful as a consistent marker of ER
4. No activation of complement cascade
implantation 5. Downregulation of T-cell activity nated expression or repression of key molecules • Poor intra-patient consistency between cycles, i.e.
4. JAK/STAT intracellular signaling pathway 6. Th1–Th2 homeostasis (Fig. 3.10): • Allows the blastocyst to be received, attached, and poor reproducibility
STAT-3 proposed for trophoblast invasiveness ↓ TH1 (pro-inflammatory)—IL 1, IL 12, IL 15, IL 17, invaded via implantation • Poor reliability in predicting good ER or pregnancy
5. Endocrine control: IFN gamma, and TNF-α • Through mediation by immune cells, cytokines, growth outcome
a. Neuropeptides—Corticotropin-releasing hormone— ↑ TH2 (protective cytokines)—IL 5, IL 6, IL 10, and IL 13 factors, chemokines, and adhesion molecules • May have a prolonged (>5 days) presence in the luteal
induces stromal decidualizing effect of progester- 7. Immunomodulators such as haptoglobin, uter- • Opens on day 19 or 20 of the menstrual cycle or days 4 phase
one via IL-1, IL-6, PGE-239 iglobin, and PG-E or 5 after progesterone presence • Failure to delineate the brief (24–48 hours) window of
b. Leptin—Product of OB gene which ↑ integrin • Days 7–10 after LH surge receptivity.
αvβ3 expression • In an ART cycle, WOI can be induced by supplying E2
6. Serum marker:
SUMMARY OF EMBRYO IMPLANTATION and P4 to synchronize with embryo transfer
• Human leukocyte antigen G (HLA-G) is a major Embryonic implantation thus involves the following: • Remains open for an extended period at lower estro-
Biochemical Markers of Endometrial
histocompatibility complex (MHC) Class 1b gen levels but rapidly closes at higher levels due to Receptivity
1. Anchoring of conceptus in the maternal uterine wall
gene—modulates cytokine secretion to maintain aberrant expression of implantation related genes.
2. Embryo maternal cross talk involving various growth Biochemical markers of ER have been discussed along
local immunotolerance and control trophoblastic
factors, cytokines, and hormones with markers of implantation.
cell invasion Dynamics of E and P in
3. Trophoblast migration and differentiation
Embryo–maternal signaling through hCG (Fig. 3.9). 4. Establishment of vascular supply Regulating ER (Fig. 3.11) IMPACT OF OVARIAN STIMULATION
5. Leukocyte influx and activation Over-expression of ESR 1 Prog resistance → LPD, RPL,
Mechanisms of Immunotolerance 6. Promotion of tolerance of fetal alloantigens encoded
ON ENDOMETRIAL RECEPTIVITY
infertility, or persistent of PR in glandular epithelium is
of Pregnancy by paternal gene seen in endometriosis. (FLOWCHART 3.1)
Fetus is a semi-allograft as it has genetic make-up from 1. Supraphysiological serum estrogen:45
maternal and paternal genes.
ENDOMETRIAL RECEPTIVITY (ER) Morphological Markers of ER • Endometrial advancement
Immunomodulation is required to prevent maternal Endometrial receptivity is defined as a state when the 1. Transformation of fibroblast-like endometrial stro- • Stromal glandular dyssynchrony
immune system from rejecting the embryo. endometrium allows the blastocyst to attach, penetrate, mal cells into larger and rounded decidual cells • Advanced downregulation of steroid receptors
22 The Infertility Manual Molecular Mechanisms: Endometrial Receptivity and Implantation 23
Flowchart 3.1: Flowchart suggesting model for the favorable ef- Flowchart 3.2: Various models of “Omics.”6 Flowchart 3.3: Possible results of endometrial biopsy in ERA.
fect of injury (endometrial biopsy/curettage/hysteroscopy) induced
inflammation on implantation. TNF-α, tumor necrosis factor-α;
GRO-α, growth-regulated oncogene-α; IL15, interleukin-15; MIP-1B,
macrophage inflammatory protein 1B; NK, natural killer.46
Fig. 3.12: Timing of endometrial biopsy for ERA. HRT, hormone
replacement therapy.
Genomics, transcriptomics, and proteomics:
Need endometrial tissue biopsy, i.e. invasive
Interferes with process of implantation 7. What is role play of “Omics” in modern ART?
Secretomics of aspirated endometrial fluid—enables 8. Explain the potential “ERA” holds in maximizing
analysis of complex array of chemokines, cytokines, and implantation rates in IVF cycles.
growth and signaling factors.50
REFERENCES
Potential Uses of Endometriomics 1. Galliano D, Bellver J, Diaz-Garcia C, Simón C, Pellicer A.
ART and uterine pathology: how relevant is the maternal
• Nutritional environment of developing embryo
side for implantation? Hum Reprod Update. 2015;21:13-38.
• Markers for assessment of ER
2. Zegers-Hochschild F, Mansour R, Ishihara O, Adamson GD,
• Timing of embryo transfer de Mouzon J, Nygren KG, et al. International Committee
ENDOMETRIAL RECEPTIVITY ARRAY48-50 • Effect of therapeutic interventions for Monitoring Assisted Reproductive Technology: world
• Alternative stimulation regimens report on assisted reproductive technology, 2005. Fertil
• Developed by Diaz-Gimeno et al. (2011)
• Insight about diagnosis of RIF Steril. 2014;101:366-78.
• A bioinformatic predictor of endometrial dating
• Assessing impact of pathological conditions 3. Gardner DK, Surrey E, Minjarez D, Leitz A, Stevens J,
• Based on the transcriptomic signature of human ER, Schoolcraft WB. Single blastocyst transfer: a prospective
specifically when the human endometrium is recep- randomized trial. Fertil Steril. 2004;81:551-5.
tive to blastocyst adhesion FUTURE DIRECTIONS
4. Yang Z, Liu J, Collins GS, Salem SA, Liu X, Lyle SS, et al.
• A total of 238 genes that are differentially but consist- One of the main objectives in reproductive medicine has Selection of single blastocysts for fresh transfer via standard
2. Early and increased progesterone: ently expressed in optimal models been the search for markers predictive of ER. In light of morphology assessment alone and with array CGH for
• Premature luteinization • High reproducibility—transcriptomic profile valid the Omics revolution, the use of microarray or proteomic good prognosis IVF patients: results from a randomized
• Glandular maturation arrest over next 3 years technology will offer the possibility to better understand pilot study. Mol Cytogenet. 2012;5:24.
• Value greater than 1.5 ng/mL is detrimental • Sensitivity—99.758%, specificity—88.57% 5. Koot YE, Teklenburg G, Salker MS, Brosens JJ, Macklon
the biomarkers of ER leading to better implantation
3. Gonadotropin-releasing hormone (GnRH) and GnRH • Classifies endometrial sample as “receptive” or “non- NS. Molecular aspects of implantation failure. Biochimica
rates and will usher a new era of successful pregnan-
et Biophysica Acta. 2012;1822:1943-50.
agonists → antiproliferation receptive” cies. Till until then, our goal in the future should be to
• Nonreceptive result given a “personalized” WOI → 6. Mahajan N. Endometrial receptivity array: clinical
make repetitive implantation failure an exceedingly rare application. J Hum Reprod Sci. 2015;8:121-9.
Inflammatory Events in Endometrium personalized embryo transfer to improve reproductive occurrence. 7. Marshall JR, Hammond CB, Ross GT, Jacobson A, Rayford P,
outcome
Odell WD. Plasma and urinary chorionic gonadotropin during
THE SCIENCE OF OMICS • Good tool to provide favorable outcome in RIF
PROBABLE QUESTIONS early human pregnancy. Obstet. Gynecol. 1968;32:760-4.
• Accurate, reproducible, no inter-cycle variability 8. Armstrong EG, Ehrlich PH, Birken S, Schlatterer JP,
Omics technology has been used to identify biomarkers of • Defining a receptive window will avoid embryo wast- Siris E, Hembree WC, et al. Use of a highly sensitive and
human ER (Flowchart 3.2). age and emotional, physical, and financial distress. 1. Explain the dynamics of human endometrium as it
specific immunoradiometric assay for detection of human
Analysis of gene expression pattern is done by three undergoes through the menstrual cycle.
chorionic gonadotropin in urine of normal, nonpregnant,
approaches,47 which are as follows: 2. Explain in detail the steps involved in implantation.
Endometrial Biopsy Result 3. What is the role played by molecules mediating the
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1. Macroarray 1984;59:867-74.
2. Microarray
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(DDPCR)—whole genome analysis • Large variation + variable expression of genes 5. Explain the concept of window of implantation. 10. Fox C, Morin S, Jeong JW, Scott RT Jr, Lessey BA. Local and
Endometrial receptivity array (ERA) is one such • More importantly, the mRNA/gene products/proteins 6. Elaborate the role of various markers of endometrial systemic factors and implantation: what is the evidence?
c ustomized microarray. need to be analyzed receptivity. Fertil Steril. 2016;105(4):873-84.
24 The Infertility Manual Molecular Mechanisms: Endometrial Receptivity and Implantation 25
11. Braude P, Bolton V, Moore S. Human gene expression 27. Giess R, Tanaescu I, Steck T, Sendtner M. Leukemia 44. Quinn CE, Casper RF. Pinopodes: a questionable role in 48. Diaz-Gimeno P, Ruiz-Alonso M, Blesa D, Simón C.
first occurs between the four and eight cell stages of inhibitory factor gene mutations in infertile women. Mol endometrial receptivity. Hum Reprod Update. 2009;15(2): Transcriptomics of the human endometrium. Int J Dev
preimplantation development. Nature. 1988;332:459-61. Hum Reprod. 1999;5:581-6. 229-36. Biol. 2014;58:127-37.
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DNA deletion in human oocytes and embryos. Mol Hum stimulating factor-1 and c-fms expression in human of perimplantation endometrium in patients having Conejero JA, Alamá P, et al. The accuracy and reproducibility
Reprod. 1999;5(9):845-50. endometrial tissues and placenta during the menstrual cycle excessively high oestradiol concentrations after ovarian of the endometrial receptivity array is superior to histology
13. Heikinheimo O, Gibbons W. The molecular mechanisms of and early pregnancy. J Clin Endocrinol Metab. 1991;73:746-51. stimulation. Hum Reprod. 2001;16:435-40. as a diagnostic method for endometrial receptivity. Fertil
oocyte maturation and early embryonic development are 29. Polan ML, Simon C, Frances A, Lee BY, Prichard LE. Role 46. Granot I, Gnainsky Y, Dekel N. Endometrial inflammation Steril. 2013;99:508-17.
unveiling new insights into reproductive medicine. Mol of embryonic factors in human implantation. Hum Reprod. 50. Boomsma CM, Kavelaars A, Eijkemans MJ, Amarouchi K,
and effect on implantation improvement and pregnancy
Hum Reprod. 1998;4(8):745-56. 1995;10(Suppl 2):22-9. outcome. Reproduction. 2012;144:661-8. Teklenburg G, Gutknecht D, et al. Cytokine profiling
14. Sathananthan H, Menezes J, Gunasheela S. Mechanics 30. Inagaki N, Stern C, Mc Bain J, Lopata A, Kornman L, Wilkinson D. 47. Dominduez J, Pellicer A, Simon C. Human endometrial in endometrial secretions: a non-invasive window on
of human blastocyst hatching in vitro. Reprod Biomed Analysis of intra-uterine cytokine concentration and matrix receptivity: a genomic approach. Reprod Biomed Online. endometrial receptivity. Reprod Biomed Online. 2009;18:
Online. 2003;7(2):228-34. metalloproteinase activity in women with recurrent failed
15. Bonnet R. Beitrage zur embiologie der weiderkauer, 2003;6(3):332-8. 85-94.
embryo transfer. Hum Reprod. 2003;18:608-15.
gewonnen am schfei. Arch Anat Physiol. 1884;8:170-230. 31. Lessey BA, Gui Y, Apparao KB, Young SL, Mulholland J.
16. Enders AC, Schlafke S. Cytological aspects of trophoblast-
Regulated expression of heparin-binding EGF-like growth
uterine interaction in early implantation. Am J Anat.
factor (HB-EGF) in the human endometrium: a potential
1969;125(1):1-29.
paracrine role during implantation. Mol Reprod Develop.
17. Lindhard A, Bentin-ley U, Ravn V, Islin H, Hviid T, Rex S,
2002;62:446-55.
et al. Biochemical evaluation of endometrial function at
32. Song H, Lim H, Paria BC, Matsumoto H, Swift LL,
the time of implantation. Fertil Steril. 2002;78(2):221-33.
Morrow J, et al. Cytosolic phospholipase A2alpha is crucial
18. Achahe H, Revel A. Endometrial receptivity markers, the
journey to successful embryo implantation. Hum Reprod for on-time embryo implantation that directs subsequent
Update. 2006;12(6):731-46. development. Development. 2002;129:2879-89.
19. Hey NA, Graham RA, Seif MW, Aplin JD. The polymorphic 33. Okamoto N, Uchida A, Takakura K, Kariya Y, Kanzaki H,
epithelial mucin MUC1 in human endometrium is Riittinen L, et al. Suppression by human placental protein
regulated with maximal expression in the implantation 14 of natural killer cell activity. Am J Reprod Immunol.
phase. J Clin Endocrinol Metab. 1994;78(2):337-42. 1991;26:137-42.
20. Meseguer M, Aplin JD, Caballero-campo P, O’Connor JE, 34. Horcajadas JA, Riesewijk A, Martin J, Cervero A, Mosselman S,
Martín JC, Remohí J, et al. Munan endometrial mucin Pellicer A, et al. Global gene expression profiling of human
MUC1 is up-regulated by progesterone and down- endometrial receptivity. J Reprod Immunol. 2004;63:41-9.
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2001;64(2):590-601. 1994;78:191-201.
21. Carson DD, Lagow E, Thathiah A, Al-Shami R, Farach- 36. Daftary GS, Taylor HS. Molecular markers of implantation:
Carson MC, Vernon M, et al. Changes in gene expression clinical implications. Curr Opin Obstet Gynecol. 2001;13:
during the early to mid-luteal (receptive phase) transition in 269-74.
human endometrium detected by high-density microarray 37. Godkin JD, Dore JJ. Transforming growth factor beta and
screening. Mol Hum Reprod. 2002;8(9):871-9. endometrium. Rev Reprod. 1998;3(1):1-6.
22. Illera MJ, Cullinan E, Gui Y, Yuan L, Beyler SA, Lessey BA. 38. Vu TH, Werb Z, Matrix metalloproteinases: effectors
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23. Lessey BA, Castelbaum AJ, Sawin SW, Sun J. Integrins as
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Margioris AN, Coukos G, et al. Corticotropin releasing
unexplained infertility. Fertil Steril. 1995;63(3):535-42.
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24. Kumar S, Zhu LJ, Polihronis M, Cameron ST, Baird DT,
Schatz F, et al. Progesterone induces calcitonin gene maternal tolerance. Nat Immunol. 2001;2(11):1018-24.
expression in human endometrium within the putative 40. Herrler A, von Rango U, Beier HM. Embryo maternal signalling
window of implantation. J Clin Endocrinol Metab. how the embryo starts talking to its mother to accomplish
1998;83(12):4443-50. implantation. Reprod Biomed Online. 2002;6(2):244-56.
25. Cavagna M, Mantese JC. Biomarkers of endometrial 41. Hunt JS, Fishback JL, Andrews GK, Wood GW. Expression
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Hum Reprod. 2003;9(4):189-98. human endometrium. Immunobiology. 2004;209:569-74.
C HA PT E R
Infertile Couple—Initial Investigations
4 EVALUATION OF OVARIES
Ovarian Reserve
Ovarian reserve is the number and quality of remaining
Infertile Couple—Initial Investigations 27
in urine, endometrial biopsy, and transvaginal ultrasonog-
raphy have been used to detect ovulation.
Women with irregular cycles are tested for FSH,
LH, estradiol, thyroid-stimulating hormone (TSH), and
prolactin.
Chaitra Nayak follicles and oocytes in both ovaries at a given age.3 It helps
High FSH and LH with low estradiol indicate primary
in prognosticating patients with higher risk of deceased
ovarian failure; low FSH, LH, and estradiol indicate
Chapter Outline ovarian reserve.
hypo
gonadotropic hypogonadism; and high TSH or
• History and Physical Examination • Evaluation of Male Infertility Risk factors for decreased ovarian reserve include the
pro
lactin with all other tests being normal indicates
• Evaluation of Ovaries following:1
hypothyroidism or hyperprolactinemia, respectively.
1. Women over 35 years of age
2. Family history of early menopause Evaluation of Fallopian Tubes
INTRODUCTION Contd...
3. Women who have undergone single ovary/ovarian
About 26% of infertile couples have tubal factor infertility.
Female history1 Male history2 surgery, chemotherapy, or radiotherapy
Infertility is the inability to conceive after 1 year of regular Methods to evaluate the fallopian tube infertility are com-
Surgeries: Pelvic surger- Medication history: Expo- 4. Unexplained infertility
unprotected intercourse. plementary and include hysterosalpingography (HSG),
ies with indication and sure to gonadotoxins includ- 5. Poor response to gonadotropin stimulation
Incidence is reported in 10–15% of the population. saline infusion sonogram (SIS), and laparoscopy.3
intraoperative findings ing heat, current medications, 6. Candidates wanting assisted reproductive technology
Earlier evaluation is indicated if:1,2 and allergies (ART)
1. Age of the female partner is more than 35 years Investigations: Details of Pelvic surgery including Hysterosalpingography3
2. There is history of amenorrhea or oligomenorrhea or previous USG, hormonal in- varicocelectomy and sexually Antral Follicle Count3 • It uses either oil- or water-based contrast media to
irregular cycles vestigations, or Pap smear transmitted diseases delineate uterine cavity and tubes and is done from
3. Diagnosed with or suspected for male subfertility, Antral follicle count (AFC) is 2–10 mm in mean diameter in
Endocrine abnormalities: Social history: Use of day 6 to 11 of menstrual cycle.
including cryptorchidism Thyroid disorder, hirsutism, anabolic steroids, recreation- the greatest two-dimensional plane.
• It is a useful modality to look for tubal patency and can
4. Stage III or IV endometriosis or uterine or tubal or galactorrhea al drugs, smoking and alcohol It is menstrual cycle dependent and measured in the
show proximal and distal tubal occlusion, hydrosal-
consumption first 5 days.
pathology pinx, tubal phimosis, and peritoneal spill.
Family history: Birth Occupation history: Low AFC (<5–7 in total) has poor response to ovarian
• Delayed spill of the dye is indicative of peritubal adhe-
HISTORY AND defects, early menopause, Exposure to e nvironmental stimulation.
sions or fimbrial phimosis.
intellectual disability, or pollutants, chemicals, High AFC is seen in patients with polycystic ovary syn-
PHYSICAL EXAMINATION amenorrhea exposure to radiation drome (PCOS). AFC is cycle dependant.
• Proximal occlusions are commonly due to tubal spasm
or minor adhesions. Sensitivity is 65% and specificity
Occupation/recrea- Developmental history: is 83%.
Female history 1
Male history 2
tional history: Exposure Delayed onset of puberty, Serum AMH3 • Contraindications: contrast allergy, active pelvic infec-
Menstrual history: Age Sexual history: Coital
to tobacco, alcohol, and cryptorchism, or genital
of menarche, length and frequency, dyspareunia AMH is produced by the granulosa cells of early follicles, tion and pregnancy.
recreational drugs malformations
duration of menstrual erection and ejaculatory which are gonadotropin independent, and measurement
bleed, dysmenorrhea, inter- disorders
Physical examination of the female partner looks
of AMH is done independent of the day of menstrual HyCoSy3
menstrual bleed cycle. Low serum AMH levels (<0.5–0.7 ng/mL) is seen
Obstetric history: Details Reproductive history: for body mass index (BMI), thyroid disorders, breast in poor responders, and higher levels of AMH is seen in • It involves infusion of saline or contrast media under
of previous pregnancies; Duration of infertility and prior secretions, signs of androgen excess, hirsutism, and women with PCOS. ultrasound guidance to look for tubal patency.
mode of conception, course fertility insulin resistance. A detailed pelvic examination is also • Presence of fluid in the pouch of Douglas is indicative
of pregnancy and delivery of at least one patent tube. It however cannot differen-
required. Serum FSH
Contraceptive history: Medical history: Repeated tiate between unilateral or bilateral tubal patency.
Duration and type of con- respiratory infections, diabe- Serum follicle-stimulating hormone (FSH) is the com-
traception used tes mellitus, neurological, or Diagnostic Evaluation monly used and most indirect marker of ovarian reserve.
Laparoscopy3
systemic illness of the Female Partner1,3 It is a better predictor of poor ovarian reserve.
Previous treatment his- Past history: Childhood • A dilute solution of Indigo Carmine or Methylene blue is
tory: Details of ovulation mumps or significant illness The age of the female partner and duration of married life introduced transcervically and tubal patency is looked
induction (OI), intrauter- in the past, development are significant factors in the history and guide the relevant Evaluation of Ovulation1,3
for, as evidenced by spillage of the dye from the tubes.
ine insemination (IUI), or history investigations to be done. Majority of women with regular menstruation have ovula- • In the same sitting, corrective procedures such as per-
in vitro fertilization (IVF) The ovaries, tubes, and uterus are mainly evaluated in tory cycles. Serum progesterone more than 3 ng/mL in the itubal adhesiolysis, opening fimbrial phimosis, and
Contd... the infertile woman. mid-luteal phase, detection of luteinizing hormone (LH) hysteroscopic tubal cannulation under laparoscopic
28 The Infertility Manual Infertile Couple—Initial Investigations 29
guidance can be done, which is not possible with Semen is collected by a masturbation sample after an Depending on the results of the repeat semen analy- percent of azoospermic men have Y chromosome micro-
HSG. abstinence period of 2–7 days. sis and physical examination, further evaluation including deletion.
• It is regarded as the gold standard to look for tubal Any spillage of sample, difficulties in ejaculation, or appropriate endocrine or ultrasound examination is done.
patency, and in addition to the above-mentioned exam- admixture of urine must be reported. Retrograde ejacula- High FSH and LH with low testosterone indicate pri- AZFa AZFb AZFc
inations, confirmation of hydrosalpinx can also be done. tion will require a post-ejaculate urine examination. mary testicular failure, while low FSH, LH, and testoster- Incidence Less Less Commonest, 60%
• Tubal cannulation can be done either by fluoroscopy one indicate hypogonadotropic hypogonadism. common common
or hysteroscopy if proximal tubal occlusion is detected Azoospermia is the absence of sperms in the pellet
either on HSG or laparoscopy with chromotubation. A
WHO Semen Analysis 2010 Parameters after centrifuging the semen sample for 15 min at 3,000 g.
Histology Sertoli Germ cell Hypospermatogenesis
cell only arrest
set of specialized catheters are used to cannulate the Criteria Reference value The current assessment of morphology of sperms is Prognosis Poor Poor Good with ICSI
fallopian tubes transcervically. Volume ≥1.5 mL based on the Kruger or strict criteria and is used to iden-
tify couple who may benefit from intracytoplasmic sperm
Total sperm number ≥39 million/ejaculate
Evaluation of Uterus1,3 Sperm concentration ≥15 million/mL
injection (ICSI) rather than in vitro fertilization (IVF) PROBABLE QUESTIONS
and hence helps in tailoring appropriate treatment for
Total motility ≥40% 1. How do you evaluate an infertile couple?
Ultrasonography (USG) infertility.
Progressive motility ≥32% 2. What are the indications for early evaluation in an
Two-dimensional transvaginal USG is the simplest and Normal morphology ≥4% infertile couple?
commonest evaluation of pelvis with sensitivity of 56% and Additional Sperm Function Tests3 3. What is ovarian reserve and how is it measured?
Vitality ≥58%
specificity of 89% for uterine anomalies. However, it fails to Sperm DNA fragmentation This can occur during sper- 4. Discuss the evaluation of irregular cycles in the
differentiate between intrauterine lesions like endometrial matogenesis or transport of sperms in the genital tract. female partner.
polyps and submucosal fibroids.
Grading of Varicocele Higher incidence of DNA fragmentation is seen in infer- 5. What are the methods available for evaluation of Fal-
tile males but may also be seen in men with normal sperm lopian tubes?
Hysterosalpingography (HSG) Grades Varicocele parameters. 6. Write a note on evaluation of the infertile male.
I Palpable only with Valsalva maneuver Low DNA fragmentation is associated with better 7. What are the WHO semen analysis parameters?
It delineates the cavity of the uterus and is a useful modal-
II Palpable even without Valsalva maneuver chances of conception. 8. How do you evaluate a case of azoospermia?
ity to differentiate uterine anomalies like sub-septate and
bicornuate uterus. Polyps, synechiae, and fibroid are best III Seen by visual inspection Genetic testing In patients with primary testicular fail-
detected by a three-dimensional sonography or magnetic ure, karyotyping and Y chromosome microdeletion are REFERENCES
resonance imaging (MRI). indicated.
What if a Semen Analysis is Abnormal?2,4 1. Diagnostic evaluation of the infertile female: a committee
opinion. Practice Committee of American Society for
Sonosalpingograph can differentiate between endo- Karyotyping may reveal chromosomal abnormali-
metrial polyps, submucosal fibroids, and synechiae and When the first semen analysis is abnormal, a repeat semen Reproductive Medicine. Fertil Steril. 2012 Aug;98(2):302-7.
ties such as Klinefelter syndrome or translocations of 2. Diagnostic evaluation of the infertile male: a committee
has a high positive predictive value for the same. analysis is asked for and the male partner is examined by a
chromosomes. Non-mosaic Klinefelter accounts for
opinion. Practice Committee of American Society for
urologist or andrologist, who in turn takes a complete his- Reproductive Medicine. Fertil Steril. 2012;98(2):294-301.
11% of azoospermic males, and preimplantation genetic
Hysteroscopy tory and does a detailed physical examination.
diagnosis (PGD) needs to be discussed in such couple 3. Roux I, Tulandi T, Chan P, Holzer H. Initial investigation
of the infertile couple. Textbook of Assisted Reproductive
It is the method of choice for detecting intrauterine pathol- going for ART.
Techniques, 4th edition. Volume 2: Clinical Perspectives,
ogies as it also gives an opportunity to operate on the same. Examination of Male Partner Y chromosome microdeletion is checked for in three 2012. pp. 31-40.
However, it is an invasive modality involving surgery and specific regions of the long arm of Y chromosome, namely, 4. WHO laboratory manual for the examination and
The examination of male partner includes the following:
hence better alternatives are available. AZFa, AZFb, and AZFc (AZF—azoospermic factor). Seven processing of human semen Fifth edition.
1. Height, weight, and BMI
Three-dimentional Ultrasound 2. Secondary sexual characters including hair distribu-
tion, body habitus, breast development, and signs of
It is excellent in detecting congenital Müllerian anomalies appropriate androgenization
and other uterine pathologies with a high correlation with
3. Palpation and measurement of testes (Prader orchi-
MRI, hysteroscopy, or laparoscopy especially when per-
dometer)
formed during the luteal phase.
4. Presence and consistency of vasa and epididymis
5. Examination of the penis and location of the opening
EVALUATION OF MALE INFERTILITY4 of urethral meatus
Semen analysis is the cornerstone of evaluation of male 6. Presence or absence of varicocele with grade
infertility. 7. Serum FSH, LH, TSH, and prolactin.
C HA PT E R
Counseling in Infertility
5 invasive medical treatments; they also have to face com-
plex decisions associated with choice of techniques,
disposal of embryos, the possibility of complications
associated with treatments (often related to the cause of
infertility) that may result in pregnancy or birth com-
CULTURAL ISSUES RELATED
TO INFERTILITY
Counseling in Infertility 31
certain scenarios where the woman is not working, the state
of pseudo-waiting for motherhood can increase her stress.
Shreedevi Kumar
plications.5 Their frequent visits to hospital for consul-
tation, physical examination, investigations, and treat- Feeling the Pressure from Family Members
Chapter Outline
• Infertility as a Cause and Effect of Stress • Financial Aspects of Infertility Treatment
ment procedures are time consuming; hence couples, and Relatives
• Treatment-induced Stress • Gender more specifically women, have to make adjustments
• Psychological Aspects of Infertility • Types of Counseling and compromises in their p rofessional and personal In a culture where children are highly valued, the state of
• Social Issues Related to Infertility • Special Considerations during Third-party Reproduction
lives. Many couples also drop out of treatment either childlessness may induce stigma for the whole family, let
• Cultural Issues Related to Infertility alone the couple.
after their first experience of failure or after repeated
negative results, which causes feelings of hopelessness
and helplessness.
In Male Sterility
INTRODUCTION individuals become patients, undergoing a range of medi-
cal treatments for being childless and assume passive As many myths about male infertility continue to exist, it
Around 80–168 million of people are affected by infer- creates a highly stigmatizing situation across many cul-
tility in the world today. It is estimated that one in every
patients role—all a quest of parenthood.4 As a result of this PSYCHOLOGICAL ASPECTS
and associated treatment challenges, infertility has not tures. It is worthwhile to ascertain his feelings about the
10 couples are experiencing primary and/or secondary remained a medical condition alone but has become the
OF INFERTILITY diagnosis as well as what are his partner’s feelings and
infertility.1 Its wide variance in incidence rate contributes cause and effect of social and emotional conditions. When the wish to have a child remains unaccomplished, those of their families.
to significant and unique psychosocial consequences it can result in emotional and physical stresses in majority
impacting individual and their social well-being. of the population. They frequently experience a range of
Opinion about Options to a
Whether driven by biological need, social compul- INFERTILITY AS A CAUSE AND Biologically Linked Child
psychological feelings such as anger, humiliation, denial,
sion, or psychological desire, the pursuit of a child has EFFECT OF STRESS confusion, guilt, blaming, anxiety, and depression often For many couples (in some cultures), a biologically linked
compelled men and women to seek a variety of remedies,2 leading to poor self-esteem. Stress becomes an added
Around the world, infertility is recognized as a stressful child is the primary focus to each partner; hence, thinking
therefore seeking assistance to have children through experience, which can potentially threaten individual, factor, as those already impacted struggle to take compli- and planning for an alternative way to have a child is sel-
medically advanced interventions, such as in vitro fertili- marital, familial, and social stability. This may be due to cated decisions about how long or even how far to endure dom discussed or appreciated that delays the timely deci-
zation (IVF) and intracytoplasmic sperm injection (ICSI) the unexplained or involuntary childlessness, disrupted sion making.
in pursuit of a child. Infertility may even trigger feelings
have increased manifold. For most couple, assisted repro- reproduction, the diagnosis of infertility itself, or the of failure, embarrassment, or personal disappointments,
ductive techniques are the last, best option for having a demands of the medical system to treat the condition of which in turn lead to strained relationships with spouse, FINANCIAL ASPECTS
child and occur after long months, and sometimes years of infertility. Some of the common concerns are discussed in family, and the social circle. OF INFERTILITY TREATMENT
treatment, often at tremendous emotional, physical, and Figure 5.1.
financial cost.3 Couples seeking assisted reproductive technique (ART)
Numerous claims of success of assisted reproduc- SOCIAL ISSUES R
ELATED TO may also experience considerable burden of cost involved
tive technologies have brought increased expectation
TREATMENT-INDUCED STRESS INFERTILITY with treatment. A number of infertility treatments may not
and aspiration in the minds of childless couple who go Couples not only have to understand complex biologi- be covered under insurance plans and therefore becomes
As infertility happens to be a life-altering phenomenon
into a process which has been termed as “medicaliza- cal processes and treatment procedures; or face delays unaffordable; surrogacy and adoption may be even more
creating an isolating and stigmatizing environment that
tion of infertility.” It is a phenomenon wherein healthy and failures, and the effects of intensive, protracted, and convoluted and high priced.
occurs not only within the context of the individual’s or
couple’s life but also with their social milieu.
GENDER
Sharing and Confinement in a Social Circle Although infertility affects both men and women equally,
More often than not, the subject of childlessness is so there may be some cases with regional variations existing in
our population.6 The distress is more obvious in men specif-
stigmatizing that one partner or the couple is unable to talk
ically when a male-factor infertility is diagnosed, whereas
about it with anyone around them, resulting in isolation.
women report stronger negative reactions to overall infer-
tility.7 These stronger response to infertility by women than
Career Compromise those of men is probably due to multiple factors: some
In majority of the cases, woman does not want to pursue medical procedures that are invasive in nature are exclu-
Fig. 5.1: Infertility factors generating stress
career interests or goals in anticipation of parenthood. In sive to infertile women and many of the medical treatments
32 The Infertility Manual Counseling in Infertility 33
for infertility given in the form of hormonal medications are While infertility experience is not easy to handle for TYPES OF COUNSELING domain of the physician, and mental health professionals
administered on women irrespective of the cause of infer- anyone, some cope very well on their own. However, should also have fairly adequate knowledge of the medical
tility. These medications have unavoidable side effects that Today, the role of the infertility counselor has expanded
when the individual or couple finds that infertility is tak- issues involved for providing counseling.
can lead to certain psychological symptoms such as mood to deal with various psychosocial challenges of assisted
ing a toll on their functioning, their coping mechanisms Decision-making counseling involves helping the
swings whereas men typically receive none of these power- reproduction, which includes assessment, treatment, sup-
are failing and impairs their quality of life one should con- couple to understand the various treatment options avail-
ful medications as part of their treatment.8 port, education, research, and consultation.
sider counseling. able for them and about parenting. They are facilitated to
choose the best suitable treatment, after carefully consid-
Diamond and colleagues9 illustrated five distinct phases Psychological Assessment/Screening
of infertility: Infertility Counseling ering all available options. It is even more crucial when
Assessing people presenting with infertility for their clini- egg/sperm donation and surrogacy is being thought of.
They are Dawning, Mobilization, Immersion, Resolution, Infertility counseling as an emerging specialty for the
and Legacy. cal intervention is the primary responsibility of the medi- The implications on and rights of the unborn child are
identification of psychological distress and to provide cal doctor or assisted reproductive treatment specialist of
1. During the Dawning phase, couples become progressively to be kept in mind. This method of counseling is aimed
aware that they are not able conceive naturally, hence expert care and suitable intervention in conjunction with the infertility clinic. On the other hand, screening aspiring
complex medical treatment have been suggested by pro- particularly for those couple who intend to use donated
explore various options available and then come forward couple for their psychosocial vulnerabilities, social sup-
to seek medical assistance from a specialized doctor. fessionals in the field, legislated, and/or recommended on sperm, eggs, or embryos. Genetic counseling can also be
ports, coping abilities, and distress is the responsibility of a
2. Mobilization indicates the first step into the medical the basis of evidence-based research. In the field of repro- part of this mode of counseling.
treatment wherein the couple start with initial diagnos-
mental health professional. Various tools have been devel-
tic testing. A definitive diagnosis can result in shock,
ductive medicine, infertility counseling began to grow oped to help identify couples likely to be distressed with
disbelief, and denial, particularly in the case of sec- as an allied profession in accord with the major medical IVF procedures though they are not widely used. More Support Counseling
ondary infertility. As the couple faces the first of what advancements, particularly assisted and third-party repro- reliable tools such as SCREEN IVF, Fertility Quality of Life
will probably be many losses, it may ignite differences Support counseling, as the name suggests, supports infer-
duction. Although the psychological impact of infertil- (FertiQoL) provide pathways to develop interventions.
and invite problems in their relationship. tile patients in distress. The source of distress may be the
ity was addressed in literature in the beginning of 1930s, However, current roles of counseling in ART centers go far
3. Immersion phase contains complex and demanding individual whose needs to have a wanted child are not met
process, as the couple undergoes numerous tests and infertility counseling to emerge as a recognized profession beyond screening12 by adding therapeutic interventions in
from the partner, immediate family, or the society. These
intense treatment. This stage endures the feelings of being and mental health speciality it took nearly 30 years.10 various phases of treatment.
also include group therapy, and the counseling may need
in “limbo” or “not parents yet” because of the obstacles to Boivin and colleagues11 from several European nations However, infertility medical procedures involve coun-
move ahead to the next stage of the life cycle, i.e. par- to extend even after the couple stops treatment for infer-
in 2001 published infertility counseling guidelines under the seling to be part of the treatment. The following types of
enting. Late in the Immersion phase, couples may think counseling are commonly performed, although in practice tility. The aim is to provide emotional support during the
auspices of the European Society of Human Reproduction
of alternative methods of building family that they would time of particular stress—for instance, when treatment
have been avoiding or did not prefer to consider: decisions and Embryology (ESHRE). These guidelines illustrate the these approaches will normally overlap. (Source: Human
Fertilisation and Embryology Authority, Code of Practice, fails in achieving a pregnancy or the pregnancy ends in
about donor gametes, donor embryos, or adoption. rationale and objectives for providing counseling and psy-
4. The Resolution phase consists of three subphases 1998, part 6.) miscarriage or bio-chemical pregnancy.
chosocial care to infertility patients in both general and spe-
that are overlapping: (1) wind-up medical treatment, cific situations, e.g. infertility-induced distress, planning and It is important to keep in mind that most high-tech and
(2) accepting the loss of not having a genetically Information Gathering and Analysis high cost fertility treatment such as IVF or ICSI not neces-
ending treatment, and third-party reproduction, and so on.
shared (or related) child, and (3) refocusing on other sarily offer an excellent success rates. There are potential
viable options such as prenatal adoption, traditional Imparting relevant information about the medical aspects
adoption, or childlessness.
chances for disappointment connected with these proce-
Major goals of infertility counseling are as follows: of treatment is the primary responsibility of the repro-
5. The Legacy phase encompasses after effects of the dures and on its outcome. Support counseling can also be
•• Improving the quality of life of couple with infertility ductive specialist. However, patients directly approach
infertility experience including the marital, sexual, and during and after treatment used in certain situations where the patient has twins or
parenting problems that may emerge as an outcome or consultants refer the patient to counselors (as part of
•• Decreasing the negative impact of infertility triplets.
of infertility, particularly when partners have not pre- a mandatory referral process) to gain more information
•• Facilitating the pre-eminence of infertility experience
pared adequately to handle the significant losses of it. with regard to the social and emotional implications of the
so as to minimize long-term impairment or distress
•• Ensuring the healthy adjustment of all participants, infertility treatment. Counselor should help the individual Therapeutic Counseling
When is it Time to See especially in families formed through complex (third to collect all the information and make sense of it while
Therapeutic counseling also incorporates support coun-
an Infertility Counselor? party) reproduction or adoption and for children with a deciding about the treatment and parenting options.
seling, but it includes some aspects beyond it. In a broader
legacy of parental infertility12
After a thorough evaluation of the couple, a cause for their sense, it aims to help people cope with the consequences of
infertility may be found or in about 15% of couple may Implication Counseling and infertility and fertility treatment. The Human Fertilization
The objectives of infertility counseling is to:
be unexplained. All couple have to be explained about Decision-making Counseling and Embryology Authority (HFEA) brochure says that the
their available treatment options and should be helped in •• Help the couple to express their emotions
•• Identify individual specific causes of distress Implication counseling intends to facilitate the person or purpose of the counseling is to help people to modify their
deciding on the best line of action for their particular case.
•• Enable coping strategies, offer preparation for procedure people concerned to understand the implications of the expectations and to accept the reality. It may also help
Few situations in life are as challenging and over-
•• Facilitate decision making recommended course of treatment for themselves, for couples resolve the problems in their relationship caused
whelming as coping with infertility. Yet, maximum empha-
•• Explore the impact of infertility on the lives of people their family, and for any children born as a result. While by the stresses of fertility issues and treatment. As the
sis is projected on the physical aspects of infertility and the
as well as of significant others information about treatments and procedures is the therapy is little more intensive, it can explore wider issues
emotional aspects most often go unnoticed and untreated.
34 The Infertility Manual Counseling in Infertility 35
that may be deterring a successful outcome. Therapeutic Introducing Infertility CONCLUSION 3. Covington SN. Patient support in the ART. 424-33.
counseling aims at helping the patient return to normal 4. Greil AL. Infertile bodies: Medicalization, metaphor, and
Counseling to Patients Over a period of time, the focus of counseling on the agency. In: MC Inhorn, F Van Balen (Eds). Infertility
activities of daily living and focus on coping mechanisms,
acceptance of infertility, alternative life-goal options, and In the introduction part of counseling, it is ideal to estab- psychological aspects of infertility is not on individual Around the Globe: New Thinking on Childlessness,
psychopathology alone, but spread to more holistic/ Gender, and Reproductive Technologies. Los Angeles, CA:
resolution of particular issues. lish an open communication with the patients on their University of California Press; 2002. pp. 101-18.
interactive views of infertility and to the impact of
notions, expectations, and reservations about counseling. 5. Barnes M, Roiko A, Reed R, Williams C, Willcocks K.
advancing assisted reproductive technologies. Besides,
SPECIAL CONSIDERATIONS DURING Subsequently, the counselor need to be aware of the
there has been a shift from a singular focus on the indi-
Outcomes for women and children following assisted
reluctance and bias of the patient as it is a natural response. conception. Implications for perinatal education, care and
THIRD-PARTY REPRODUCTION vidual to holistic approach in the form of assessments support. J Perinat Edu. 2012; 21 (1):18-23.
Because, for many, it may be the first ever time meeting with
followed by interventions aimed at groups, be it cou- 6. Thonneau P, Marchand S, Tallec A, Spira A. Incidence
It is a mandatory requirement for all those who have a counselor and they might feel intruded and stigmatized
ples or families. While clinical experiences indicate that and main causes of infertility in a resident population
decided to undergo treatment to meet a counselor before on having to come to share with someone their intimate
most of infertile men and women not necessarily expe- (1,850,000) of three French regions (1988–1989). Hum
commencing IVF/ICSI or donor gamete treatment (includ- matters such as wish for a child and procreation. Based on
rience significant levels of psychological trauma or psy- Reprod. 1991; 6:811-16.
ing donors and their partners) to consider all the implica- how the counseling session was introduced and conveyed, 7. Connolly KJ, Edelmann RJ, Cooke ID. Distress and marital
tions and receive information, education, direction. The chopathology, the use of advanced medical technology
the patients may fear rejection or they might perceive problems associated with infertility. J Reprod Infant Psych.
counselor can also cover some aspects of psychological and/or third-party reproduction involving a plethora
themselves to be weak for breaking down in front of their 1987;5:49-57.
interventions. of additional stressors may increase psychological dis- 8. Daniluk JC. Gender and infertility. In: Leiblum SR (Ed).
counselor. On the contrary, they may even feel comfort-
tress during specific period of the treatment cycle. The Infertility: Psychological Issues and Counseling Strategies.
able and relieved for having someone to listen to their view
World Health Organization has reported the challenges
Oocyte/Embryo Donation points and grief. Hence, at the minimum, infertility coun-
of lack of heterogeneity regarding assisted reproduc-
New York: John Wiley & Sons; 1997. pp. 103-25.
9. Diamond R, Kezur D, Meyers M, et al. Couple Therapy for
seling should provide a safe and supportive environment
The decision to proceed with oocyte or embryo donation is tive technologies, unavailability of quality of infertility Infertility. New York: Guildford Press; 1999.
for the patients to express, explore, and understand their 10. Covington SN. The role of the mental health professional in
a complex phenomenon and counseling plays an impor- services, and inconsistent standards in the developing
feelings about their infertility experience and treatment. reproductive medicine. Fertil Steril. 1995; 64:895-7.
tant role in aiding the decision. Psychological evaluation world.12 Similar challenges exist regarding counseling
During the first counseling session, infertile patients 11. Boivin J, Appleton TC, Baetens P, Baron J, Bitzer J, Corrigan
and counseling by a qualified mental health professional and mental health services in terms of the wide variation
will not be in a position to say exactly what they expect E, et al. Guidelines for counselling in infertility: Outline
is recommended strongly for the donor and the partner.13 of attitude toward counseling services, quality of care, version. Hum Reprod. 2001; 16:1301-4.
from the consultation service. Thus, a definition of infer-
The psychological assessment is necessary to explore read- standard practices, and training. Hence, there should be 12. Covington SN, Burns LH. Infertility Counseling: A
tility counseling fulfills some important functions; one of
iness, any risks of emotional or financial coercion involved a serious focus on both medical and “evidence-based” Comprehensive Handbook for Clinicians, 2nd edition.
them is to act as a corrective to unrealistic expectations
and to understand the knowledge about the treatment. psychosocial research, which can give direction to the Cambridge, UK: Cambridge University Press; 1999.
such as the assumption that removing ‘‘barriers in the
identification of clinical issues, on assessment parame- 13. Malhotra J, Malhotra N, Patel N, Arora P, Bora NM.
Counseling aims at providing psycho-education make mind’’ will automatically improve pregnancy prospects.
ters, and most applicable therapeutic interventions that Surrogacy. Principles and practice of assisted reproductive
informed and satisfying decisions, implications and out- Many couples postulate that in the first attempt itself technology. In: Rao KA, Carp H, Fischer R, Deepika (Eds).
are beneficial and effective in counseling practice.
come of the treatment, and supports in accepting the deci- the treatment will be successful and the thought of end- Infertility. Vol 1. New Delhi: Jaypee Brothers Medical
sion positively. ing treatment or putting a limit on it is unfathomable. It Publishers (p) Ltd.; 2014. pp. 807-18.
becomes an essential component in counseling to help PROBABLE QUESTIONS 14. Gunasheela D, Patil J, Ashwini S. Oocyte sharing program.
them reconsider this outlook. In addition, the infertility In: Rao KA, Carp H, Fischer R, Deepika (Eds). Principles
Surrogacy 1. Stress as a cause and effect of infertility—explain.
and practice of assisted reproductive technology. Infertility.
counselor should encourage them to become active par- 2. Role of counseling in infertility—explain.
Surrogacy is a complex process, which not only involves Vol 1. New Delhi: Jaypee Brothers Medical Publishers (p) Ltd.;
ticipants in the treatment process, making them aware of
financial and legal mandates but psychological aspects 2014. pp. 798-06.
their right and responsibility to choose the treatments that
also plays a vital role. The surrogate mother’s preparedness REFERENCES 15. Wischmann T, Stammer H, Gerhard I, Verres R. Couple
are best for them. counselling and therapy for the unfulfilled desire for
to enter into the program, her expectations, treating her Infertility counselors should also help the couple 1. Butler P. Assisted reproduction in developing countries—
a child: the two-step approach of the ‘Heidelberg
with respect and dignity, and ability to “walk away” after facing up to the issues. Progr Reprod Health. 2003;63:1-8.
probe and consider alternatives as a next line of interven- 2. Burns LM, Covington SN. Psychology of Infertility. Infertility infertility consultation.’ In: B Strauss (Ed). Involuntary
delivery are important psychological factors that need to Childlessness: Psychological Assessment, Counselling and
tion. This is important for couples particularly with poor Counseling: A Comprehensive Handbook for Clinicians,
be assessed. The role of counseling in surrogacy is to pre- Psychotherapy. Seattle: Hogrefe & Huber Publishers; 2002.
prognosis and for whom parenthood is highly valued or 2nd edition. Cambridge, UK: Cambridge University Press;
pare the people involved contemplating this last resort of 1999. pp. 127-50.
the stigma of childlessness is significant.
treatment and to take into account all the factors that will
An example is the Heidelberg Model,15 which advocated
have an influence on the future lives of each of them. The
counseling ensures that they be comfortable and confi- solution-focused counseling, a technique that was found
dent with their decisions and have trust in each other and useful for the couples with infertility, particularly those
explains risks involved as it could have a profound effect who were with high distress and experienced worsening of
on them and their families.14 mood and sexual problems over the course of treatment.
C HA PT E R
Thyroid Disorders and Infertility
6 3. Metastatic differentiated thyroid cancer
4. Metastatic tumors within the thyroid gland.
Signs and symptoms (Table 6.1)
Table 6.1: Signs and symptoms of hypothyroidism
•
oligomenorrhea.
Thyroid Disorders and Infertility 37
Menstrual disturbances in hypothyroidism are three
times that of normal population. Most c ommon—
Infertility and hypothyroidism:
Causes of infertility:
GB Ashwini
and hyperthyroidism. ■■ Altered peripheral estrogen metabolism
Hypothyroidism Hyperthyroidism ■■ Hyperprolactinemia
Chapter Outline
Hair loss Hair loss ■■ Defects in hemostasis
• Thyroid Disorders and Infertility
Dullness Exophthalmos ■■ Disturbances in gonadotropin-releasing hor
Goiter Goiter mone (GnRH) secretions
Reduced heart rate Palpitations ■■ Both gonadotropins and T4 are crucial to
INTRODUCTION 3. Androgens: plasma levels of testosterone and andros
Fatigue Tremors
tenedione are higher and the secretion rates of these achieve the best fertilization and blastocyst
Thyroid abnormalities are not uncommon in women of Cold insensitivity Heat intolerance development.15
are increased too.3-5
child bearing age. So its screening and adequate manage Dry skin Sleep disturbances Patients with overt thyroid failure are detected
4. Mean levels of luteinizing hormone (LH) in follicu
ment is vital and ineludible part of infertility parapherna Weight gain Weight loss before referral for fertility treatment therefore caus
lar and luteal phases are significantly increased.6 Memory problems Shortness of breath
lia. The incidence of hypothyroidism in women is on the ing a bias in the estimation of prevalence of infertil
Constipation Diarrhea
rise, and the causes of such a rise are not known in detail. ity incidence in hypothyroidism.
Likewise, the current guidelines for detection and manage Clinical Manifestations Related to Fertility Irregular menses Increased appetite
Depression Irregular menstruation
ment of hypothyroidism both in pregnancy and in women • Menstrual irregularities: menstrual disorders are 2.5 fold Mood swings Muscle weakness Clinical Hypothyroidism
with infertility are complex, confusing, and conflicting. greater in thyrotoxic women than in general popula Sweating • Measurement of TSH and FT4 remains the common
This chapter makes an attempt to simplify and compare tion. Common manifestations being amenorrhea, oli Anxiety/nervousness
est practice for diagnosing thyroid disorders.
the various current guidelines and evidence to screen and gomenorrhea, hypomenorrhea, and anovulation.7
• Measurement of serum antithyroid peroxidase anti
treat thyroid disorders in both male and female infertility. • Most thyrotoxic women remain ovulatory.8 Clinical and Subclinical Hypothyroidism bodies and antithyroid globulin antibodies allows for a
• There no proper evidence to support prompt treatment
THYROID DISORDERS AND INFERTILITY of subclinical hyperthyroidism in infertile women. and Female Fertility firm etiological diagnosis of both clinical hypothyroid
ism (CH) and subclinical hypothyroidism (SCH).
• Treatment of clinical hyperthyroidism in infertile • Hormonal changes:
Normal thyroid function is vital for the optimal function • Levothyroxine replacement remains the gold standard
women usually corrects menstrual disturbances and it Decreased rate of metabolic clearance of estrone
ing of the hypothalamic–pituitary gonadal axis. Hence, it is indicated in both infertile and fertile women. treatment for CH.
and androstenedione along with increased periph
plays a major role in female and male fertility. • Treatment of clinical hyperthyroidism may include • Very effective when administered orally and has a long
eral aromatization.9,10
antithyroid drugs radioactive iodine or surgery. half-life permitting once a day dosage.
Plasma binding activity of SHBG is decreased,
Thyroid Disorders and Female Infertility • Radioactive iodine treatment in doses used for hyper which in turn leads to decreased plasma concen
• The aim of therapy should be to reach normal serum
thyroidism has no significant harmful effects on the TSH (0.3–2.5 mIU/L) concentrations.
There are three categories of thyroid disorder: tration of total testosterone and estrogen, and free
gonads. • Where possible levothyroxine should be administered
1. Hyperthyroidism—hyperactivity of the thyroid gland fractions of these hormones are increased.11
• It is customarily advised for prophylactic reasons separately from other interfering medications and
2. Hypothyroidism—under activity of the thyroid gland Serum prolactin concentration is often increased
to avoid conception until at least 6 months after its and this in turn is due to hypothalamic TRH, which supplements such as calcium carbonate and ferrous
3. Thyroid autoimmunity
administration. not only increases TSH but also prolactin levels. sulfate, etc. A 4-hour difference in administration is
Galactorrhea is also one of the common manifesta desirable.
Hyperthyroidism and Female Infertility Causes of Thyrotoxicosis • Initiation or discontinuation of estrogens and andro
tions.12
• Incidence in women: 0.5–2% • Menstrual disturbances: gens should be followed by reassessment of serum thy
• The prevalence of hyperthyroidism in 2.1% of infertile
Common causes
Hypothyroidism can lead to disturbances in cycle rotropin at steady state.
women compared to 3% in a fertile control population.1 1. Graves disease length (both reduction and prolonged menstrual • Starting replacement dose should be decided based
• Epidemiological evidence regarding potential link 2. Toxic multinodular goiter cycle) and amount of bleeding (heavy flow and on factors such as patient’s weight (1.6–1.8 µg/kg),
between infertility and thyrotoxicosis is scarce and 3. Solitary toxic adenoma reduced flow). body mass index, pregnancy status, history of infertil
inconclusive. 4. Thyroiditis The abnormal bleeding is attributed to estrogen ity, etiology of hypothyroidism, serum TSH levels, age
• Hormone changes: breakthrough bleeding which in turn is due to (requirement decreases with age).
Uncommon causes
1. Increase in concentrations of sex hormone binding absence of ovulation.13 • The dose for replacement should be with an initial full
globulin (SHBG). 1. Thyroid-stimulating hormone (TSH) secreting Defects in hemostasis (decreased levels of factors replacement or alternatively partial replacement with
2. Serum estradiol: two to threefold higher estrogen pituitary adenoma VII, VIII, IX, and XI) can contribute to polymenor gradual increase in the dose titrated upward or down
levels in all phases of menstrual cycle.2 2. Struma ovarii rhea and menorrhagia.14 ward using serum thyrotropin as the goal.
38 The Infertility Manual Thyroid Disorders and Infertility 39
Subclinical Hypothyroidism (SCH) Flowchart 6.1: Management of SCH in infertile female. olycystic ovarian syndrome and premature ovarian
p Flowchart 6.2: Hyperthyroidism and male infertility.
failure.21-23
• Various professional bodies have defined SCH in dif
ferent ways (Flowchart 6.1).
• ATA defines SCH as a TSH level greater than the upper Association with Pregnancy
limit of normal range (4.5–5.0 mIU/L) with normal FT4 Pregnancy rates remain the same in euthyroid women
levels. with and without TAI inferring that their presence alone
• European thyroid association (ETA) defines it as mild does not alter implantation of the embryo.24,25
and severe SCH mild being TSH values between 4 and However TAI is associated with single and recurrent
10 and severe being greater than 10 with normal FT4 miscarriage. Most of the studies point to a 3–5 times
levels.16 higher miscarriage rate, independently whether con
• Incidence: 4–10% (17) 90% being mild variety. ception occurred naturally or was after fertility treat
• When women attempting pregnancy are consid ment.26-29
ered, the upper limits of normal for pregnancy in first Risk of progression to hypothyroidism is increased if:30-32
trimester (2.5 mIU/L) should be considered for defin
ing SCH.17 1. First measurements of TSH level are higher than
• Hence for purposes of infertility SCH is defined as a 2.5 mIU/L.
2. Thyroid peroxidase antibody titres are raised higher tion and maintain the TSH values less than 2.5 mIU/L,
TSH value of greater than 2.5 mIU/L with normal FT4
than 2,000 IU/mL. and one may consider LT4 replacement prior to COH
values.
• If anti-TPO antibodies are detected, TSH levels should 3. Glands appearing hypoechogenic. and ART even with normal serum TSH levels.
• Insufficient evidence that SCH (defined as TSH
>2.5 mIU/L with normal FT4) is associated with be checked, and treatment should be considered if the
Medical interventions that can be considered to reduce
infertility.17 TSH level is over 2.5 mIU/L (Grade B). Hyperthyroidism in Male Infertility
the miscarriage risk in women with TAI consist of immu
• Fair evidence that SCH (defined as values >4 mIU/L) is nomodulation or thyroxin administration.33 (Flowchart 6.2)
associated with miscarriage. Table 6.2: Subclinical hypothyroidism in infertile female
population (thyroid autoimmunity).
Few small studies showed a significant improvement Hormonal changes
• Fair evidence that treatment of SCH (TSH >4) is asso
in the live birth rate in women (with TAI) who received
ATA 2011 ES 2012 ETA 2014 ASRM 2015
ciated with improved pregnancy rates and decreased
thyroid hormone.34-36 Clinical Manifestation Related to Fertility
miscarriage rates.17 Subclinical Should Should Should Should
hypothyroid- be treated be be be treated 1. Erectile dysfunction incidence upto 70%, correctable
Recommendations of American Society for Reproduc ism plus TAI (TSH >2.5 treated treated (TSH >2.5 Effect of COS/ART on Thyroid Function after specific correction of thyrotoxicosis40
tive Medicine for management of SCH in infertile female mIU/L) mIU/L)
2. Spermatogenic abnormalities41
population (Table 6.2). Subclinical – Should Should Can be • The need for a rapid increase in T4 was identified in
3. Low total sperm count 43%
hypothyroid- be be treated to hypothyroid treated women to maintain euthyroidism.
• Currently available data support that it is reasonable ism (no TAI) treated treated maintain
4. Lineal motility defects 86%
The timing of such increased requirement was even
to test TSH in infertile women attempting pregnancy. TSH levels 5. Progressive motility abnormal 62%
more rapid and pronounced when conception had
If TSH concentrations are over the nonpregnant lab below 2.5
been achieved after assisted reproductive technology Treatment for Hyperthyroidism
reference range (typically >4 mIU/L), patients should ASRM, American Society for Reproductive Medicine; ATA, American
be treated with levothyroxine to maintain levels below Thyroid Association; ES, Endocrine Society; ETA, European Thyroid (ART) procedures, probably because of the higher E2
Association. levels reached in this clinical setting.37 Radioiodine (RI)—remains the mainstay of treatment.
2.5 mIU/L (Grade B).
• Muller et al.38 were the first to describe and show an Reproductive performance remains normal in men after
• Given the limited data, if TSH levels prior to pregnancy
RI therapy.41
are between 2.5 and 4 mIU/L, management options Thyroid Autoimmunity (TAI) increase of serum TSH postcontrolled ovarian hyper
include either monitoring levels or treating with stimulation (COS) and decrease in serum FT4. They
• The prevalence of TAI is 5–10 times higher in females related this to the rapid 10 fold increase in serum
Hypothyroidism in Male Infertility
levothyroxine to maintain TSH less than 2.5 mIU/L
than males and this is due various factors like genetic Estradiol. Less common in men than in women. Effects on reproduc
(Grade C).
• During the first trimester of pregnancy, it is advisable to factors, estrogen-related effects and chromosome X • In women with and without TAI,39 the above changes tive function not well delineated.
treat when the TSH is greater than 2.5 mIU/L (Table 6.2). abnormalities. It is the most common autoimmune were found to be more profound in women with TAI
disorder in women, affecting 5–20% in women of Hormone changes
• While thyroid antibody testing is not routinely than women without it.
recommended, one might consider testing anti- reproductive age group.19,20 • In women with TAI, the impact of COS on thyroid func • Decrease in SHBG and total testosterone and free tes
thyroperoxidase (TPO) antibodies for repeated TSH • Most studies so far have showed an higher preva tion tends to be permanent and may lead to SCH in preg tosterone.
values greater than 2.5 mIU/L or when other risk lence of TAI in women visiting fertility clinics espe nancy, which in turn can alter the pregnancy outcome. • Prolactin elevation is common in women, however,
factors for thyroid disease are present (Grade C). cially so among infertility women with endometriosis, Hence, it is important to closely monitor thyroid func men with primary hypothyroidism rarely exhibit ele
40 The Infertility Manual Thyroid Disorders and Infertility 41
vated serum prolactin concentrations except when Commonest parameter of semen analysis affected 9. Redmond GP. Thyroid dysfunction and women’s autoimmunity: an unfortunate combination? J Clin Endo
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WB Saunders; 1986. pp. 424-40. High prevalence of autoimmune thyroiditis in patients
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SCH defined as TSH values above 2.5 and normal free levels in women with thyrotoxicosis. J Endocrinol. 2004;150:363-9. 36. Negro R, Formosa G, Mangieri T, Pezzarossa A, Dazzi D,
T4 levels need to be started on LT4 when aiming for preg 1972;53:173-4. 23. Abalovich M, Laura M, Carlos A, Silva G. Subclinical Hassan H. Levothyroxine treatment in euthyroid pregnant
nancy. 7. Benson RC, Dailey ME. The menstrual pattern in hypothyroidism and thyroid autoimmunity in women with women with autoimmune thyroid disease: effects on
hyperthyroidism and subsequent posttherapy hypothy infertility. Gynecol Endocrinol. 2007;23:279-83. obstetrical complications. J Clin Endocrinol Metab. 2006;
In the presence of thyroid autoimmunity consider LT4
roidism. Surg Gynecol Obstet. 1955;100:19-26. 24. Grassi G et al. Thyroid autoimmunity and infertility. 91:2587-91.
before COS/ART. 8. Sturgis SH, Lerman J, Stanbury JB. The menstrual pattern Gynecol Endocrinol. 2001;15:389-96. 37. Davis LB, Lathi RB, Dahan MH. The effect of infertility
Hyperthyroidism in men is associated with erectile dys in thyroid disease. J Clin Endocrinol Metab. 1952;12: 25. Poppe K, Glinoer D, Tournaye H, Devroey P, Steirteghem medication on thyroid function in hypothyroid women
function in upto 70%, which is reversible post treatment. 846-55. AV, Kaufman L, et al. Assisted reproduction and thyroid who conceive. Thyroid. 2007;17:773-7.
C HA PTE R
42 The Infertility Manual
38. Muller AF, Verhoeff A, Mantel MJ, De Jong FH, Berghout
A. Decrease of free thyroxine levels after controlled ovarian
hyperstimulation. J Clin Endocrinol Metab. 2000;85:545-8.
39. Poppe K, Glinoer D, Tournaye H, Schiettecatte J, Devroey P,
van Steirteghem A, et al. Impact of ovarian hyperstimulation
on thyroid function in women with—and without thyroid
43. Krassas GE. The male and female reproductive system in
hypothyroidism. In: Braverman LE, Utiger RD (Eds). Werner
and Ingbar’s The Thyroid—A Fundamental and Clinical
Text. 9th edition. Philadelphia: Lippincott Williams &
Wilkins. pp. 824-9.
44. Carani C, Isidori AM, Granata A, Carosa E, Maggi M,
Ultrasound in Infertility
7
Arunima Halder, Divyashree PS
autoimmunity. J Clin Endocrinol Metab. 2004;89:3808-12. Lenzi A, et al. Multicenter study on the prevalence of
40. Meikle AW. The interrelationships between thyroid sexual symptoms in male hypo- and hyperthyroid patients. Chapter Outline
dysfunction and hypogonadism in men and boys. Thyroid. J Clin Endocrinol Metab. 2005;90:6472-9.
• Physics of Ultrasound • Cyclical Changes in the Endometrium
2004;14(1):S17-25. 45. Krassas GE, Papadopoulou F, Tziomalos K, Zeginiadou T, • Evaluation of the Pelvic Structures • Ultrasound-Guided Procedures
41. Abalovich M, Levalle O, Hermes R, Scaglia H, Aranda C, Pontikides N. Hypothyroidism has an adverse effect on • Changes in the Pattern of Endometrium during the
Zylbersztein C, et al. Hypothalamic-pituitary-testicular axis human spermatogenesis: a prospective, controlled study. Menstrual Cycle
and seminal parameters in hyperthyroid males. Thyroid. Thyroid. 2008;18:1255-9.
1999;9:857-63. 46. Trummer H, Ramschak-Schwarzer S, Haas J, Habermann H,
42. Krassas GE, Perros P. Thyroid disease and male reproductive Pummer K, Leb G. Thyroid hormones and thyroid antibodies
function. J Endocrinol Invest. 2003;26:372-80. in infertile males. Fertil Steril. 2001;76:254-7.
PHYSICS OF ULTRASOUND1 EVALUATION OF THE PELVIC
• Medical ultrasonographic imaging comprises using STRUCTURES3
high-frequency sound waves that are allowed to The pelvic structures can be evaluated using the transab-
bounce off tissues to generate an image. dominal, transvaginal, and transrectal probe. However,
• Frequency refers to the number of cycles of compres- advantages of the transvaginal probe are as follows:
sions and rarefactions that are produced in a sound
wave per second. (1 cycle/sec = 1 Hz). • The transvaginal probe when introduced into the
• Wavelength is the distance between one peak to the next. vagina comes in proximity to the pelvic structures, i.e.
Wavelength is inversely proportional to the frequency. the uterus, tubes, and the ovaries.
• Higher frequency probes (5–10 MHz) have poor pen- • Since it has a higher frequency, resolution is better.
etration and better resolution. They are used for super- • The inconvenience of undergoing ultrasound in a full
ficial structures. Lower frequency probes (2–5 MHz) bladder can be avoided.
provide better penetration, hence used for deeper The disadvantages of the Transvaginal Ultrasound (TVS)
structures. However, resolution is poor in this case.
are as follows:
• Whenever the sound wave reaches an interface, part
• Cannot be used in a virgin.
of the sound wave is reflected back to the probe. This
• Cannot be used in those having any vaginal problems
is obtained as an echo. When the pulse wave hits gas
or vaginismus.
or solid, the difference in density is so great that the
entire sound energy is reflected back, making it diffi-
cult to see the structures underneath. This is called as Methodology of TVS
acoustic shadowing. Prerequisites for the TVS are as follows:
• The Thermal index is the biological effects of heat pro- • Bladder to be emptied
duced during an ultrasonographic examination.2 • Patient in lithotomy position
Thermal index = Wp /Wdeg , • Cover the patient
• Basic probe movements. (refer Table 7.1)
Wp: Relevent (attenuated) acoustic power at the depth Counsel regarding the procedure and allay anxiety
of interest. Commonly used terms while performing TVS are as
Wdeg: Estimated power necessary to raise the tissue follows:
equilibrium temperature by 1°C.
Thermal index is clinically relevant while imaging the 1. Scanning angle: Maximum angle that can be covered
embryo, especially while using the colour Doppler and by the ultrasound beam. The ideal scanning angle
the spectral Doppler. In both these cases, the ultrasound varies from 80°to 180°.
power output is higher, which may lead to increased ther- 2. Frame rate: No of static images that can be captured
mal index. Hence, it is advisable not to use the same while by the scanner in unit time. More the frame rate, bet-
evaluation of early pregnancy (keeping in mind the theo- ter the resolution. Also the images look more real time
retical rise in thermal index). with higher frame rate.
44 The Infertility Manual Ultrasound in Infertility 45
Table 7.1. Basic probe movements. Table 7.2. Systematic scan. Table 7.3. Various phases of menstrual cycle.
Basic probe •• Ultrasound jelly over the probe for acoustic coupling During menstruation Thin echogenic endo 1–4 mm thickness, intraluminal blood or sloughed endometrium may
movements Use •• Cover probe with condom be seen
1 Inward and outward To slide tissues over one •• Patient in lithotomy position Early proliferative phase Thicker endometrium (5–7 mm), more echogenic
another, to rule out adhesions •• Marking on the probe facing the patient’s anterior or the roof
Late proliferative Multilayered appearance with an echogenic basal layer and hypoechoic inner functional
•• Mobility of the tissue planes are noted as the probe
2 Spanning side to side To survey the complete pelvis (periovulatory phase) layer, separated by a thin echogenic median layer arising from the central interface or lumi-
enters the vagina nal content (upto 11 mm)
3 Clockwise and To visualise a structure in a •• Visualise the uterus in the centre
anticlockwise different plane •• Minor adjustments of the uterus to visualise the entire Post ovulation Loss of the layered appearance (7–16 mm)
4 Anteroposterior Survey of the pelvis, visualisa- uterus with the cervix in the sagittal section Secretory phase May become thicker or may show a slight drop in the thickness. Increased echogenicity
tion of the POD and structures •• Rotate the probe in the anticlockwise direction to 90° to could be related to stromal edema and glands distended with mucus and glycogen
that are difficult to locate obtain the transverse plane
•• Keeping the probe in the same manner, take the probe
to either lateral wall to visualise the ovaries one frame of the dual screen. Now the probe is rotated • It is not only possible to classify the lesions, but also to
3. Scanning depth: Lower the frequency, better the pen- •• Screen the ovaries in the longitudinal and transverse planes to 90° to obtain the transvered diameter and the stored detect if it is benign or malignant.
etration, larger the area under scan. So can be used •• Check mobility of the pelvic structures against each in the second frame. The largest longitudinal (X), • Adnexal lesions include ovarian lesions and extraovar-
for initial survey. Higher the frequency, lesser pene- other using the sliding sign transverse (Y) and AP (Z) diameters are measured. The ian lesions.
tration, better resolution, more superficial structures •• Reach the posterior fornix to visualise the POD
ovarian volume is calculated as (X × Y × Z × 0.523). • If a lesion is arising from the ovary, it will be sur-
seen. Increasing the scanning depth is better once the • Antral follicle count (AFC): The antral follicle count is rounded by the ovarian tissue either partially or com-
region of interest is identified. done by taking a two-dimensional (2D) sweep along pletely. This is called as the “beak” or the “rim” sign.
3. Endometrium
4. Focal zone: The area where the image is sharpest. the entire ovary. However, when there are a large num-
One should define the endometrium on the basis of
Detected by an arrow head on the side of the image. It
• Symmetry of shape ber of follicles, a 2D sweep may be inaccurate and a Terms and Definitions
is always present at the area of interest. three dimensional (3D) may be useful in such case.
• Central line of the endometrium The International Ovarian Tumor Analysis Group has pro-
5. Zoom: HD zoom is used for a larger image for better • Stromal echogenicity is assessed with respect to the
• Endomyometrial junction (EMJ)
visualisation of the anatomical details. Image should myometrium vided a consensus opinion for various terms and their def-
• Pathology within the endometrium initions for various morphological features of the adnexal
be zoomed large enough to fill up 3/4th of the screen.
• The phase of menstrual cycle it appears to be in lesions.8
6. Gain is used to make an image brighter or dimmer by
The endometrium may be described as hypoechoic,
Evaluation of the Tubes • Septum
altering the intensity of the beam returning back to
the scanner. Optimum gain is when the urinary blad- isoechoic, or hyperechoic with respect to the myometrium. A normal fallopian tube not usually seen on a pelvic A septum is defined as a thin strand of tissue run-
der looks anechoic. Other structures are compared ultrasound except on an occasion when it is blocked and ning across the cyst cavity from one internal surface
based on the urinary bladder once gain is optimised. enlarged in size and contains fluid within (hydrosalpinx).5 to the contralateral side.
CHANGES IN THE PATTERN OF A septum may be complete or incomplete.
A systematic method should be followed while per-
ENDOMETRIUM DURING THE Pathologies of the Uterine Body An incomplete septum is one that does not traverse
forming transvaginal ultrasonography. (Table 7.2).
MENSTRUAL CYCLE from one internal surface to the contralateral side
1. Congenital Anomalies: Incidence of congenital
Evaluation of the Uterus 4 in all scanning planes.
Measuring the Endometrium uterine anomalies is 0.1–3.8% in normal women but A cyst with only incomplete septa is considered as
The uterus can be evaluated under the following headings: reaches 6.7% in infertile women.6 unilocular, e.g. hydrosalpinges.
Measured from outer margin to the outer margin of ante- 2. Fibroids: Fibroids are benign tumors of the uterus
1. Serosa: The serosa defines the outer contour of the To measure the thickness of a septum, one should
rior layer to the outer margin of the posterior layer of that are monoclonal in origin. Found in 20–40% of
uterus. In a normal uterus, the serosa should appear measure the thickest part of the thickest septum
the endometrium in the sagittal uterine plain, perpen- women greater than 30 years and are more common
“smooth” and “continuous.” Any irregularity in the (except where it joins the cyst wall).
dicular to the longitudinal axis of the endometrium in its in the nulliparous women
uterus with loss of continuity will denote the presence • Solid
broadest part (1–2 cm below the fundus), excluding the 3. Adenomyosis: Abnormal growth of the endometrial Solid means exhibiting high echogenicity. This may
of an abnormality in the uterus. Also the presence of
“sliding sign” (movement of adjacent structures in junctional zone. Various Phases of endometrium during tissue within the myometrium. suggest the presence of tissues such as myome-
opposite directions while moving the probe in and menstrual cycle are described in Table 7.3. trium, ovarian stroma, myoma, and fibroma.
The sonographic characteristics of various uterine
out) can denote the absence of adhesions. Solid lesions all exhibit blood flow. However,
lesions are mentioned in (Tables 7.4 to 7.7).
2. The myometrium: The myometrium is evaluated for: Evaluation of the Ovaries absence of blood flow is not informative.
• The thickness • Papillary projections
• Homogenecity The ovaries are described as follows: Ultrasound of the Adnexal Lesion Solid papillary lesion is any solid projection into a
• Presence or absence of myometrial mass • Volume: On identification of the ovary, it is required to • Ultrasound is an accepted modality to identify and cyst cavity from the cyst wall with a height of greater
• Any scar tissues achieve the longest diameter of the ovary and stored in investigate an adnexal lesion. than 3 mm.
46 The Infertility Manual Ultrasound in Infertility 47
Table 7.4. Characteristics of various congenital uterine anomalies on USG.
Uterine anomalies Characteristics on ultrasound
1. Uterine aplasia •• Suspected in those with primary amenoorhea
•• On Trans-abdominal Ultrasound (TAS), absence of dimpling on the poste-
rior wall of the bladder. This indicates complete absence of the uterus and
karyotyping is advised
2. Unicornuate uterus •• Uterus is not seen in the midline
•• Normal looking uterus seen in one side on moving the probe
•• Rudimentary horn may be seen as isoechoic pear-shaped structure on the
opposite side with or without thin endometrium
•• Corresponding ovaries may be seen next to the horns
•• Transverse section will show a “beak like” projection of the endometrium.
•• 3D: Banana-shaped appearance of the uterus
3. Uterus diadelphus •• Both cornu of the uteri may be placed near to each other like any other
(uterus diadelphus has two separate duplication anomaly
uteri and two cervices) •• Or may be at near the lateral pelvic wall and appear as unicornuate uterus. Fig. 7.2: Septate uterus.
•• Both ovaries seen near either cornu
4. Bicornuate uterus (Figs. 7.1 and 7.4) •• Widening of the fundus Fig. 7.1: Bicornuate uterus.
•• Division of the endometrial cavity and indentation of the fundus
•• Volume Ultrasonography (USG) showing the coronal view of the ultrasound
(Figs. 7.1 and 7.4)
Fundus shows dimple
Straight line joining the top of the endometrial cavity, fundus dimple is
<5 mm above this line
Endometrial cavities are convex medially
Angle between two cavities is obtuse
5. Septate–subseptate uterus (Fig. 7.2) Volume USG findings (Fig. 7.2):
•• No/minimal indentation
•• 5 mm uterine wall seen above the line joining the tips of the cavity
•• Angle between the two cavities is acute
•• Medial margins of the cavities are straight
•• The distance between the line joining the two cavities and the deepest point
in the endometrium is greater than 10 mm
6. Arcuate uterus (Fig. 7.3) •• On 3D USG, there is concavity at the fundus (Fig. 7.3) Fig. 7.4: Bicornuate uterus (3D picture).
•• Externally the fundus is flat
•• Distance between the line touching the tips of the endometrial cavity and
the deepest point at the endometrium is <10 mm • Amount of fluid in the POD is measured in the sagittal
•• Angle between the cavities is obtuse
plane and the largest AP diameter is recorded.
7. Hypoplastic uterus •• Infantile uterus with uterine body: Cervix ratio 1:1 (in normal adults it is 2:1) • Refer to (Fig. 7.5) for classification of adnexal lesion
•• 3D USG shows small uterus with long cervix
Fig. 7.3: Arcuate uterus. provided by the IOTA group.
8. T-shaped uterus •• T shape on 3D ultrasound • Sonographical features of ovarian lesions are men-
tioned in (Table 7.8).
Table 7.5. Differential diagnosis between bicornuate, septate–subseptate, and arcuate uterus. The largest projection is measured in two planes per-
Bicornuate Septate/subseptate Arcuate pendicular to each other, i.e. the height and the base. Ultrasound Features of Polycystic Ovaries
Characteristics (Figs. 7.1 and 7.4) (Fig. 7.2) (Fig. 7.3) The number of separate papillary projections and • Volume of ovaries greater than 10 cc.9
External contour Concave Flat/convex Flat/convex the presence of blood flow within them have to be • Antral follicular count of 12 per ovary. In view of
Fundus endo tip distance <5 mm >5 mm >5 mm documented. increased resolution and the quality of the new
Angle between the cavities Obtuse Acute Obtuse • Internal wall is described as being smooth or irregular. ultrasound machines, it has been recently suggested
The wall is said to be irregular if it has a solid papillary that an average number of 26 or more follicles per
Endometrial tip—deepest point distance – >10 mm <10 mm
projection. ovary is a more reliable threshold for detecting PCO.10
Medial wall of cavities Convex Straight Straight
• In case of a cystic solid tumor, largest solid component • Follicles may be peripherally arranged (string of pearl
Myometrial dipping Present Absent – is measured separately in three dimensions. appearance) or may be generally distributed.
48 The Infertility Manual Ultrasound in Infertility 49
Table 7.6. Sonographic differences between fibroids and focal adenomyosis. Table 7.8. Sonographical classification of ovarian lesions.
Characteristics on USG Fibroids Focal adenomyosis Unilocular lesions
Margins Well defined Not well defined Characteristics Intraovarian Extraovarian Schematic diagram
Texture Hypoechoic, homogenous rounded solid Coarse heterogenous echogenicity •• Absence of Follicular cysts Paraovarian cyst
lesions. Heterogenous when degenerated. septa •• Has thick walls •• Sliding over ovarian tissue
Appearance Peripheral hypoechoic rim seen due Alternate hypo and hyperechoicaeas described as •• Absence of •• Scanty, high resistance flow •• Rim absent, thin walls
to displacement of myometrial fibers. Swiss cheese appearance/rain in forest appearance. solid parts •• Disappears in subsequent •• Avascular lesion
Calcifications may be seen. Echogenic spots and anechoic areas (myometrial •• Absence menstrual cycle
cysts) give salt and pepper appearance. of papillary •• Simple cyst of ovary (Fig. 7.6)
Mass effect Seen Not seen structures •• No echogenic rim. Thin walled
•• Usually larger than 5 cm
Vascularity Capsular vascularity around the fibroid Penetrating vascularity. Capsular vascularity is
•• Doppler shows no flow
never seen in adenomyoma Fig. 7.6: Simple cyst.
•• Does not disappear in subsequent
Junctional zone Junctional zone is not affected Junctional zone altered, echogenic flecks at the menstrual cycle
endomyometrial junction (EMJ) seen •• Endometriomas (Figs. 7.7 and
3D-PD scan Circumferentially arranged blood vessels Radial or penetrating vessels with endometrial 7.13)
strands seen penetrating into the myometrium •• Echogenic flecks in the wall
•• Pain on pressure
•• Streaming sign
Table 7.7. Various endometrial pathologies and their ultrasonographic appearance. •• Ovary is adherent usually
posteriorly
Sl No. Endometrial pathology 2D features Doppler features
•• Fluid level seen
1 Acute endometritis •• Thick isoechoic endometrium •• Increased vascularity of the endometrium •• May contain solid areas
•• Altered junctional zone seen in the early proliferative phase Fig. 7.7: Endometriotic cyst.
•• Adhesions common
2 Chronic endometritis •• Persistently thin endometrium •• Hypovascular endometrium even in the Multilocular lesions
•• Disrupted EMJ secretory phase when it is expected to
•• Echogenic flecks in the endometrium and be vascular Characteristics Intraovarian Extraovarian
myometrium •• At least one Multiple follicles (Fig. 7.8) Hydrosalpinx (Fig. 7.9)
•• Micropolyps septum •• Septae are straight tight and •• Changes shape with rotation
•• Calcifications in the ovary and calcified •• No measur- vascular of the probe
pelvic nodes able solid •• Incomplete septae, low level
3 Endometrial •• Endometrial thickness >14 mm •• Regularly placed vessels with components echoes
hyperplasia •• Normal histology normal branching pattern (Resistivity or papillary
•• EMJ well maintained Index (RI) 0.55) projections
4 Endometrial Polyp •• Solid echogenic lesions in the endometrial •• Characteristic feeding vessel either Fig. 7.8: Multiple follicles.
cavity single or maximum of two closely
•• Best visible in the periovulatory phase placed vessels
•• Bright edge sign seen •• RI >0.45
5 Synechiae •• Lines bridging between layers of endometrium •• Sparse subendometrial vascularity7
Fig. 7.9: Hydrosalpinx.
Serous cyst Peritoneal inclusion cyst
•• Loose, curved, thin avascular •• Change in shape with com-
septae pression
•• Flapping Snail sign
Fig. 7.5: Classification of adnexal lesions (IOTA).8 •• Ovary may be close by
Contd...
50 The Infertility Manual Ultrasound in Infertility 51
Contd... by 2D and 3D ultrasonography, whereas the vascular Table 7.9. Blood flow in a mature follicle.
Table 7.8. Sonographical classification of ovarian lesions. changes can be well assessed by the power Doppler.
Blood flow covered (in a single
Unilocular lesions Follicular study can be done under the following
cross area slice)(%) Grade of vascularity
Septated fluid collection headings:
•• Bowel shadows within the fluid In the early follicular phase under the effect of endog- <25 1
•• Rim absent enous and exogenous FSH, follicles grow in size and one of 25–50 2
Unilocular-solid/multilocular solid cyst them attains the dominant status by day 5.
Hemorrhagic cyst (Figs. 7.10 and 7.12) •• Pelvic collection Characteristics of a leading follicle capable of giving a 50–75 3
•• Seen on day 2 scan,convex fibrin •• Pelvic hematoma healthy mature ovum are as follows: >75 4
strands
•• Scanty, high resistance blood flow • Greater than 10 mm in diameter Adapted from Bhal et al.12
•• Corpus luteum (Fig. 7.11)
• Rate of growth: 2–3 mm per day
•• Thick crenulated walls
•• Varied echogenicity and • Pencil line like thin walls
• Resistivity Index (RI): 0.4–0.48
appearance • No internal echogenicity
Fig. 7.10: Hemorrhagic cyst. • Peak Systolic Velocity (PSV) greater than 10 cm/s
•• RI <0.5, PSV >5 • Shape may be round to polygonal (due to pressure
•• Flushed with blood flow effect from adjacent follicles) Importance of Flow Parameters on
Characteristics of a mature follicle: Ovum Quality
• Size: 16–18 mm • Decreased blood flow to the follicle signifies increased
• Thin walls oocyte hypoxia.
• Round in shape • This leads to the abnormality in the chromosomal
• No echogenicities in the lumen organisation on the metaphase spindle leading to seg-
• Thin hypoechoic rim surrounding the follicle (devel- regation disorders.
ops 36 hours before rupture) • Mosaicism in embryos
• Cumulus-like shadow (about 35–40% cases) (develops Nargund et al in their study concluded that the prob-
36 hours before rupture) ability of getting a grade 1 or 2 embryos is 75% if ePSV was
greater than 10 cm/s, 40% if PSV less than 10 cm/s, 24 % if
Doppler features of a mature follicle (Figs. 7.14 and 7.15)
no perifollicular flow was seen.13
• Blood vessels cover at least three fourth of the follicular In another study by Coulam et al it was suggested that
circumference (grades 3–4 of vascularity) Table 7.9. a threshold value of ≥10 cm/s for PSV in at least one grade
Fig. 7.11: Functional corpus luteum with ring of vascularity around it. Fig. 7.12: Hemorrhagic cyst.
• Increase in the stromal echogenicity, area, and volume.
• Increased stromal vascularity.
• High uterine artery resistance.
Follicular Monitoring11
During an IVF or an IUI cycle, the follicular and the endo-
metrial maturity have to be assessed prior to giving the
hCG trigger. Since the advent of the transvaginal ultra-
sonography, it, being noninvasive by nature, has taken an
upper hand in monitoring of a stimulated cycle as com-
pared with the blood estrogen levels.
The development of the follicles and endometrium,
ovulation and leutinisation is a delicate interplay between Fig. 7.15: The Doppler findings of a preovulatory follicle. Peri-follicular
various morphological, biochemical, and vascular Fig. 7.14: Polycystic ovary. Volume exceeding 10 cc, increased stromal Blood Flow (PFBF) is seen to be greater than 75% (arrow). PSV is greater
Fig. 7.13: Endometriotic cyst with ground glass appearance. echogenicity, and numerous antral follicles. than 10 cm/s.
changes. The morphological changes can be well assessed
52 The Infertility Manual Ultrasound in Infertility 53
Table 7.10. Various procedures under ultrasonographic guidance.
Intra Uterine
Procedure Insemination (IUI) Ovum Pick Up (OPU) Embryo Transfer (ET)
Advantage Better visualisation Minimal access Improved visualisation of the endometrial cavity
less recovery time better pregnancy rates especially in older women
easy access to ovaries cathteter does not curl up or bend
Disadvantage Cost of machinery Chance of infection and Learning curve
expertise in USG bleeding requirement of expensive machine
Evidence Ultrasound-guided Minimizing the risk of infec- The authors believe that ultrasound-guided embryo
IUI does not produce tion and bleeding at trans- transfers should be used in clinically difficult em-
better results than blind vaginal ultrasound-guided bryo transfers and in older women, as it appears
insemination because ovum pick-up: Results of to improve the pregnancy rate over clinical touch
the PR per cycle is a prospective web-based transfers. Embryo transfer: Ultrasound-guided
similar.19 world-wide survey.18 versus clinical touch.20
Fig. 7.17: Zone 4 vascularity of a late follicular phase endometrium
Fig. 7.16: Triple line pattern of late follicular phase endometrium. suggestive of receptive endometrium.
Complications Bullet sign: Easy passage of the drop of media con-
taining embryo followed by the air bubble
• Pelvic infection (0.1–0.6%)18
3–4 follicle predicted a pregnancy with a sensitivity of 91%, • Zone 4: Blood vessels reaching the endometrial Minor bleeding (1.4–18.4%)18 Assessment of Early Pregnancy
specificity of 36%, positive predictive value of 13%, and cavity. Injury to the iliac vessels
negative predictive value of 97%.14 These blood vessels show an RI of 0.49–0.59 Puncture of an endometrioma or hydrosalpinx Before the Gestational Sac Appears
Bleeding from the uterine vessels (Midluteal Scan)
CYCLICAL CHANGES IN THE Features of Follicular Rupture
The embryo implants on the Day 5 of fertilization. In
ENDOMETRIUM15 • Decrease or disappearance of the preexisting follicle Embryo Transfer response to this, and the release of progesterone from
• Presence of a corpus luteum • Full bladder the corpus luteum, the endometrium becomes thick and
The changes in the endometrium with respect to the men-
• Free fluid in the POD • Lithotomy position decidulalised.
strual cycle has been mentioned above. The endometrial • Secretory pattern of the endometrium • Asepsis Also there is increased blood flow due to neoangio
growth is similar in case of stimulated and nonstimulated
• Cervical and vaginal cleaning with saline and media genesis.
cycles. It is seen that in gonadotropin stimulated cycles,
ULTRASOUND-GUIDED PROCEDURES • Transabdominal ultrasound to visualise the endome- The USG features that may help to predict pregnancy
the endometrial growth is thicker.
(TABLE 7.10) trial cavity are as follows:
2D features of endometrium with good receptivity • Asymmetric endometrial thickening.
• Soft catheters preferred over rigid ones
(assessing the anatomic maturity)
Ovum Pick up • Canula is seen as a hyperechogenic line in the cavity. • Corpus luteum seen in one of the ovaries.
• Optimum endometrial thickness of 8 mm • The uterocervical angle may be measured and the Colour Doppler:
• Multilayered endometrium (Fig. 7.16)
Technique angulation in the catheter may be adjusted a ccordingly. • Localised area of vascularity on the thickened endo-
• Endometrial grading16 • IV General anesthesia/local anesthesia + sedation. • Use of stylet in case of difficult cervical passage makes metrium (described as comet sign).
Grade A: Multilayered endometrium with interven- • Lithotomy position. Embryo Transfer (ET) easy. • High velocity, low resistance vessels with PSV greater
ing area more echogenic than the myometrium. • Aseptic precautions. • Tip of the catheter is placed approximately 1–1.5 cm than 6 cm/s.
Grade B: Multilayered endometrium with echo- • Cleanse TVS probe. Cover with sterile probe cover from the fundus so that the embryos are released in the • Corpus lutem vascularity with low Resistivity Index
after instilling with gel. mid portion of the uterine cavity. less than 0.5.
genic area hypoechoic to the myometrium.
• Biopsy guide on the probe to be fixed along with the 3D USG shows the asymmetrical area of thickening
Grade C: Homogenous hyperechoic endometrium
Doppler features of a receptive endometrium (assess-
biopsy guide line on the scanner. Optimisation of the Embryo Transfer more clearly.
• Introduce TVS probe such that the follicle of interest is
ing the functional maturity)17 (Fig. 7.17) • Pretransfer assessment of the uterine cavity
• Zone 1: Blood vessels reaching the EMJ.
in line with the biopsy line.
• Thorough but gentle removal of the cervical mucus Early Pregnancy Scan
• Pick up needle is introduced through the fornix.
• Zone 2: Blood vessels reaching the hyperechoic endo- • Suction applied upto 110–120 mmHg. • Avoid touching the fundus to avoid uterine contrac- Scans are done during early pregnancy for the following
metrial edge. • Follicles aspirated one after the other and the proce- tions and endometrial damage reasons:
• Zone 3: Blood vessels reaching the internal endome- dure is repeated on the opposite side. • Confirmation of delivery of the embryos by • Confirmation of pregnancy
trial hypoechoic zone. • Probe is removed and checked for bleeding. Confirming location of the catheter tip • To look for any abnormal/ectopic pregnancy
54 The Infertility Manual Ultrasound in Infertility 55
• To look for viability PROBABLE QUESTIONS prediction of oocyte recovery and preimplantation embryo 17. Bhandari H, Agrawal R, Weissman A, Shoham G, Leong M,
• To look for multiple gestation quality. Hum Reprod. 1996;11:2512-17. Shoham Z. Minimizing the risk of infection and bleeding
• To determine the correct gestational age 1. Describe the ultrasound findings of a mature follicle. 13. Coulam CB, Goodman C. Colour Doppler indices of at trans-vaginal ultrasound-guided ovum pick-up: results
2. Write a short note on systematic scan. follicular blood flow as predictors of pregnancy after of a prospective web-based world-wide survey. J Obstet
Parameters to note in case of an early pregnancy scan21 Gynaecol India. 2015;65(6):389-95.
3. Enumerate the sonographic differences between in-vitro fertilization and embryo transfer. Hum Reprod.
1. Gestational sac: Gestational sac is a round, anechoic 1999;14(8):1979-82. 18. Ramón O, Matorras R, Corcóstegui B, Meabe A,
fibroid and focal adenomyosis. Burgos J, Expósito A, et al. Ultrasound-guided artificial
structure, 2–3 mm in diameter. It has thick hyper- 14. Fleischer AC. Sonographic assessment of endometrial
4. What is the IOTA classification for adnexal lesions? insemination: a randomized controlled trial. Hum Reprod.
echoic margins and is placed eccentrically in the disorders. Semin Ultrasound CT MR. 1999;20:259-66.
Describe various adnexal lesions under the same. 15. Gonen Y, Casper RF. Prediction of implantation by the
2009;24(5):1080-84.
uterus. The echogenic margin is representative of 5. Describe cyclical changes in ovaries and endome- 19. Kan AKS, Abdalla HI, Gafar AH, Nappi L, Ogunyemi BO,
sonographic appearance of the endometrium during
the choriodecidual reaction and is called the Double trium in COS. Thomas A, Ola-ojo OO, Embryo transfer: ultrasound-guided
controlled ovarian stimulation for IVF. J In Vitro Fert versus clinical touch. Hum Reprod. 1999;14(5):1259-61.
Decidual Ring sign. Presence of a gestational sac is a
Embryo Transf. 1990;7:146-52. 20. Honeymeyer U, Kurjak A, Monni G. Normal and Abnormal
confirmatory diagnosis of pregnancy. 16. Applebaum M. The ‘steel’ and ‘teflon’ endometrium— Early Pregnancy in Donald School Text Book of Ultrasound
2. Yolk sac: Appears after 5 weeks. It is the first struc- REFERENCES ultrasound visualisation of endometrium in IVF patients in Obstetrics and Gynecology; Jaypee Medical Publishers;
ture to appear within the gestational sac. Yolk sac is and outcome. Presented at the third world congress of pp. 106-29.
1. ICU Sonography. Basics physics of ultrasound and the
a c ircular, thick walled echogenic structure with an Doppler phenomenon. [online]. Available from http:// ultrasound of obstetrics and gynecology. Ultrasound 21. Muzio BD, Galliard F. Yolk sac. 2017. Available from https://
anechoic center. The yolk is not seen in pseudosac, www.criticalecho.com. Obstet Gynecol.1995;6:191-8. radiopaedia.org/articles/yolk-sac.
anembryonic pregnancy or decidual cyst and is pre- 2. Morgan MA. (2005–2017) Thermal Index.[online]. Available
sent only in an intrauterine gestational sac.22 Fetal from https://radiopaedia.org/articles/thermal-index.
pole of 1.2–2 mm may be seen and may have cardiac 3. Panchal S, Nagori C. Ultrasound in Infertility and
flutter. Gynecology. Text and Atlas. 1st edition. New Delhi: Jaypee
Brothers; 2015.
3. Crown rump length (CRL): By around 6 weeks, the
4. Hall DA, Yoder IC. Ultrasound evaluation of the uterus.
fetal pole is around 4–8 mm. The cardiac activity can In: Callen PW (Ed). Ultrasonography in Obstetrics and
be seen clearly within the fetal pole. Gynecology. 3rd edition. Philadelphia: Pa Saunders; 1994.
4. Functional corpus luteum may be seen in one of the pp. 586-614.
ovaries. 5. Advanced Women’s Imaging. Pelvic gynaecological ultrasound
[online]. Available from www.advancedwomensimaging.
Ectopic gestation: com.au/pelvic-gynaecologic-ultrasound.
Features of tubal ectopic pregnancy on USG (Table 7.11): 6. Raga F, Bonilla-Musoles F, Blanes J. Congenital
• No intrauterine gestational sac Mulleriananomalies: diagnostic accuracy of three
• Symmetric endometrial cavity dimensional ultrasound. Fertil Steril. 1996;65(3):523-8.
• A pseudo sac may be seen in the uterus 7. Sohail S. Uterine sonomorphology on Asherman
syndrome on transvaginal ultrasound. Pak J Med Sci.
• Adnexal complex mass lesion with or without a gesta-
2005;21(4):451-4.
tional sac with or without cardiac activity. 8. Timmerman D, Valentin L. Terms, definitions and
• Doppler suggestive of low resistance vascularity measurements to describe the sonographic features of
around the lesion. “Ring of fire” appearance may be adnexal tumors: a consensus opinion from the International
seen. Ovarian Tumor Analysis (IOTA) group. Ultrasound Obstet
• Free fluid in the pelvis Gynaecol. 2000;16:500-5.
9. Rotterdam ESHRE/ASRM- Sponsored PCOS Workshop
• Functional corpus luteum on the same side of the
Group. Revised 2003 consensus on diagnostic criteria and
mass lesion long term health risk related to polycystic ovary syndrome.
• Tenderness on pressing the probe on the ipsilateral Fertil Steril. 2004;81(1):19-25.
side. 10. Lujan ME, Jarrett BY, Brooks ED, Reines JK, Peppin AK, Muhn
N, et al. Updated ultrasound criteria for polycystic ovary
syndrome: reliable thresholds for elevated follicle population
Table 7.11. Difference between pseudosac and true sac. and ovarian volume. Hum Reprod. 2013;28(5):1361-8.
PanchalS, Nagori CB. Follicular monitoring. Donald School
Features Pseudosac True sac J Ultrasound Obstet Gynecol. 2012;6(3):300-12.
Position in the uterine Central Eccentric 11. Bhal PS, Pugh ND. The use of transvaginal power Doppler
cavity ultrasonography to evaluate the relationship between
Ring sign Absent Present perifollicular vascularity and outcome in in-vitro fertilization
treatment cycles. Hum Reprod.1999;14(4):939-45.
Response to uterine Change in No change in 12. Nargund G, Doyle PE. Ultrasound derived indices of
peristalsis shape shape follicular blood flow before HCG administration and the
CH A PTE R
Unexplained Infertility
8 •
•
Mid luteal progesterone greater than 3 ng/mL—
for pregnancy.
Endometrial biopsy obsolete nowadays.
mentioned above.
POSSIBLE CAUSES OF SUBFERTILITY
Unexplained Infertility 57
Unexplained infertility is a diagnosis, which should be
indicate ovulation, greater than 10 ng/mL—optimum made by excluding all the possible causes for infertility as
Navya Vavuluru
ASSESSMENT OF TUBES AND UTERUS THAT HAVE BEEN PROPOSED AS UNDER-
Chapter Outline • HSG is considered as standard investigation for evalu LYING “UNEXPLAINED INFERTILITY”6
• Incidence • Possible Causes of Subfertility that have been Proposed as ation of tubes and cavity. It is quite reliable when per
• Diagnostic Criteria Underlying “Unexplained Infertility” formed in correct technique under supervision. Disturbances in endocrinological balance, immunology,
• Assessment of Male Factor • Chances of Conception
• Assessment of Ovulation • Sensitivity 44–75%, specificity 55%. genetic, and reproductive physiology.
• Management
• Assessment of Tubes and Uterus • Different Modalities of Management • Sonosalpingography can be done in case of endome
• Assessment of Ovarian Reserve
• Laparoscopy and Hysteroscopy
• Prognosis trial polyps, intrauterine adhesions, and submucous Peritoneal Factors
myomas.
• Transvaginal ultrasound—used to assess uterine • Increased phagocytic activity of peritoneal leuco
architecture, ovaries-antral follicular count. cytes—which engulf oocyte/sperm
DEFINITION • If semen analysis is abnormal for first time, repeat • Minimal endometriosis
analysis after 4–6 weeks in a laboratory that adheres to • Antibodies to chlamydia
Diagnosis of unexplained infertility was made in couples WHO guidelines. ASSESSMENT OF OVARIAN RESERVE
in whom all standard investigations that is ovulation, tubal • Despite of its limitations semen analysis remains most • Women with advanced age, previous surgery, endo Ovarian and Endocrine Factors
patency, and semen analysis are normal but not yet con important tool in investigating male infertility. metriosis, and chemotherapy are at risk of diminished
ceived after 1 year of unprotected intercourse/6 months • If any abnormality noted patient referred to urologist • Tonic increase in LH secretion
ovarian reserve.
if female greater than 35 years. Diagnosis made by exclu as part of multidisciplinary approach. • Hyper prolactinemia
• This can be tested by day 2/3-follicle stimulating hor
sion. Conception is delayed by chance as their fecundity • Abnormalities in follicle growth
mone (FSH), estradiol, antral follicle count (AFC).
is lower due to subtle undetected defects in reproductive • Reduced growth hormone secretion/sensitivity
process.1
ASSESSMENT OF OVULATION4 • These tests are not absolute indicators of fertility, but
we can correlate the response of ovulation induction • Genetic and cytological abnormalities in oocytes
• The ovulatory defects account for 15% of couples with • Poor quality oocytes
medications.
INCIDENCE infertility. • Increased age
• Detailed history, general, local, speculum, bimanual
15–30% among infertile couple.2 LAPAROSCOPY AND HYSTEROSCOPY • Luteinized unruptured follicle (LUF)
examinations, investigations including endocrino
logical tests, and ultrasound should be noted care • In the present era, laparoscopy is the main tool in the
DIAGNOSTIC CRITERIA fully. armamentarium of infertility work up, which serves as Tubal Factors
ASRM 20123 • Additional tests like basal body temperature (BBT), both diagnostic and therapeutic modality. • Abnormal peristaltic movements—bidirectional
urinary LH, mid luteal progesterone can be done. • This test reserved for selected cases to recognize endo movement
1. Semen analysis: World Health Organization (WHO)
• BBT cumbersome to patient, not reliable always, metriosis, pelvic adhesions, hydrosalpinx, and other • Lost ciliary activity of cells inside lumen of fallopian
5th Edition criteria to be followed
retrograde method hence not practical. peritoneal factors. tube
2. Assess ovulation: Menstrual cycle, serum p4 in mid
• Minimal hydrosalpinx that is not detected by ultra
luteal phase, luteinizing hormone (LH) surge kit Table 8.1. The world health organization’s 5th edition Established Not consistently sound
3. Evaluate tubal patency and uterine cavity— of “normal semen analysis” values are shown below.5 correlation with correlated with Not correlated
hysterosalpingography/hysterolaparoscopy Semen analysis parameter Normal values pregnancy pregnancy with pregnancy
4. Transvaginal ultrasonography (TVS) Volume 1.5 mL or more •• Semen •• PCT (postcoital •• Endometrial Endometrial Factors
5. If indicated tests for ovarian reserve (unexplained analysis test) dating
pH ≥7.2 •• Tubal patency •• Cervical mucus •• Varicocele • Reduced expression α1,4 and β3 integrins (adhesion
infertility) and laparoscopy
Sperm concentration 15,000,000/mL or more by HSG penetration test assessment molecules)
6. Sperm function tests, antisperm antibodies tests are (hysterosal- •• ASA (antisperm- •• Falloposcopy
Total motility 40% or more • Abnormal secretion of adhesion molecules such as
not routinely recommended pingogram)/ antibodies) •• Chlamydia cathedrins and integrins
Progressive motility 32% or more laparoscopy •• Hysteroscopy testing
• Altered Th 1/Th 2 ratio
ASSESSMENT OF MALE FACTOR4 Morphology 4% or more normal forms •• Ovulation •• Lap findings of
• Uterine perfusion problems
(strict criteria) mild endometriosis
• The male factor accounts for 30% of infertile p
opulation. •• Zona free hamster • Nonreceptive endometrium due to altered gene
Vitality 58% or more live
• Detailed history, general and local examination, egg penetration expression
White blood cells <1,000,000/mL test
semen analysis should be noted. • Luteal phase defects
58 The Infertility Manual Unexplained Infertility 59
Cervical Factors 3. Intra uterine insemination (IUI) with super ovulation • The chances of potential side effects and the risks of e vidence of a difference in live birth rate between the
4. In vitro fertilization (IVF) multiple pregnancies are to be discussed in detail with two treatment groups.
• Altered cervical mucus 5. Use of aromatase inhibitors the couple.10 But because of low cost and easy avail • IUI in natural cycle versus TI or expectant manage
• Immunological factors ability this drug remains always a first choice. ment in stimulated cycle, there was evidence of an
Expectant Therapy and Lifestyle • Tailored expectant management of at least 6 months increase in live births for IUI.14
Male Factors in couples with unexplained infertility is not associ
Modifications • Mohamed Aboulghar et al. concluded that overall PR
• Fertilization ability of sperm—ability to negotiate uter ated with doubts regarding quality of care or trust on per cycle in cycles 4–6 of controlled ovarian stimulation
otubal junction, capacitation and acrosome reaction, • Unfortunately it is not possible to predict which couple their physician.11 (COH)/IUI was significantly lower than in first three
and penetrate zona pellucida will conceive spontaneously or in what time frame as • As there is good prognosis of natural conception within cycles and higher PR was achieved in the group receiv
• Egg fertilized with morphologically abnormal sperm after 3 years of marital life 60% will conceive spontane 1 year in couples with unexplained infertility, expect ing IVF/ICSI after three failed cycles of COH/IUI. These
• High DNA fragmentation ously and after 5 years of marriage 80% will conceive. ant management with good medical assistance for points are very much helpful in counseling the couple
• The chance of spontaneous pregnancy with expectant 6–12 months does not compromise ongoing birth rates
management is low but never zero. while management of their unexplained infertility.15
Embryological Factors • As the age of female partner greater than 35 years and
and is equally effective as starting medically assisted • Most of the recent trials concluded that among the
reproduction immediately.11
• Poor quality embryos duration of infertility greater than 3 years its always subjects undergoing COH/IUI, there is no much sta
• Fertilization failure wise to start aggressive therapy than expectant therapy. tistically significant difference between single and
• Aneuploidy resulting in increased miscarriage rates • Expectant management has role where the resources IUI and COS with Gonadotropins double IUI, therefore double IUI is not performed
are limited, female partner age is less than 28–30 years, • IUI is the procedure where washed sperms are placed routinely.16
where the duration of marriage is less than 2 years.4,8 • In a randomized controlled study by Tayfun Bagis
CHANCES OF CONCEPTION inside the uterine cavity around the time of ovulation.
• Most of the studies concluded that cigarette smok • It can be performed in natural cycles with LH kits or cc et al. eligible patients were divided in to two arms
84% will conceive in first year. ing, abnormal body mass index (BMI), excess caffeine induced cycles or in cycles where gonadotropins are where one arm received single IUI 36 hours after trig
If age is less than 20 years—76% chances of conception intake, and alcohol consumption reduce fertility in ger and other arm received double IUI 18 and 40 h
used.
If age is less than 30 years—57% chances of conception the female partner and affect the sperm parameters • Nat Athaullah et al. concluded in Cochrane, that there after trigger.
If age is less than 40 years—40% chances of conception adversely. is insufficient evidence to suggest that oral ovulogens • This study did not find any difference in live birth rate
If age increases by 1 year after 30 years pregnancy • Couple as such should be counseled to control and
are inferior or superior to injectable agents in treat (LBR) between single and double IUI groups in stimu
chances of pregnancy decreases by 9%. stop all the addictions while they are wishing for the
ment of unexplained subfertility and there was insuffi lated OH cycles with multifollicular development.17
92% may conceive in second year.7 pregnancy.
cient evidence to prefer gonadotropins when compar
• Expert advice along with involvement of dieticians,
ing pregnancy or live births.12
MANAGEMENT nutrition counselors, psychological therapists, and
• According to National Institutes of Health (NIH)
In Vitro Fertilization
physicians should be considered in modifying life style
Once there is cause which is well known and obvious then sponsored study fast track and standard treatment • IVF with or without intra cytoplasmic sperm injection
which is very helpful for couple in achieving the goal.
the treatment of the couple becomes easy. In unexplained (FASTT) trial, compared with conventional treat is widely accepted, successful, expensive but invasive
Expectant management always can be provided as a first
infertility, the disease itself is not defined, so the treatment ment which includes CC/IUI, followed by gonado modality of treatment for unexplained infertility.
option for a couple with unexplained infertility in whom
remains empirical. An addition of 1 month after average tropins/IUI if failed then for IVF, when women is • IVF increases the number and quality of oocytes
the female partner is young and the problems of oocyte
age of reproductive period decreases the chances of preg younger than 40 years an accelerated approach that available for fertilization, it also facilitates sperm–
depletion are not at all an immediate concern because of
nancy by 2%. As female partner age increases by 1 year less includes cycles with CC/IUI if failed immediately oocyte interaction and enhances the chances of
than 30 years, pregnancy rate is reduced by 9%.1 its low cost even though it has low fecundity rates. IVF after eliminating cycles with gonadotropins/
fertilization.
Aim of the treatment in these cases has to increase IUI resulted in shorter time to pregnancy and is also
the pregnancy rate above natural rate of 1.5–3%, improve Clomiphene Citrate cost-effective.13
• IVF also aids in documenting the occurrence of fertili
zation and also helps in evaluating the embryo quality,
ment in gamete quality of both male and female part • CC is the commonest and first modality of treatment • Veltman-Verhulst et al. concluded in cochrane review,
ner, increase in gamete number which facilitates gamete as the chances of pregnancy reduces every year after
for unexplained infertility. included 14 trials including 1,867 women, and con
interaction. Treatment and prognosis depend on duration age of 40 years, the women should be referred to IVF
• CC has been used in unexplained infertility as it is time cluded that there is no difference in live births in IUI
of infertility, women’s age, and the previous pregnancy versus timed intercourse (TI) or expectant management after a short trial of gonadotropins and IUI.1
tested, low technology, and cost-effective, easily avail
history. both in natural cycle, IUI versus TI or expectant manage • Zabeena Pandian et al. included six randomized con
able drug. From all the randomized controlled trials, it
was concluded that the number of clomiphene cycles ment both in stimulated cycle. trolled trials in final analysis concluded that IVF may
be more effective than IUI and super ovulation in cou
DIFFERENT MODALITIES needed for one additional pregnancy was 40 when • An increase in live birth rate was found for women who
compared to placebo.9,10 were treated with IUI in a stimulated cycle compared ples suffering from unexplained infertility.18
OF MANAGEMENT with those who underwent IUI in natural cycle. • Lauren et al. concluded in a meta-analysis which
• Edwards Hughes et al. concluded in Cochrane 2010
1. Expectant management and life style changes that there is no clinical benefit of using CC for unex • In cases of IUI in a stimulated cycle versus TI or expect included eleven studies with 901 couples that use of
2. Use of clomiphene citrate (CC) plained infertility. ant management in a natural cycle, there was no ICSI favors increase in fertilization rates and decrease
60 The Infertility Manual Unexplained Infertility 61
Flowchart 8.1: Overview of management of unexplained infertility6 between both the drugs. Although the current evidence on 5. Cooper TG, Noonan E, von Eckardstein S, Auger J, Baker the fast track and standard treatment (FASTT) trial. Fertil
these drugs is weak, meta-analysis concluded that the HWG, Behre HM, et al. World Health Organization Steril. 2010;94(3):888-99.
reference values for human semen characteristics. Human 14. Veltman-Verhulst SM, Cohlen BJ, Hughes E, Heineman MJ.
pregnancy rates are comparable with these two groups of
Reprod Update. 2010;16(3): 231-45. Intra-uterine insemination for unexplained subfertility.
drugs.1 6. Glenn LS, Sandro E, Ashok A (Eds). Unexplained Infertility Cochrane Database Systc Rev. 2012; Issue 9. Art. No.:
The optimal treatment strategy needs to be based on Pathophysiology, Evaluation and Treatment. New York: CD001838.
individual patient characteristics such as age, treatment Springer; 2015. 15. Aboulghar M, Mansour R, Serour G, Abdrazek A,
efficacy, resources available, side-effect profile such as 7. Eimers J, te Velde E, Gerritse R, Vogelzang E, Looman C, Amin Y, Rhodes C. Controlled ovarian hyperstimulation
multiple pregnancies and cost considerations. Habbema J. The prediction of the chance to conceive in and intrauterine insemination for treatment of unexplained
subfertile couples. Fertil Steril. 1994;61(1):44-52. infertility should be limited to a maximum of three trials.
8. Guzick DS, Sullivan MW, Adamson, GD, Cedars M, Falk Fertil Steril. 2001;75(1):88-91.
PROGNOSIS RJ, Peterson EP, et al. Efficacy of treatment for unexplained 16. Gezginç K, Görkemli H, Çelik Ç, Karatayli R,
infertility. Fertil Steril. 1998;70(2):207-13. Çiçek M, Çolakoglu M. Comparison of single versus double
Prognosis is good 9. Randall J, Templeton A. The effects of clomiphene citrate intrauterine insemination. Taiwanese J Obstet Gynecol.
• Female age less than 30 years upon ovulation and endocrinology when administered 2008;47(1):57-61.
to patients with unexplained infertility. Obstetr Gynecol 17. Bagis T, Haydardedeoglu B, Kilicdag E, Cok T,
• Infertility less than 2 years
Survey. 1992;47(2):129-30. Simsek E, Parlakgumus A. Single versus double intrauterine
• Previous pregnancy {1.8 times greater chances of preg 10. Hughes E, Brown J, Collins JJ, Vanderkerchove P. Clomiphene insemination in multi-follicular ovarian hyperstimulation
nancy} citrate for unexplained subfertility in women. Cochrane cycles: a randomized trial. Human Reprod. 2010;25(7):
Prognosis is worse Database Syst Rev. 2010;Issue 1. Art. No.: CD000057. 1684-90.
• Female age greater than 35 years 11. Kersten FAM, Hermens RPGM, Braat DDM, Tepe E, 18. Pandian Z, Gibreel A, Bhattacharya S. In vitro fertilisation
• Infertility greater than 3 years Sluijmer A, Kuchen becker WK, et al. Tailored expectant for unexplained subfertility. Cochrane Database Syst Rev.
management in couples with unexplained infertility does 2012; Issue 4. Art. No.: CD003357.
not influence their experiences with the quality of fertility 19. Johnson L, Sasson I, Sammel M, Dokras A. Does
in the risk of total fertilization failure rates in couples PROBABLE QUESTIONS care. Human Reprod. 2015;1-9. intracytoplasmic sperm injection improve the fertilization
12. Athaullah N, Proctor M, Johnson N. Oralversus injectable rate and decrease the total fertilization failure rate in couples
with well-defined unexplained infertility.19 1. What is unexplained infertility? What is the percent ovulation induction agents for unexplained subfertility. with well-defined unexplained infertility? A systematic
• ICSI should be preferred as first option for the women age of couple that falls under this category? What is Cochrane Database Syst Rev. 2002; 3. Art. No.: CD003052. review and meta-analysis. Fertil Steril. 2013;100(3):
with advanced maternal age. Fertilization failure per the standard fertility testing means? 13. Reindollar R, Regan M, Neumann P, Levine B, 704-11.
centages are high with conventional IVF than with 2. Explain the possible reasons for unexplained Thornton K, Alper M, et al. A randomized clinical trial 20. Van Voorhis B. Outcomes from assisted reproductive
ICSI; therefore, most of the IVF centers perform ICSI to evaluate optimal treatment for unexplained infertility: technology. Obstet Gynecol. 2006;107(1):183-200.
infertility.
as a routine procedure in IVF. 3. How do you evaluate and counsel a couple with unex
• IVF procedure may be associated with some unwanted plained infertility?
side effects like multiple pregnancies, ovarian hyper 4. What are the various modalities of management avail
stimulation syndrome (OHSS), ectopic pregnancies, able to treat a case of unexplained infertility?
and increased perinatal mortality and morbidity 5. What is the evidence-based management in a case of
which are very rare.20 unexplained infertility?
• Based on the data available, the following recommen 6. IUI versus IVF in unexplained infertility—debate
dation may be beneficial for the couples suffering from 7. IVF is the modality of management in a case of unex
unexplained infertility—expectant management to plained infertility—justify with evidence.
be followed for certain period of time before stepping
to interventional treatment if all the parameters for REFERENCES
expectant management are satisfied (Flowchart 8.1).
1. Ray A, Shah A, Gudi A, Homburg R. Unexplained infertility:
• IUI may not be beneficial without super ovulation, CC/
an update and review of practice. Reprod BioMed Online.
IUI is more effective than HMG/IUI, IVF provides the 2012;24(6):591-602.
best option for these couples even though it is cost- 2. Practice Committee of the American Society for
effective.8 Reproductive Medicine. Effectiveness and treatment for
unexplained infertility. Fertil Steril. 2006;86(5):S111-4.
3. Gianaroli L, Racowsky C, Geraedts J, Cedars M,
Aromatase Inhibitors Makrigiannakis A, Lobo R. Best practices of ASRM and
ESHRE: a journey through reproductive medicine. Fertil
Many authors and studies compared different aromatase Steril. 2012;98(6):1380-94.
inhibitors with clomiphene. All these studies and meta- 4. Quaas A, Dokras A. Diagnosis and treatment of unexplained
analysis showed that there is no significant difference infertility. Rev Obstetr Gynecol. 2008;1(2):69-76.
SE C T I O N
2
Endocrinological Disorders
of Infertility
C HA PTE R
Menstrual Cycle
9
J Sneha
Chapter Outline
• Follicular Phase
INTRODUCTION The cyclical changes that occur in the uterus (prepa-
ration for implantation of embryo) and ovary (production
1. Menstrual cycle constitutes the cyclical changes of eggs) for the purpose of sexual reproduction constitutes
which occur in the uterus and ovary for the purpose the menstrual cycle.
of sexual reproduction. It is divided into two phases based on the ovarian and
2. The events which occur sequentially in the ovary to endometrial events:
prepare the right number of follicles for ovulation 1. Ovarian cycle: The follicular phase, phase of ovulation,
constitute the follicular phase. luteal phase, and luteal–follicular transition phase.
3. An average of about 400 ovulations occur between 2. Uterine cycle: Menstruation, proliferative phase, and
secretory phase.
menarche and menopause.
4. The sequence of changes which occur in the uterus
and ovary post ovulation constitute the luteal phase. FOLLICULAR PHASE
5. Menstruation is the process of shedding of the endo- The events that occur sequentially in the ovary to prepare
metrium due to fall in progesterone production from the right number of follicles for ovulation constitute the
corpus luteum. follicular phase (Figs. 9.1 and 9.2).
Fig. 9.1: Changes in germ cell number in relation to age.
66 The Infertility Manual Menstrual Cycle 67
Fig. 9.3: Primordial follicle.
Fig. 9.2: Stages of follicular growth.
It usually terminates with one surviving mature follicle Primordial follicle Composed of a single layer of spindle- Fig. 9.4: Preantral follicle.
in the ovary over a space of 10–14 days.1 shaped granulosa cells surrounding an oocyte (arrested in
The follicle destined to ovulate passes through the fol- diplotene stage of prophase of meiosis 1) (Fig. 9.3).
lowing phases over a span of approximately 85 days.
Preantral follicle Composed of multiple layers of granu-
1. Primordial follicle losa cells, theca interna, and theca externa surrounding an
2. Preantral follicle oocyte (Fig. 9.4).
3. Antral follicle Antral follicle Fluid in the intercellular spaces coalesce
4. Preovulatory follicle to form an antrum within the follicle (Fig. 9.5).
Development of Follicle Recruitment of Follicle
The primordial germ cells originate from yolk sac endo- A follicular cohort is recruited to grow in each cycle (Fig. 9.2).
derm, hindgut, and allantois of the developing embryo The early growth of follicles upto the antral phase is
and migrate to gonadal ridge by 5–6 weeks period of follicle-stimulating hormone (FSH) independent. Even-
gestation. tually, they become FSH dependent and undergo atresia
The maximum oocyte number of 6–7 million is reached unless recruited by FSH.
Fig. 9.5: Antral follicle. Fig. 9.6: Two-cell two-gonadotropin hypothesis.
by 16–20 weeks period of gestation (Table 9.1).2 The follicular development is characterized by:3
Follicles begin to grow and undergo atresia (Fig. 9.1). 1. Oocyte increasing in size of.
At birth, there are 2 million oocytes. 2. Multilayered granulosa cells that become cuboidal in Two-cell Two-gonadotropin Hypothesis 2. Enlargement of granulosa cells with acquisition of
shape. lipid inclusions.
At puberty, there are 3,00,000 oocytes. Selection of Dominant Follicle The growing follicle pro- 3. Increased vascularity and assumption of vacuoles by
An average of about 400 ovulations occur between 3. Aromatization of androgen to estrogen induced by FSH. duces increasing levels of estrogen, which act at the hypo-
4. Increase the FSH receptor content of the follicle theca cells.
menarche and menopause. thalamus and pituitary level to produce a negative f eedback 4. Resumption of oocyte meiosis.
induced by FSH and estrogen together. effect on FSH. The falling FSH levels induce atresia of the
5. Theca cell layer forms from the surrounding stroma.
Table 9.1: Development of follicles (age and number of smaller follicles.5 The growth of the dominant follicle is Ovulation
oocytes in the ovary). favored due to:
Two-cell, two-gonadotropin hypothesis In early-stage The increasing estradiol production in the preovulatory
ovarian follicles theca cells, luteinizing
hormone (LH) 1. Greater content of FSH receptors in the follicle;
Age Number of oocytes in the ovary follicle crosses the peripheral threshold to induce a posi-
receptors and granulosa cells contain only FSH receptors 2. Enhancement of FSH action due to higher intrafolli-
16–20 weeks 6–7 million tive feedback at the hypothalamic pituitary level, hence
(Fig. 9.6). cular estrogen level.
the LH surge (Fig. 9.7).
Birth 2 million For the transition from negative to positive feedback of
LH acts on theca cells to produce androgens.
Androgens diffuse into the adjacent granulose cells
Preovulatory Follicle LH release;
Puberty 30,000
where they undergoes aromatization to estrogen, influ- Features include 1. Estradiol concentration must be greater than 200 pg/mL
Menopause 1,000
enced by FSH.4 1. Hyperemic appearance. 2. Sustained for 50 hours
68 The Infertility Manual Menstrual Cycle 69
Fig. 9.7: Pre-ovulatory follicle.
Timing of ovulation include Luteal cells are of two types:8
1. 10–12 hours post the peak in LH concentration 1. Large cells derived from granulosa cells, produce more
2. 24–36 hours post the peak in estradiol levels.6
progesterone than small cells and peptides (oxytocin,
inhibin, relaxin, gonadotropin-releasing hormone
The key events that occur during ovulation include
(GnRH), growth factors, and prostaglandins).
1. Meiosis resumption in the oocyte.
2. Small cells derived from theca cells are abundant in
2. Granulosa cells luteinization.
number, contain LH and hCG receptors.
3. Prostaglandins and progesterone syntheses that are
required for dissolution of the follicular wall and Regression of corpus luteum occurs 9–11 days after
release of oocyte. ovulation unless rescued by hCG of pregnancy.
4. Ensure adequate LH receptors for a normal luteal
phase. The Luteal–Follicular Transition
Initiation of luteinization and progesterone production in The phase of menstrual cycle between the fall in estradiol Fig. 9.8: Endometrial changes at different phases of menstrual cycle.
the granulosa cells of follicle is mediated by the action of LH. and progesterone production in the late luteal phase till
the selection of dominant follicle.9 This is characterized by
increased secretion of FSH due to: Proliferative phase of endometrium begins with the in thickness occurs only upto 3 days post ovulation. This is
Luteal Phase rise in estradiol produced by the growing follicles. The due to the effect of progesterone which include:12
1. Fall in estradiol, progesterone levels;
The normal luteal phase lasts for 14 days. changes which occur include: 1. Decreases the mitosis and DNA synthesis.
2. Fall in inhibin levels;
The remnants of the follicle form the corpus luteum in 3. Increase frequency of GnRH release from hypothalamus. 2. Decreased estrogen receptor expression.
1. The narrow tubular glands initially lined by colum-
the ovary. The granulosa cells enlarge and appear v acuolated nar epithelium become pseudostratified and mitosis 3. Increased enzyme activity of 17B hydroxysteroid
due to the presence of the yellow pigment “lutein.” dehydrogenase and sulfotransferase (increased
Endometrial Changes During the becomes prominent.
After the cessation of LH surge, capillaries penetrate the 2. The dense stromal component is converted into a loose formation of estrone sulfate from estradiol).
granulosa cell layer that peak by day 8 or 9 after ovulation. Menstrual Cycle (Fig. 9.8)
syncitium-like tissue as a result of stromal edema. The changes taking place in the endometrium include:
This coincides with peak levels of serum estradiol and The endometrium during the menstrual phase is charac- 3. The spiral vessels extent upto the basement membrane 1. Individual components of tissue continue to grow
progesterone. terized by a thin and compact tissue of nonfunctional stra- of the epithelium and forms a loose capillary network. leading to increasing endometrial gland tortuosity
For the optimal luteal phase, the following are required: tum basalis, overlying which is the residual stratum spon-
These changes cause thickening of single layer of and spiral vessels coiling.
giosum.10 Endometrial regeneration originates in:
1. Adequate development of follicles in the preovulatory endometrium from 0.5 mm at the beginning to 3.5–5 mm 2. Glandular cells acquire glycogen intracellular vacu-
phase. 1. Endometrial epithelial stem cells in the base of the toward the end of proliferative phase.11 oles (secretory changes) over a period of 7 days post
2. Continuous tonic LH support in the postovulatory endometrial glands; The changes in endometrium that take place during the ovulation.
luteal phase.7 2. Stromal stem cells in the basalis layer. luteal phase constitute the secretory phase. Further increase 3. Increase in stromal edema.
CH APT E R
70 The Infertility Manual
In the absence of fertilization and implantation, the
corpus luteum starts regressing in 14 days. This is charac-
terized by
1. Decreasing levels of estrogen and progesterone;
2. Shrinking of endometrial thickness.
2. Define menstrual cycle. Describe the changes occur-
ring in the ovary during each menstrual cycle.
3. Physiology of ovulation.
4. Describe the changes occurring in the uterus during
each menstrual cycle.
Normal and Abnormal Puberty
10
5. Neuroendocrine control of menstrual cycle. Meghana Nyapathy
3. Vasomotor changes in spiral artery that undergo
vasoconstriction and relaxation alternatively. The fre- Chapter Outline
quency and amplitude of the contractions increase REFERENCES
• Gonadarche • Precocious Puberty
progressively causing endometrial blanching, endo- 1. Gougeon A. Dynamics of follicular growth in the human: • Adrenarche
metrial ischemia, and menstruation. A model from preliminary results. Hum Reprod. 1986;1:81.
4. Progesterone stabilizes the lysosomal membranes and 2. Baker TG. A quantitative and cytological study of germ
retains the lytic enzymes such as acid phosphatase. cells in human ovaries. Proc Roy Soc Lond. 1963;158:417.
3. Oktay K, Newton H, Mullan J, Gosden RG. Development INTRODUCTION absence of adrenarche.1,3 Though gonadarche occurs with
With progesterone withdrawal, the lysosomal mem- the maturation of HPO axis and adrenarche is independent
of human primordial follicles to antral stages in SCID/hpg In humans, puberty is defined as the period of life when
brane is lysed causing release of the enzymes into
mice stimulated with follicle stimulating hormone. Hum of the same, the two are often temporarily related.2
epithelial, endothelial, and stromal cells that digest Reprod. 1998;13:1133. the person attains the capability to reproduce. It is char-
What triggers puberty is a compelling unanswered ques-
the cellular components. This leads to the release of 4. Kobayashi M, Nakano R, Ooshima A. Immunohistochemical acterized by maturation of the genital organs, increase in
prostaglandins, tissue necrosis, vascular thrombosis, tion in reproductive endocrinology. The factors responsible
localization of pituitary gonadotropins and gonadal steroids linear growth velocity, development of secondary sexual
and red blood cells extravasation. confirms the two cells two gonadotropins hypothesis of for the onset of puberty and menarche are as follows:
characteristics, and the occurrence of menarche.1
5. Matrix metalloproteinases also contribute to the steroidogenesis in the human ovary. J Endocrinol. 1990; 1. Genetics—Variations in or near locus(6q21) of the
126:483. The phases of puberty are as follows:
endometrial tissue breakdown.13 LIN28B gene and NEGR 1, activating mutations in
5. Montgomery Rice V, Limback SD, Roby KF, Terranova PF. 1. Gonadarche
Endometrial changes at different phases of menstrual KISS-1 or KISS 1 receptor.4,5
Differential responses of granulosa cells from small and a. Thelarche
cycle. large follicles to follicle stimulating hormone (FSH) during 2. Overall health—Early onset of puberty seen in over-
b. Menarche
the menstrual cycle and acyclicity: Effects of tumour weight and obese children. Prepubertal body compo-
Neuroendocrine Control of Menstrual Cycle necrosis factor-a. Hum Reprod. B 1998;13:1285. 2. Adrenarche
sition in healthy boys and girls influences the rate of
6. Ecochard R, Gougeon A. Side of ovulation and cycle
1. GnRH is secreted by the hypothalamus into the characteristics in normally fertile women. Hum Reprod.
progression of puberty.7
hypothalamo–pituitary portal system in a pulsatile 2000;15:752.
GONADARCHE 3. Social environment—Improved socio-economic sta-
fashion. The GnRH secretion is modulated by kisspep- 7. Chaffi n CL, Dissen GA, Stouffer RL. Hormonal regulation It includes the growth and maturation of gonads. This leads tus has brought down the age of puberty.
tin, neurokinin B, endogenous opiods/ dynorphin, of steroidogenic enzyme expression in granulosa cells 4. Environmental exposures—Endocrine disruptors like
to increased production of sex steroids, thus enabling the
and gonadotropin inhibitory hormone. during the periovulatory interval in monkeys. Mol Hum
Reprod. 2000;6:11. folliculogenesis and ovulation. Gonadarche is responsible dioxin and dioxin-like compounds, polychlorinated
2. The gonadotropins (LH and FSH) are secreted by the biphenyls, DDT, and other pesticides such as bisphe-
8. Retamales I, Carrasco I, Troncoso JL, Las Heras J, Devoto L, for thelarche and menarche in girls.
anterior pituitary gonadotropes, which are basophilic Vega M. Morpho-functional study of human luteal cell nol influence the timing of sexual development.2,3
cells under the influence of pulsatile GnRH stimulation. subpopulations. Hum Reprod. 1994;9:591.
3. The gonadotropins acting through receptors are 9. Roseff SJ, Bangah ML, Kettel LM, Vale W, Rivier J, Burger ADRENARCHE The exact trigger for puberty is unknown, but three dis-
responsible for the follicular selection, dominance, HG, et al. Dynamic changes in circulating inhibin levels tinct changes occur:
during the luteal-follicular transition of the human
It occurs before gonadarche. It includes the maturation
ovulation, and functioning of corpus luteum. They also a. Onset of nocturnal sleep-related augmentation of
menstrual cycle. J Clin Endocrinol Metab. 1989;69:1033. of the adrenal cortex with increased production of adre-
stimulate the synthesis of estrogen, progesterone, and pulsatile LH release.
peptides in the ovary. These exert negative feedback at
10. Ludwig H, Spornitz UM. Microarchitecture of the human nal androgens-dehydroepiandrosterone (DHEA) and
endometrium by scanning electron microscopy: Menstrual dehydroepiandrosterone sulfate (DHEAS). Adrenarche is b. Decrease in the sensitivity of the hypothalamus to
the hypothalamic–pituitary level due to which there is a desquamation and remodeling. In: Bulletti C, Gurpide E
responsible for the appearance of sexual hair (Pubarche).2 estrogen and testosterone (increased sensitivity to FSH
decrease in gonadotropin secretion, which is responsi- (Eds). The Primate Endometrium, New York: The New York
ble for a single dominant follicle developing in a cycle. Academy of Sciences; 1991. p. 28. The order of the appearance of the signs of puberty is and LH which is secondary to the CNS maturation).
4. The increase in estradiol levels beyond the physi- 11. Bergeron C, Ferenczy A, Shyamala G. Distribution of as follows: c. Gonadotropins
ologic threshold for negative feedback in the preovu- estrogen receptors in various cell types of normal, 1. Increase in the linear growth, followed by breast bud-
hyperplastic, and neoplastic human endometrial tissues. ding (thelarche) (Fig. 10.1).
latory phase initiates the positive feedback effects on
hypothalamus and pituitary. This is responsible for
Lab Invest. 1988;58:338.
2. The appearance of pubic hair (pubarche) (Fig. 10.2). PRECOCIOUS PUBERTY
12. Falany JL, Falany CN. Regulation of estrogen sulfotrans
the preovulatory gonadotropin surge. tferase in human endometrial adenocarcinoma cells by 3. The onset of menses (menarche).
Precocious puberty is defined as the manifestation
progesterone. Endocrinology. 1996;137:1395.
In humans, the attainment of adrenarche and gonadarche of secondary sexual characteristics before the age of
PROBABLE QUESTIONS 13. Fata J, Ho ATV, Leco KJ, Moorehead RA, Khoka R. Cellular
is mutually independent, and gonadarche is seen even in the 8 years.1,2,6
turnover and extracellular matrix remodelling in female
1. Describe the stages of development of ovarian reproductive tissues: Functions of metalloproteinases and
follicle. their inhibitors. Cell Mol Life Sci. 2000;57:77.
72 The Infertility Manual Normal and Abnormal Puberty 73
Eitiology of Precocious Puberty1,2,8,9 5. Laboratory studies:
a. GnRH stimulation tests—these tests help to differ-
Eitiology of precocious puberty entiate GnRH-dependent precocious gonadarche
from premature thelarche (GnRH-dependent
A. GnRH dependent:
premature gonadarche has a predominant LH
1. Idiopathic: Progressive, Non-progressive
2. Congenital CNS lesions: hypothalamic hamartomas, response whereas premature thelarche has a pre-
septo-optic dysplasia, arachnoid cysts, suprasellar dominant FSH response). The upper limit of LH
cysts, KISS 1 receptor activity mutation. after GnRH stimulation test by ICMA (immuno-
3. Acquired CNS disorders: Post-inflammatory, post- chemiluminometric) assay is 3.3 IU/L for girls.
radiation therapy, abscess, hydrocephalus, trauma, Random LH concentrations more than 1.0 IU/L
tumours like ependymomas, astrocytomas pineal
tumours, neurofibromatosis using third-generation gonadotropin assays in
4. Chronic exposure to androgens girls with features of precocious puberty helps in
5. Other conditions like William- Beurensyndrome, diagnosing central precocious puberty.
maternal uniparental disomy of chromosome 14, b. A peak LH/peak FSH greater than 1 can also be
Histiocytosis-X used as a diagnostic tool to differentiate true CPP
6. Severe primary hypothyroidism(rare)
7. Increased activation of KISS 1R.8
from premature thelarche. There is a predominant
FSH response in cases of premature thelarche.11
B. GnRH Independent: c. Thyroid function tests (TSH and freeT4) if there is
1. Autonomous functional ovarian follicular cyst (most
a clinical evidence of hypothyroidism.
common cause)
2. Mc-cune Albright syndrome d. Serum concentrations of estradiol, testosterone,
Fig. 10.1: Tanner staging-breast development. Fig. 10.2: Tanner staging-pubic hair development.
3. Feminising disorders, estrogen secreting tumours: and hCG, late afternoon cortisol, DHEA-S, and
Based on information found in disorders of puberty. Blondell RD, Foster MB, Based on information found in Disorders of puberty. Blondell RD, Foster MB,
Dave KC. Am Fam Physician 1999 Jul 1;60(1):209-218, wikepedia. Dave KC. Am Fam Physician 1999. Jul 1;60(1):209-218, wikepedia.
ovarian (granulosa cell tumours dysgerminomas, 17OHP to determine the peripheral source of sex
cystadenoma, theca cell and lipoid cell tumours) steroid production, which might lead to early puber-
4. Aromatase mutation tal development in children with gonadotropin-
5. Exposure to isogenous sex steroids, endocrine
Flowchart 10.1: Schematic representation of neuroendocrinology Flowchart 10.2: Classification of abnormal puberty. independent precocious puberty (identified by
disruptors
of puberty 6. Prenatal exposure to cigarette smoking and low birth normal basal and stimulated serum LH levels).
weight 6. Bone age (BA), in most of the cases, advanced in
C. Isolated premature menarche: Estrogen secreting patients with GnRH-dependent precocious gona-
cyst, tumours darche but is not significantly advanced in cases of
D. Premature thelarche:variant of normal development premature adrenarche or premature thelarche.
E. Virilizing disorders: congenital adrenal h yperplasia,
7. Magnetic resonance imaging of the brain and pituitary
Cushing syndrome, inherited glucocorticoid
resistance (in cases of GnRH-dependent precocious gonadarche).1
8. Pelvic ultrasound to be done to look for the pubertal
development of the uterus and ovaries. Ultrasound
Evaluation of Precocious Puberty assessment of the adrenal glands for adrenal tumours
1. Complete medical history with questions pertain- or adrenal hyperplasia. The two reliable indicators of
ing to physical manifestations of puberty, age, and true CPP on ultrasound are uterine length of more
the order of their appearance, history of neurological than 3.5 cm and uterine volume of more than 1.8 mL,
The sequence of pubertal development is like in nor- symptoms, previous treatment. which are useful in differentiating CPP from prema-
mal puberty, but it begins at an earlier age. 2. Complete family history since some disorders show ture adrenarche and premature thelarche.11,12
autosomal dominant inheritance, e.g. familial central 9. Liquid chromatography-tandem mass spectrometry
Peripheral Precocious Puberty precocious puberty caused by mutations in MKRN (LC-MS/MS) can be done to look for disorders of
3 systems, familial male precocious puberty, spastic steroidogenesis.
Occurs independently of pulsatile GnRH secretion. The
paraplegia with precocious puberty.10
deviation from normal, of the gonadal or adrenal ster-
Central Precocious Puberty oid secretion, or exposure to exogenous steroids induces
3. Complete physical examination with particular atten- Treatment of Precocious Puberty
tion to development of breast, genitalia, and body
Due to either early maturation of the HPO axis or incom- the pubertal changes. In most of these cases, pubertal habitus. The stage of puberty should be documented. A. Gonadotropin-dependent precocious puberty
plete suppression of the hypothalamic GnRH pulse gen- development is not complete and fertility potential is 4. Look for evidence of thyroid or other endocrine 1. Appropriate management of the intracranial
erator. It is seen more often in girls than in boys. compromised. dysfunction. lesion if detected.
74 The Infertility Manual Normal and Abnormal Puberty 75
Flowchart 10.3: Algorithm for evaluation in a girl presenting with precocious puberty. Good predictors of height outcomes include The hypoestrogenic state induced by the GnRH agonist
(1) younger chronological age (CA) and BA at the time of treatment in girls with precocious puberty does not have
initiation of therapy, (2) greater height SD score for CA at any effect on the post-pubertal uterine size.21
initiation of therapy, and (3) predicted adult height is more There is a partial suppression of AMH by GnRH agonist
using Bayley-Pinneau tables.12,16 treatment suggesting partial gonadotropin dependence
The following treatment regimens are generally of AMH. The AMH levels revert to pretreatment levels in
expected to suppress the pituitary–gonadal axis: about 6 months after discontinuation of the treatment.22
As the pubic hair development is controlled by adrenal
• Buserelin 6.3 mg every 2 months.
androgens, GnRH agonist treatment is not found to have
• *Goserelin 3.6 mg every month or 10.8 mg every
any significant benefit on premature pubarche.15
3 months.
GnRH agonist treatment causes a brief initial flare of
• *Histerelin 50 mg implant every year.
gonadotropin release, followed by pituitary desensitiza-
• *Leuprolide 3.75–7.5 mg every month or 11.25 mg once
tion and downregulation. To counteract this effect, three
in 3 months.
doses of GnRH antagonist given every 72 hour at the ini-
• *Triptorelin 3.0–3.75 mg every month or 11.25 mg once
tiation of GnRH agonist is seen to induce decreased flare
in 3 months.17
response as indicated by urinary gonadotropin levels.15,23
The dose of GnRHa to treat central precocious puberty 2. Medroxy-progesterone acetate (MPA) can be used in
is 75–100 µg/kg, which in practice corresponds to 3.75 mg the treatment of precocious puberty if it is not pos-
given IM or SC every 28 days. The adequacy of the admin- sible to use GnRH analogs (cost-effective). It can be
istered dose can be checked by the serum LH measure- used as injectable MPA (50 mg/month).24 The disad-
ment 30–60 min after each repeated injection of the vantage with this treatment is that MPA does not help
agonist. The peak LH level should be less than 2.0 IU/L, in improving the final height of the child. The mode
consistent with prepubertal norms after acute GnRH of action of MPA: (a) Inhibits gonadotropin release by
agonist stimulation. The treatment with GnRH agonist acting on hypothalamus, (b) inhibits 3-beta hydrox-
can be continued until the epiphyses are fused or until ysteroid dehydrogenase 2 enzyme and thus inhibits
the pubertal and chronological ages are appropriately steroid production by the gonads, and (c) if used for
matched. Use of GnRH agonists for the management of a long duration, causes the suppression of ACTH,
precocious puberty is not found to have any significant hypertension and development of cushingoid fea-
long-term adverse effects on the hypothalamic-pituitary tures due to its glucocorticoid mimetic action.15,24,25
ovarian (HPO) axis function. The treatment should be 3. Cyproterone acetate is an antiandrogen with antig-
monitored at 3–6 months intervals with serial physical onadotropic properties. The action of cyproterone
examinations to detect any p rogressive pubertal devel- acetate is the same as that of medroxy-progesterone
2. Criteria for treatment of precocious puberty: f. In the setting of parental anxiety, psychologi- opment, BA also should be evaluated periodically. Long- acetate (MPA) except for its ACTH and cortisol sup-
a. Sexual maturation progresses to the next cal stress.12 term follow-up is recommended for monitoring of adult pressing effect, which can cause iatrogenic adrenal
stage within 3–6 months. The various treatment modalities available are as height in relation to the expected target height and repro- insuffiency in some patients.15,26 Cyproterone acetate
b. Growth velocity is accelerated to greater than follows: ductive function. causes regression of secondary sexual characteristics
6 cm/year. In most cases, growth velocity GnRH analog is useful to improve adult height only in but does not benefit height gain.
1. Long-acting GnRH agonist treatment—By suppress-
should be monitored for 3–6 months before cases of central precocious puberty with an early onset.18 4. Early metformin therapy (8–12 years) in low birth
ing the pituitary–gonadal axis, GnRH agonist therapy
making a decision to treat. Although there are no general concerns about GnRH weight girls can help to delay menarche, reduces total,
can prevent progressive pubertal development, and
c. BA is advanced by 1 year or more (more than agonist therapy and safety, careful prolactin monitoring visceral and hepatic adipose tissue deposition, helps
increase the final height. The effect of GnRH agonist
2.5 SD for chronological age). The presence of is required in these children. Around 3.8% of treated chil- to improve post menarcheal height gain and helps to
accelerated bone maturation rate and deteri- treatment on height gain is good if the treatment is
dren developed hyperprolactinaemia (more with triptore- mitigate the occurrence of adolescent PCOS in these
oration in adult height prediction (AHP) with initiated early before significant BA advancement lin than with leuprolide acetate).19 low birth weight-precocious puberty girls.27
time is an indication for treatment.13 has occured.14,15 The continuous GnRH exposure with GnRH agonist therapy can cause weight gain in chil- 5. Thyroxine supplementation in children with
d. Estradiol (>10 pg/mL) long-acting GnRH analogs causes desensitization of dren having central precocious puberty. These children hypothyroidism.
e. When predicted adult height is below the tar- the pituitary better than pulsatile GnRH. This results should be monitored for excessive weight gain and obesity 6. GH and GnRH analog combination therapy: GnRH
get level or is found to be decreasing on serial in decreased gonadotropin production and reversal should be prevented by diet and exercise program in the analog can cause growth suppression in some cases
measurements during follow-up. of pubertal changes.15 course of the treatment.20 and addition of GH is found to be beneficial in such
76 The Infertility Manual Normal and Abnormal Puberty 77
children. While the role in children with underlying 3. McCune-Albright syndrome is treated with drugs Flowchart 10.4: Algorithm for evaluation in a girl presenting with delayed puberty.
GH deficiency is clear, the beneficial effect on GH suf- that inhibit steroidogenesis or hormone action.
ficient children is not much.28 Aromatase inhibitors such as letrazole and anastra-
7. Oxandrolone, a nonaromatizable anabolic-androgen zole; antiestrogens like tamoxifen have been used in
steroid (AAS) and GnRH analog, when administered the treatment. Biphosphonates can be useful in the
together is an useful alternative to GH in preventing management of fibrous dysplasia of the bone causing
pain and fractures.
GnRH agonist associated growth decelearation.29
4. Fulverstrant, a pure estrogen receptor blocker (trade
When to stop GnRH agonist treatment: Age of inter-
name-Faslodex), has shown promising results in
ruption of agonist treatment ranges from 10.1 years to
the treatment of McCune Albright syndrome. It is
11.3 years though Few studies show GnRH agonist being given as monthly intramuscular injections at a dose
used till 13.8 years. The most commonly used end-point of 4 mg/kg, which helps in reducing vaginal bleed-
to stop GnRH agonist treatment is adult height or near- ing and the rate of skeletal maturation. No difference
adult height as compared to the pretreatment expected has been noted in the predicted adult height. Further
height. The difference in the height between pre and post- trials are required.32
treatment is positive and is a gain of about 2.9–9.8 cm.30 5. Aromatase inhibitors, selective estrogen receptor
B. Treatment of gonadotropin-independent preco- modulators (SERMs), and anti-androgens are useful
cious puberty: in the management of gonadotropin-independent
In these cases, treatment is primarily aimed at the under- precocious puberty.1,15
lying pathology.
1. Functional tumors involving ovaries and adrenals are Delayed Puberty
treated surgically. Adjuvant chemotherapy and/or Delayed puberty is the result of delayed gonadarche. For
radiotherapy may be required in some cases. girls, lack of breast development by 13 years or menarche
2. Glucocorticoids are used to treat children with con- by 16 years may be viewed as delayed. 1Eitiology of delayed
genital adrenal hyperplasia.1,31 puberty:
Eitiology of delayed puberty.1,2,11
Evaluation of Delayed Puberty secondary (hypogonadotropic) hypogonadism.
1. Constitutional delay Measurement should be performed on an early
1. Thorough medical history with evaluation of the pre-
A. Sporadic B. Familial morning blood sample using an ultasensitive pae-
vious growth pattern and neurological symptoms,
a. Chronic Illness—GI diseases such as inflammatory bowel disease, celiac disease; renal failure, hepatic d
isease diatric assay. GnRH agonist stimulation is neither
family history of delayed puberty, physical examina-
b. Haematological abnormalities such as sickle cell disease, hemolytic anaemia helpful nor necessary for the diagnosis.
tion and measurement of BA is a prerequisite in the
c. Pulmonary disease such as asthma, cystic fibrosis b. Serum prolactin levels.
management of delayed puberty. History should also
d. Malignancy c. Serum TSH and free T4 particularly if growth
include dietary and exercise habits, previous serious
e. Endocrinopathies such as diabetes mellitus, hypothyroidism, hyperprolactinemia, and so on. velocity is slowed or BA is grossly delayed.
illnesses, and medications that could have delayed
2. GnRH-dependent (hypothalamic hypogonadotropichypogonadism): Mutations in KAL1, GnRH1, NELF, KISS1, d. Other pituitary hormone tests to rule out com-
the onset or retarded the pace of pubertal progression.
and KISS1R mutations; nutritional disorders such as malnutrition, anorexia nervosa, energy expenditure. bined pituitary deficiencies in patients with con-
2. BA—Children with constitutional delay show a BA
3. Pituitary-dependent (pituitary hypogonadotropichypogonadism): GnRHR gene mutation, developmental firmed hypogonadotropic hypogonadism.
of between 12 and 13.5 years, which generally does
anomalies due to intrauterine CNS vascular events, maternal cocaine use, and valproate toxicity. e. Serum DHEA-S to distinguish constitutional delay
not progress further without the exposure to gonadal
4. Hypothalamic and pituitary dependent hypogonadotropichypogonadism: Congenital adrenal hypoplasia, of puberty from congenital GnRH deficiency.
steroids (required for epiphyseal closure). BA is also
intracranial tumors, hyperprolactinemia, Prader-Willi syndrome, histiocytosis-X, heriditaryhaemochromatosis. f. Other tests include haemogram, liver function
helpful in distinguishing between functional and
5. Gonad-dependent: gonadal dysgenesis (Turner syndrome, pure gonadal dysgenesis 46XX and 46XY), Klinefelter tests, and so on.
permanent hypogonadism, in the interpretation of
syndrome, autoimmune and nonautoimmune premature ovarian failure, Down syndrome, Noonan syndrome
hormonal evaluation and in monitoring the effect of g. Ultrasound examination to look for dysgenetic
6. Anatomical abnormalities: Mayer-Rokitansky-Kuster-Hauser syndrome
therapeutic intervention.13 gonads, renal abnormalities like agenesis, horse
7. Chemotherapy and radiotheraphy
3. Laboratory studies: shoe kidney, and so on.33
8. Exposure to high molecular weight phthalates like di(2-ethyl hexyl)phthalate metabolites delays pubarche11
a. Serum FSH, LH, and estradiol concentrations to h. ECHO in Turner syndrome (coarctation of aorta
9. Autoimmune disorders with multiple endocrine abnormalities
differentiate primary (hypergonadotropic) from and bicuspid aortic valve).33
78 The Infertility Manual Normal and Abnormal Puberty 79
i. MRI of the brain in patients with hypogonado- 2.5mg/day) along with growth hormone in patients 6. Zimlich R, RN. AAP offers guidelines on evaluating early during and after suppression with GnRH agonist. Fertil
tropic hypogonadism, neurological signs and with Turner syndrome starting at 8 years of age has puberty. Contemporary Paediatrics guidelines. Jan 2016. Steril 2012;98(5):1326-30.
7. Soliman A, De Sanctis V, Elalaily R. Nutrition and pubertal 23. Roth CL, Brendel L, Ruckert C, Hartmann K. Antagonistic
symptoms, and hyperprolactinaemia. been found to be useful in achieving the expected
development. Indian J Endocrinol Metab. 2014;8(7): and agonistic GnRH analogue treatment of precocious
j. Karyotype in girls with hypogonadotropic hypog- adult height. The side effect is virilization.36 39-47. puberty: Tracking gonadotropin concentrations in urine.
onadism to detect chromosomal abnormalities. 9. All women with confirmed hypogonadism post- 8. Calley JL, Dhillo WS. Effects of the hormone kisspeptin on Horm Res. 2005;63:257-62.
puberty will need lifelong hormone replacement reproductive hormone release in humans. Adv Biol. 2014. 24. Albanese A, Hopper NW. Suppression of menstruation in
Treatment of Delayed Puberty therapy (HRT) after induction of puberty until about pp. 1-10. adolescents with severe learning disabilities. Arch Dischild.
50 years of age.33 9. Behie AM, O’Donnell MH. Prenatal smoking and age at 2007;92:62.
1. Correction of the specific cause when possible. menarche: Influence of the prenatal environment on the 25. Rosenfield RL, Cooke DW, Radovick S. Puberty and its
10. Patients with hypogonadotropic hypogonadism
2. Vitamin A and iron supplementation in normal timing of puberty. Hum Reprod. 2015;30(4):957-62. disorders in the female. In: Sperling MA(Ed). Paediatric
constitutionally delayed children who have reduced are potentially fertile. To initiate gametogenesis, 10. De Macedo DB, Brito VN, Latronico AC. New causes of Endocrinology. 4th edition. Saunders Elsevier; 2014.
the typical approach to fertility induction is pump- central precocious puberty: the role of genetic factors. pp. 612-615.
Vitamin A intake is found to be atleast as effective as
hormone therapy in inducing puberty.34 administered gonadotropin therapy or parenteral Neuroendocrinology. 2014;100:1-8. 26. Kauli R, Galatzer A, Kornreich L, Lazar L, Pertzelan A,
combination gonadotropin therapy. 11. Kumar M, Mukhopadhyay S, Dutta D. Challenges and Laron Z. Final height of girls with central precocious
3. Sex hormone therapy should be limited to girls over controversies in diagnosis and management of gona puberty, untreated versus treated with cyproterone acetate
12 years of age having few or no signs of sexual mat- 11. Girls who do not achieve adequate breast develop-
dotropin dependent precocious puberty: An Indian pers or GnRH analogue. A comparative study with re-evaluation
ment with estrogen treatment may require breast
uration monitoring the BA at 6 monthly intervals pective. IJEM, 2015;19(2 ):228-35. of predictions by the Bayley-Pinneau method. Horm Res.
during treatment. The aims of short-term hormone implant. 12. Styne DM, Grumbach MM. Puberty: ontogeny, neuro 1997;47:54-61.
therapy are to (i) foster age-appropriate secondary 12. Nonsurgical self-vaginal dilatation or creation of a endocrinology, physiology and disorders. In: Melmed S, 27. Ibanez L, Lopez-Barmejo A, Diaz M, Marcos MV. Early
neo-vagina in cases with congenital adrenal hyper- Polonsky KS, Larsen PR, Kronenberg HM (Eds). Williams metformin therapy to delay menarche and augment
sexual development, (ii) induce a growth spurt, and Textbook of Endocrinology. 12th edition. Saunders Elsevier; height in girls with precocious pubarche. Fertil Steril.
plasia, Mayer-Rokitansky-Kuster-Hauser syndrome,
(iii) induce a normal adolescent increase in bone den- 2012. Pp. 1141-71. 2011;95(2):727-73.
and complete androgen insensitivity.2
sity without causing premature closure of epiphyses. 13. Lazar L, Phillip M. Endocrinology and metabolic clinics of 28. Pucarelli I, Segni M, Ortore M, Moretti A, Iannaccone R,
4. Oral or transdermal estrogen therapy (preferred) North America. 2012;41(4):805-22. Pasquino AM. Combined therapy with GnRH analog plus
titrated according to the response with the goal PROBABLE QUESTIONS 14. Gyorgy Bartfai RL. Clinical applications of gonadotrpin- growth hormone in central precocious puberty. J Pediatr
releasing hormone and its analogues. Hum Reprod. Endocrinol Metab. 2000;13(1):811-20.
of completing sexual maturation over a period of
1. Neuroendocrinology of puberty 1988;3(1):51-7. 29. Vottero A, Pedori S, Verna M, Pagano B, Cappa M, Loche
2–3 years. 15. Bajpai A, Menon PSN. Contemporary issues in precocious S, et al. Final height in girls with central idiopathic
2. Evaluation and management of precocious puberty.
5. Progestins should be withheld until there is sub- puberty. Indian J Endocrinol Metabol. 2011;15(7):172-9. precocious puberty treated with gonadotropin-releasing
3. Evaluation and management of delayed puberty.
stantial breast development and full contour breast 16. Bayley N, Pinneau SR. Tables for predicting adult height for hormone analog and oxandrolone. J Clin Endocrinol
4. Criteria for initiation of treatment in precocious skeletal age: Revised for use with the Greulich-Pyle hand Metab. 2006;91:1284-7.
growth has plataued or at the onset of breakthrough
puberty. standards. J Padiatr. 1952;40:423-41. 30. Carel JC, Lahlou N, Roger M, Chaussain JL. Precocious
uterine bleeding. Progestins can be safely started
17. Chiocca E, Dati E. Central precocous puberty: Treatment puberty and statural growth. Hum Reprod Update.
once menses have began or after 12–24 months of with triptorelin 11.25 mg. Sci World J. 2012. 2004;10(2):135-147.
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tered as Provera (medroxy progesterone) at a dose of 1. Fritz MA, Speroff L. Normal and abnormal growth and Ohyama K, et al. Results of long term follow-up after Zonari N. PO-0090 different aspects of precocious puberty:
5–10 mg/day or micronized progesterone in a dose of pubertal development. In: Clinical Gynecologic Endo treatment of central precocious puberty with leuprolide Clinical experience and outcome(8 cases). Arch Dischild.
200 mg/day for 10–14 days.33 After the full adult dose crinology and Infertility. 8th edition. Wolters Kluwer acetate:Evaluation of effectiveness of treatment and 2014;99:A278.
Health. Lippincott Williams and Wilkins South Asian recovery of gonadal function. J Clin Endocrinol Metabol. 32. Sims EK, Garnett S, Guzman F, Paris F, Sultan C, Eugster
of estradiol and medroxy progesterone acetate are
Edition; 2011. pp. 391-434. 2005;90:1371-6. EA. Fulvestrant treatment of precocious puberty in girls
reached, oral contraceptive pill may be used instead 2. Witchel SF, Plant TM. Puberty: Gonadarche and adren 19. Francesco M, Parrino R, Placidi G, Massai G, FedericoG, with McCune Albright syndrome. Intl J Paed Endocrinol.
of two different medications. arche. In: Strauss III JF, Barbieri RL. Yen and Jaffe’s Saggese G. Prolactin secretion before, during, and after 2012;(1):26.
6. In girls with Turner syndrome, GH and/or oxan- Reproductive Endocrinology Physiology, Pathophysiology chronic gonadotropin-releasing hormone agonist treat 33. Delayed puberty.BMJ best practice guidelines.BMJ 2016.
dralone treatment may also be required to achieve and Clinical Management. 7th edition. Elsevier: Saunders; ments in children. Fertil Steril. 2005;84(3):719-24. 34. Zadik Z, Sinai T, Zung A, Reifen R. Vitamin A and iron
2014. pp. 377-421. 20. Anik A, Catli G, Abaci, Bober E. Effect of gonadotropin supplementation is as efficient as hormonal therapy
adequate final height. GH treatment should be started
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from the time there is a dip in the normal linear and growth in girls with idiopathic central precocious 2004;60:682-7.
crinology, physiology and disorders. In: Wilson ID, Foster
growth until the age of 12–14 years. Oxandrolone may DW (Eds). Williams Textbook of Endocrinology, 8th edition. puberty. IJEM 2015;19(2):267-71. 35. Spiliotis B. Recombinant human growth hormone in the
also be added to achieve linear growth.33 W.B. Saunders; 1992. pp. 1139-221. 21. Ben-Haroush A, Goldberg-Stern H, Phillip M, De vries L. treatment of Turner syndrome. Ther Clin Risk Manage.
7. Human growth hormone (hGH) in a dose of 4. Sam AH, Dhillo WS. Kisspeptin: A critical regulator of GnRH agonist treatment in girls with precocious puberty 2008:4(6):1177-83.36.
0.375 mg/kg/wk in daily divided injections with or puberty and reproductive function. Current Drug Targets. does not compromise post-pubertal uterinesize. Hum 36. Sheanon NM, Backeljauw PF. Effect of oxandralorone
2010;11(8):971-977. Reprod 2007;22(3);895-900. therapy on adult height in turner syndrome patients
without oxandrolone is found beneficial, in girls with
5. Soliman A, De Sanctis V, Elalaily R, Bedair S. Advances in 22. Hagen CP, Soenson K, Anderson RA, Jull A. Serum levels treated with growth hormone: A meta-analysis. Int J Paed
Turner syndrome, in increasing the adult height.35 pubertal growth and factors influencing it:can we increase of antimullerian hormone in early maturing girls before, Endocrinol. 2015;18(1):1-23.
8. Oxandrolone, a nonaromatizable anabolic-androgen pubertal growth? Indian J Endocrinol Metabolism. 2014;
steroid (AAS) in a dose of 0.03–0.06 mg/kg/day (max 18(7):53-62.
C HA PT E R
Polycystic Ovarian Syndrome 11 Deepika Krishna
Table 11.2. Classification of polycystic ovarian syndrome phenotypes.7
Parameter
PCOS features
HA
Phenotype A
HA/OD/PCOM
+
Phenotype B
HA/OD
+
Phenotype C
HA/PCOM
+
Polycystic Ovarian Syndrome 81
Phenotype D
OD/PCOM
−
OD + + − +
Chapter Outline PCOM + − + +
Diagnosis and Evaluation • Management of Infertility Associated with PCOS NIH 1990 criteria × ×
• Defining PCOS • Alternative OI Agents to CC
• PCOS Phenotypes • Laparoscopic Ovarian Drilling (LOD) Rotterdam 2003 criteria × × × ×
• Etiopathophysiology • Role of Intra-uterine Insemination (IUI) AE PCOS 2006 criteria × × ×
• Associated Metabolic Features • Assisted Reproductive Technology (ART) in PCOS
• Clinical Manifestations in PCOS • In-vitro Maturation (IVM) Note: AE PCOS, Androgen Excess & PCOS Society; HA, hyperandrogenism; NIH, National Institutes of Health; OD, ovulatory dysfunction;
Management of PCOS • Adjuvants: Improvement in Metabolic Function PCOM, polycystic ovarian morphology.
• Lifestyle Modifications: First-line Treatment of Obesity in PCOS • PCOS in Adolescents
• Management of Hirsutism • PCOS and Pregnancy
• Management of Menstrual Dysfunction • PCOS and Menopause • Mutations in the candidate genes involved in fol- Neuroendocrine Dysfunction
liculogenesis, steroidogenesis, steroid hormone
effects, insulin secretion, and gonadotropin Impaired inhibition of GnRH pulse frequency by proges-
PART 1: DIAGNOSIS AND EVALUATION action.8 terone and estradiol results in excessive luteinizing hor-
II. Various hypothesis have been implicated: mone (LH) pulsatility and relative follicular stimulating
INTRODUCTION AND OVERVIEW DEFINING PCOS (TABLE 11.1) a. Fetal origin: In-utero programming hormone (FSH) deficiency.11,12
• 5–10% of women in reproductive age.6 b. Neuroendocrine dysfunction: LH hypothesis
• Polycystic ovarian syndrome (PCOS) is a common
• 30–40% women with secondary amenorrhea. c. Ovarian hypothesis
Ovarian Hypothesis
endocrine-metabolic disorder, and it represents the
• More than 70% women with anovulatory infertility. d. Adrenal hypothesis See Table 11.3.
commonest cause of normo-gonadotropic anovulation,
• 30% of overweight women. e. Insulin hypothesis
accounting for 91% of World Health Organization-II
• In up to 90% hirsute women with regular menses. Adrenal Hypothesis
(WHO-II) cohort.1
• First description by Stein and Leventhal (1935) in Fetal Origin Increased adrenal androgens due to:
women with hirsutism, obesity, amenorrhea and enlar
PCOS PHENOTYPES Barker’s hypothesis (fetal origin of adult disease): In-utero • Increased activity of P450c17.12
ged bilateral polycystic ovaries.2 See Table 11.2. hyper-androgenism exposure may disturb the epigenetic • Accelerated cortisol metabolism.
• The term “polycystic ovary syndrome” and its acronym reprogramming in fetal reproductive tissue resulting in • Hyperinsulinemia.
PCOS appeared in 1960s and gradually replaced the ETIOPATHOPHYSIOLOGY PCOS phenotype after birth.9,10 • Increased adrenocorticotropic hormone (ACTH).12
Stein-Leventhal syndrome designation. I. Genetics:
• This chapter, covered in two parts-I and II, reviews • Polygenic disorder
the diagnostic criteria, etiopathophysiology, investi- Table 11.3. Ovarian hypothesis.
• Autosomal dominant, with widely variable pen-
gations and management from a dolescents through etrance. Gene associated with PCOS maps to a Alterations in both theca and granulosa cell function Disordered folliculogenesis
women of reproductive age, pregnancy, menopause, locus on chromosome 19p13.28 •• Dysregulation of 17-hydroxylase and 17, 20-lyase •• Higher initial population of primordial follicles or a slower
and long-term sequel in women with this disorder. • X linked activities of P450c17 (rate-limiting enzyme in rate of loss by atresia13
androgen biosynthesis), resulting in ↑ activity and •• Stock-piling hypothesis10: AMH hypothesis
hyperandrogenemia12 Androgen excess → multiple small follicles → ↑AMH →
Table 11.1. Diagnostic criteria.
•• Intra ovarian factors—inhibins, retinoids, and ↓FSHaction → anovulation → → Low progesterone → ↑ LH
NIH* Consensus 19903 ESHRE/ASRM**/Rotterdam Consensus AEPCOS*** definition 20095 (androgen antimüllerian hormone (AMH) ↑ ovarian androgen +insulin androgen excess
(all required) 20034 (two out of three required) excess and one other criterion) synthesis13 •• Lacker’s model: Tracks the maturity of a cohort of follicles
Clinical and/or biochemical Clinical and/or biochemical Clinical and/or biochemical •• Androgens induces hyperinsulinaemia and as a function of time through the selection phase. If all
hyperandrogenism hyperandrogenism hyperandrogenism hyperinsulinaemia exaggerates androgen excess follicles are “low sensitivity” then normal ovulatory cycles
Oligo/amenorrhea, anovulation Oligo/amenorrhea, anovulation Oligo/amenorrhea, anovulation leading to a “vicious circle”10 occur, if majority of follicles are “high sensitivity” then arrest
Polycystic ovaries appearance on ultrasound Polycystic ovaries appearance on ultrasound •• Two-hit hypothesis12: will usually occur.14
Exclusion of other androgen excess disorders: NC-CAH, Cushing syndrome, androgen secreting tumors, hyperprolactinemia, thyroid diseases, First hit: Development of hyperandrogenism
drug-induced androgen excess. Other causes for anovulation should also been excluded. NIH*, National Institute of Child Health; ESHRE/ Second hit: Hyperandrogenism begets
ASRM**, European Society for Human Reproduction and Embryology/American Society for Reproductive Medicine; AEPCOS***, Androgen hyperandrogenism
Excess and PCOS Society.
82 The Infertility Manual Polycystic Ovarian Syndrome 83
Insulin Hypothesis Leptin Resistance (LR) Table 11.6. Diagnostic criteria for the metabolic syndrome according to NCEP ATPIII.
See Table 11.4 and Figure 11.1. Three of the following conditions
• Women with PCOS (both obese and normal weight)
Central obesity (waist circumference in cm)
have higher circulating concentrations of leptin.17
ASSOCIATED METABOLIC FEATURES Male ≥102
• Leptin inhibits insulin action in hepatocyte resulting Female ≥88
in insulin resistance. Elevated triglycerides (mg/dL) ≥150
Inositol-deficient State
• Defective suppression of appetite, which perpetuate Decreased HDL cholesterol (mg/dL)
See Table 11.5 and Figure 11.2. obesity. Male <40
Female <50
Elevated arterial blood pressure (mmHg) ≥130/85
Table 11.4. Insulin hypothesis.15 Elevated fasting blood glucose (mg/dL) ≥110
•• Insulin resistance (IR): Defined as reduced Adapted from: Third Report of the National Cholesterol Education Program (NCEPIII) Expert Panel on Detection, Evaluation and Treatment of
Insulin resistance High Blood Cholesterol in Adults (Adult Treatment Panel III, ATP III).22
glucose response to a given amount of insulin
resulting in hyperinsulinaemia (HI)
•• Occurs secondary to resistance at the insulin Hyperinsulinemia • At the ovary: Hyperleptinemia stimulates LH secre- • Phenotypes A and B (‘‘classic PCOS’’) demonstrate
receptor, decreased hepatic clearance of insulin, tion, impairs selection of dominant follicle and arrests 75–85% of insulin resistance, some form of metabolic
and/or increased pancreatic sensitivity follicle development, reduces the response to gonado- dysfunction, and increased risk of glucose intolerance
•• Seen in approximately 80% of obese PCOS and tropins, decreases oocyte maturity, poor fertilization,
30–40% of lean PCOS. Ovary Pituitary and diabetes.7,21
Liver
•• Hyperandrogenic and anovulatory pheno-
Theca cell hyperplasia disordered release poor embryo quality, and lower pregnancy rates.17,18 • South Asians have a high prevalence MetS.22
of FSH /LH
types seem to be the most insulin resistant—
irrespective of BMI or central adiposity. ↓SHBG* ↓IGBP-1** ↑Androgen production ↑LH Obesity and PCOS CLINICAL MANIFESTATIONS IN PCOS
•• In PCOS—excessive serine phosphorylation
of Insulin receptor instead of tyrosine
• Obesity is seen in 35–60% of women with PCOS.17 (TABLE 11.7)
autophosphorylation. Hyperandrogenism Oligoanovulation • Rather than PCOS leading to obesity, overweight/
•• Serine phosphorylation increases activity of obesity amplifies the clinical severity of PCOS. Investigation Protocol
P450c17 in both the ovary and adrenal, thus • Apple-shaped (android) obesity with visceral adiposity, • Patient and family history including history of any predis-
promoting androgen synthesis PCOS seen in PCOS is associated with a more adverse meta- posing factors to PCOS (low birth weight with excessive
bolic risk profile than pear shaped (gynecoid) obesity. catch-up growth or premature adrenarche/pubarche).
Fig. 11.1: Insulin Resistance leading to PCOS.
• Menstrual history: menarche and nature of menstrual
*Sex hormone binding globulin Oxidative Stress and Low-grade cycles.
** Insulin globulin binding protein Inflammation • Physical examination: Blood pressure (BP), body mass
Two features equally shared by obese and non-obese index (BMI), signs of hyperandrogenism (hirsutism,
PCOS subjects. acne and/or alopecia), or insulin resistance (acantho-
Table 11.5. Inositol-deficient state. • Oxidative damage with release of free radicals, reactive sis nigricans) and waist circumference (WC)
•• Defect in tissue availability or altered metabolism of inositol De ciency of inositol decreases oxygen species (ROS) and advanced glycation end- • Waist-to-hip ratio (WHR) can be used as a surrogate
phosphoglycans (IPGs) or IPGs m ediators leading to P13 kinase activity
products (AGEs) → endothelial dysfunction → arterial marker for central fat accumulation, with a value more
I
deficiency of myoinositol (MI) may contribute to insulin Glucose
than 0.8 suggestive of visceral adiposity.
hypertension and cardiovascular disease. 18
resistance.16 R
• Higher circulating concentrations of homocysteine, • Screening strategy for PCOS23 (Table 11.8).
•• MI causes release of calcium in the oocyte through inositol De ciency of
tri phosphate and thereby promotes meiotic progression inositol decreases malondialdehyde asymmetric dimethylarginine, and • Investigations: (Table 11.9).
resulting in better oocyte quality.16 PI3 kinase activity Translocation of nitric oxide due to oxidative stress.18
•• Enhanced MI to D-chiro-inositol (DCI) epimerization in the GLUT4 Differential Diagnosis of PCOS
ovary leads to MI deficiency in ovary, responsible for poor PI3 kinase Vitamin D Deficiency
oocyte quality • Androgen-secreting tumor
•• ↑ DCI:MI ratio in PCOS IRS More common in PCOS women and contributes to IR, • Exogenous androgens
GLUT4
hyperandrogenemia (HA), ovulatory dysfunction, obesity, • Cushing syndrome
• Myoinositol ↓→dchiro inositol ↑(glyco synth)
• IP3- 2nd mesenger for both FSH and insulin and metabolic syndrome.19 • Nonclassical congenital adrenal hyperplasia
• Acromegaly
Fig. 11.2: Inositol deficiency. Metabolic Complications • Genetic defects in insulin action
*Phosphatidylinositol-3-kinase (PI3K), • Metabolic syndrome (MetS) or the insulin resistance • Primary hypothalamic amenorrhea
**inositol phosphatidyl trisphosphate (IP3) syndrome or “syndrome X” has been identified as a • Primary ovarian failure
*** Glycogen “common soil” for developing both type 2 diabetes and • Thyroid disease
I: Insulin; R: Receptor
cardiovascular disease (CVD)20 (Table 11.6). • Prolactin disorder
84 The Infertility Manual Polycystic Ovarian Syndrome 85
Table 11.7. The spectrum of clinical manifestations in PCOS. Contd...
Table 11.9. Investigational profile in PCOS.
[I] Symptoms [II] Signs [III]Possible late sequelae21
•• Hirsutism, acne, alopecia: Hirsutism •• Acanthosis nigricans: 5–10% •• Diabetes mellitus (DM) Normal (N) and values in PCOS (may vary with
70% PCOS, Modified Ferriman– Diffuse velvety thickening and Test local laboratory assays) Additional points
•• Dyslipidemia
Gallwey score of ≥8.21 hyperpigmentation of the skin •• Hypertension Testosterone (T) Total T N: 20–80 ng/dL (0.5–3.5 nmol/L); Total T ≥200 ng/dL (≥6.94 nmol/L) warrants fur-
•• Alopecia (frontal balding): seen in Most often seen on the back of PCOS: >60 ng/dL≤150 ng/dL (>2.5 but ≤5 nmol/L) ther evaluation
•• Cardiovascular disease
severe cases of androgen excess the neck, axillae and beneath PCOS: Free T >0.75 ng/dL
the breasts and exposed areas •• Nonalcoholic fatty liver disease A total testosterone is adequate for general
•• Menstrual disturbances23: 60–85%;
(elbows, knuckles) •• Obstructive sleep apnea screening.
oligomenorrhea (8 or less cycles per
year in 47%): 47%; 19.2% amenor- Result of insulin resistance •• Quality of life reduced Free androgen FAI >8.5-PCOS Insulin suppresses SHBG, resulting in a high FAI
rhea; 2.7% polymenorrhea; 1.4% Risk of diabetes, major lipid ab- (depression, anxiety, psychologi- index (FAI): T×100/ in the presence of normal total T.
menorrhagia normalities, and HT cal and b
ehavioural disorders) sex hormone bind-
•• Normal menstrual cycle: 29.7%15 •• Polycystic ovarian morphology •• Endometrial hyperplasia ing globulin (SHBG)
(PCOM) on ultrasound observed in •• Endometrial carcinoma-2.7-fold
•• Ovulatory dysfunction and infertility: FSH 2–8 IU/I FSH and LH are measured on days 1–3 of men-
75% of women with PCOS15 risk.
24.8% (73% of anovulatory infertility)23,24 LH 2–10 IU/l strual cycle.
•• Obesity: 50% •• Ovarian cancer and breast cancer To rule out WHOI and III anovulatory disorders
(?) 50–75% of PCOS show elevated LH levels15
•• Asymptomatic—20%
LH:FSH ratio ↑LH:FSH ratio (>2:1) has been regarded as a
marker of PCOS, more so in lean than in obese
Table 11.8. Screening strategy for PCOS: Recommendations by the ESHRE Capri Workshop Group.23 PCOS.
1. Measure BMI and waist circumference at every visit. Ratio varies with assays used to measure.
2. Complete lipid profile and repeat every 2 years if normal. Hence, not a reliable diagnostic criterion
AMH N: 1.1–3.5 ng/mL or 5–15 pmol/L Serum AMH level—surrogate marker for PCOM.26
3. 2-hour 75 g oral glucose tolerance test (OGTT): repeat every 2 years if normal. Consider using HbA1c.
PCOS: >5 ng/mL or >35 pmol/l
•• Age >40 years
•• BMI >25 kg/m2 (Asian Population) (a) Prolactin (Pr) (a) N:Pr <30 ng/mL Measure if oligo/amenorrheic
•• Waist to hip ratio >0.85 (b) Thyroid stimu- (b) N:TSH 0.5–2.5 mIU/L To rule out WHO IV anovulatory disorders
•• Hyperandrogenemia with anovulation lating hormone
•• Acanthosis nigrians-stigmata of IR (TSH)
•• History of gestational diabetes mellitus (GDM) (a) Estradiol (a) Measurement is unhelpful for diagnosis (a) Estrogenization may be confirmed by
•• Family history of DM (b) Progesterone (b) M
easure mid-luteal P4 levels 7 days before endometrial assessment
4. Measure the clinical blood pressure at each visit. (P4) anticipated menses in two consecutive cycles (b) A single mid-luteal phase (day 21)
P4 >10 ng/mL or 30 nmol/L confirms ovulation.
5. Suggestion: Assess for depression, anxiety, and quality of life.
(a) 17 Alphahydroxyl (a) 1
7A OHP >200 ng/dL to do corticotrophin (a) To rule out nonclassical/late-onset congenital
progesterone stimulation test. adrenal hyperplasia
Table 11.9. Investigational profile in PCOS. (17Α OHP) 17A OHP >800 ng/dL establishes the diagnosis of (b) and (c) To rule out Cushing syndrome
Normal (N) and values in PCOS (may vary with (b) 24 h urine free late onset congenital adrenal hyperplasia
Test local laboratory assays) Additional points cortisol
(c) Overnight
Pelvic ultrasound To assess ovarian morphology and endometrial Arrangement of follicles and stromal echogenicity dexamethasone
thickness. is not relevant. suppression test
PCOM: in one or both ovaries, either 12 or Unilateral PCOM is also diagnostic. (a) Fasting insulin (FI) (a) N:FI <30 µU/mL-N; IR: >20–30 µU/mL Not routinely measured due to lack of a
more follicles, 2–9 mm or ↑ovarian volume >10 cm3 (b) 2 h Insulin (PPI) (b) N:PPI <80–100 µU/mL; IR: >100 µU/mL standardized insulin assay
(c) Fasting glucose/ (c) N: FG/FI ratio >4.5; IR <4.5 Instead OGTT done
Differential diagnosis of PCOM: Multicystic The Task Force recommends using follicular insulin ratio
ovaries: fewer cysts (6–10/ovary), larger (up to number per ovary (FNPO) for the definition of OGTT 2 h 75 g glucose Normal: <140 mg/dL
10 mm), distributed throughout the ovary with no PCOM with a threshold of ≥25, but only when Impaired: 140–199 mg/dL
stromal hypertrophy. Seen in mid-late normal pu- using maximal resolution with transducer Type 2 DM: 200 mg/dL
berty, hyperprolactinaemia, hypothalamic anovula- frequency ≥8 MHz.25 35% of PCOS exhibit impaired glucose tolerance
tion, and weight-related amenorrhea. and 10% for Type 2 DM.23
8–25% of normal women, and even 14% of Others: (a) N:H 5–11 µmol/L;
women using oral contraceptives show PCOM on (a) Serum homo- PCOS: >11 µmol/L27
scan.15 cysteine (H) (b) Triglycerides ≥150 mg/dL
(b) Lipid profile HDL cholesterol ≤40 mg/dL
Intra and inter-observer variability (c) Endometrial In women with potential long-term exposure to
Contd... sampling unopposed estrogen stimulation
86 The Infertility Manual Polycystic Ovarian Syndrome 87
PART 2: MANAGEMENT OF PCOS Table 11.11. Clomiphene citrate.
•• 1st choice for OI in anovulatory PCOS. •• CC resistance (ovulation failure): No ovulation with the
•• Non-steroidal selective estrogen receptor modulator. maximum dose of 150 mg/day, seen in 15-40%.23,24
LIFESTYLE MODIFICATIONS: FIRST-LINE • Finasteride: 5α-reductase type 2 inhibitor.
•• Mechanism of action: Binds to hypothalamic estrogen •• Options for CC resistance:24
• Combined hormonal oral contraceptive pill (OCP). receptors → blocks the negative feedback effect of Concomitant use of insulin sensitizers
TREATMENT OF OBESITY IN PCOS B. Dermatological interventions: Second line of management
estrogen → ↑ FSH and LH follicular development.
• Weight loss: of 5–10% has been shown to decrease tes- • Waxing, plucking, shaving, depilation, electroly- •• Dose: 50 mg/day for 5 days from day 2/3. Extended CC: For 7-10 days; 100 mg/day
tosterone levels, increase Sex-hormone binding globu- sis, and laser to remove current hair. •• Over response: reduce to 25 mg, no ovulation: 50 mg Use of Aromatase inhibitors
• Topical treatment: Eflornithine hydrochloride. increments with maximum dose of 150 mg/day.24 Addition of glucocorticoids
lin (SHBG), normalize menses, improve fertility, 30%
•• Outcome23,24 Pretreatment with OCPs.
reduction in visceral fat, attenuates insulin resistance, •• Clomiphene citrate failure: Ovulate but fail to conceive
Ovulation rate: 75-80%
and other metabolic aberrations.24 MANAGEMENT OF MENSTRUAL Conception rate per cycle: 22% after three or more ovulatory cycles with CC.
• Methods: •• Side effects: Vasomotor flushes, bloating, breast tender-
DYSFUNCTION Cumulative live-birth rates (for up to 6 cycles):
ness, visual symptoms (scotomas, light sensitivity, blurred/
Diet: Atkins diet with reduced glycemic index and 50-60%
low calorie diet (500 Kcal/day deficit).24 • The approach to management of menstrual dysfunc- Multiple pregnancy rate: 8% double vision), dryness of the vagina, headache, and ovar-
tion will depend on the type of menstrual dysfunc- Miscarriage rate: 20% ian hyperstimulation.
Exercises: Regular physical activity.
•• Discrepancy between ovulation and pregnancy •• Contraindications: Ovarian cyst, pregnancy, liver disease,
Pharmacological agents: Orlistat-Gastric and tion, life stage, medical history, and preferences of the
rates are due to:24 visual disorders.
pancreatic lipase inhibitor, reduces absorption of woman concerned (Table 11.10). •• Clomiphene and ovarian malignancy: With six CC cycles,
Anti-estrogenic action on the endometrium and
dietary fats. Oral dose of 120 mg three times a day cervical mucus the risk will not exceed that of other women. But >12 cycles
with food. Approved for long-term treatment of MANAGEMENT OF INFERTILITY Decreased uterine blood flow in a life time, threefold increase in ovarian cancer.24
obesity.28 Effect on tubal transport •• Congenital anomalies: the possibility of neural tube
ASSOCIATED WITH PCOS Probable detrimental effects on the oocytes. defects, cloacal extrophy, ventricular septum heart defects,
Bariatric surgery: In morbidly obese women with
Dandy-Walker syndrome, and omphalocele.23
BMI more than 40 kg/m2. • PCOS is associated with approximately 80–90% of
women who suffer from infertility due to anovulation.17
A. First-line treatment for ovulation induction (OI): Ovulation Induction Agents: • Advantage of short half-life approximately 45 hours
MANAGEMENT OF HIRSUTISM Clomiphene citrate (CC) resulting in mono-follicular growth.30
A. Anti-androgens: B. Second-line intervention: Clomiphene Citrate
• Letrozole earlier banned in India as an OI agent has
• Spironolactone: aldosterone-antagonist diuretic. Exogenous gonadotropins (GTs). See Table 11.11. now been safely welcomed/reintroduced.
• Flutamide (androgen receptor antagonist): Laparoscopic ovarian drilling (LOD). • Letrozole was proven to be safe and the incidence
limited value because of its dose-dependent C. Third-line treatment:
ALTERNATIVE OI AGENTS TO CC of major malformation was 1.2% versus 3.0% CC
hepatotoxicity. In-vitro fertilization. group.23,31
Tamoxifen
Gonadotropin (GT)
Table 11.10. Management of menstrual dysfunction: Oral contraceptive pill (OCP).
• Selective estrogen receptor modulator (SERM), potent
estrogen antagonist on breast, blood vessel but partial • Indications: In Clomiphene resistance and Clomi-
Combined hormonal oral contraceptive pill (OCP) Alternatives to OCP
agonist in uterus, bone, liver, and pituitary. phene failure.
•• In women who do not wish to conceive, given cyclically will •• Progestins: given cyclically (micronized proges- • Maximum of six cycles only (no response—resistance).
• Mechanism of action: Similar to CC.
confer regular monthly withdrawal bleeds. terone 100-200 mg daily or medroxyprogesterone
• Dose: 20-40 mg/day from day 2/3 for 5 days and with • GTs used: Urinary or highly purified preparations of
•• The estrogen component suppress LH secretion, decreases acetate 10 mg daily for 10-14 days/month).13
no ovulation, the dose can be increased to 80 mg follicle-stimulating hormone (FSH)/human meno-
ovarian and adrenal androgen synthesis, and also increases •• Levonorgestrel-releasing intrauterine system will
SHBG levels, all of which resulting in a decrease in circulating provide contraception and protect the endometrium (40 mg twice a day). pausal gonadotropin (HMG) or recombinant prepara-
free T levels. from unopposed estrogens. • Side effects: Hot flushes, vomiting, anorexia, abnormal tions of FSH.
•• The progestin component protects the endometrium from unop- •• Transdermal contraceptive patch: is a treatment uterine bleeding, and endometrial hyperplasia. • Main complications: Ovarian hyperstimulation syn-
posed estrogen. option for adolescents PCOS; however, it may be • Tamoxifen has shown reasonably good results in CC drome (OHSS) and multiple pregnancies.
•• Most commonly used OCPs: Ethinylestradiol (EE) at dose of associated with an increased risk for venous throm- failure cases.29 • Controlled ovarian stimulation (COS) responses in
20−35 μg + progestin (norethindrone, norgestimate, or des- boembolic events.13 PCOS are characterized by a reduced therapeutic
• It is first choice hormonal treatment of breast cancer in
ogestrel). •• Transvaginal contraceptive ring. margin and hence for successful outcome:
both pre- and postmenopausal women.29
•• Low-dose OCPs are metabolically safe.21 To allow slow rise of FSH to just above the FSH
•• In the absence of other risk factors, there is no evidence that threshold level (which is high in PCOS).
PCOS are at increased risk of CVD with OCP compared with Aromatase Inhibitors (AI) To avoid an explosive ovarian response because of
normal women.23 • Letrozole is the most commonly used AI for OI. exquisite sensitivity of follicles to exogenous GT.24
88 The Infertility Manual Polycystic Ovarian Syndrome 89
I. Step-up regimens II. Step-down regimen (Fig. 11.5) • Not to be done for non-fertility indications (irregular Failed to conceive despite six ovulatory cycles
cycles, hyperandrogenism) Coexistent infertility factors: advanced age, tubal
• Principle: Attainment and maintenance of follicular Principle: To achieve the FSH threshold through a loading
• Mechanism: Surgical trauma rather than the amount damage, severe endometriosis, male factor infertil-
development with exogenous FSH, without exceeding dose of FSH with a subsequent stepwise reduction with
of tissue destruction results in decrease in LH and ity, need for preimplantation genetic screening
the threshold requirement. follicular development.32,34 androgens • Protocol: Gonadotropin releasing hormone (GnRH)
• Commonly employed methods: Monopolar electro- antagonist protocol, as it is associated with shorter dura-
III. Sequential protocol (Fig. 11.6) cautery (diathermy) and laser
Conventional Step-up Dose Regimen • Armar’s rule of 4: 4 punctures/ovary with 40 W, 4 sec,
tion of stimulation, lower dosage of GTs and s ignificantly
• It combines an initial step-up GT administration fol- lower risk of moderate to severe OHSS.41
(Fig. 11.3) lowed by a step-down regimen after follicular selection.35 4 mm depth37
• Ovarian stimulation: Individualized controlled stimu-
• Reduces the risk of hyperstimulation by narrowing the • Effective thermal dosage: 60 J/cm3 of ovarian tissue38
• Cumulative conception rate of 82% after six cycles.32 lation (ICOS) based on age, BMI, antral follicle count
follicular selection window owing to atresia of the less • Seven or more punctures per ovary discouraged—
• Drawback-multiple pregnancy rate of 34% and severe (AFC), AMH, and previous response to stimulation.42
sensitive smaller follicles which are unable to grow concern about excessive destruction of ovary without
OHSS of 4.6%.32 additional benefit37 • Increased OHSS rates of 15-20% in in-vitro fertilization
with declining FSH levels.35 (IVF) cycles.43
• Outcome38: Ovulation rate: 65-80%, Pregnancy rate:
• GnRH antagonist protocol with gonadotropin releas-
Chronic Low-dose Regimen (Fig. 11.4) LAPAROSCOPIC OVARIAN 40-50%
• In 50% of LOD-treated women, adjuvant therapy will ing hormone agonist (GnRHa) trigger for follicular
• Rationale: Increasing the level of FSH in a gradual and DRILLING (LOD) be required21 maturation nearly eliminates the risk of OHSS.44
step wise fashion allows the rescue of a limited number • Advantages of LOD over medical treatment: • Segmentation strategy prevents OHSS: (a) seg-
• Creation of multiple ovarian punctures through ovar-
of follicles mirroring that of a normal hormonal cycle.33 Correction of hormonal milieu (↓ androgens) ment A: optimization of ovarian stimulation, use of
ian capsule, electro surgically, or with laser energy.
• Advantages: Safer in terms of mono-follicular develop- • Indications:36 Assessment of tubal patency and correction of GnRH antagonist protocol with GnRHa trigger, (b) seg-
ment and avoids complications of OHSS and multiple Anovulatory CC resistant PCOS/CC failure other pelvic problems simultaneously ment B: cryopreservation of embryos by vitrification,
pregnancies.33 Persistent hypersecretion of LH (>10 IU/L) One time procedure and cost effective and (c) segment C: frozen embryo transfer in the sub-
• Drawback: Long treatment cycles long of 28-35 days. High levels of androstenedione No intense monitoring required sequent cycle.45
• Outcome: Mono-ovulatory cycles (70%); Pregnancy Mono-follicular genesis • Single-elective blastocyst transfer reduces the risk of
Requiring laparoscopic assessment of pelvis
rate—20% per cycle; OHSS rate—low (<1%); multiple No multiple pregnancy multiple pregnancies.24
Intensive monitoring is not feasible with GT treat-
No OHSS • Cycle cancellation: Either due to absent/limited ovar-
pregnancy rate—<6%.32,33 ment/cannot follow-up
Clomiphene resistant patients may respond as ian response or due to OHSS.
LOD may improve the sensitivity to GTs
• IVF outcome:
The beneficial endocrinological effects of LOD
a. Oocyte and embryo quality not affected, lower fer-
appear to be sustained for about 9 years39
tilization and delayed cleavage kinetics from ferti-
• Complications:
Decrease in ovarian reserve and premature ovarian lization to 8-cell stage.46
failure-with multiple punctures and deep desicca- b. Significantly more cumulus–oocyte complexes.
tion of hilar vessels c. Poor endometrial receptivity: Suppression of
Adhesions (60%) resulting in mechanical infertility.40 Glycodelin and Homeobox A10 (HOXA10) genes,
IGFBP1, high plasma endothelin.47,48
d. The clinical pregnancy rate per started cycle was
Fig. 11.3: Conventional step-up dose protocol. Fig. 11.4: Chronic low dose step-up protocol. ROLE OF INTRA-UTERINE
similar between PCOS and non-PCOS patients
INSEMINATION (IUI) (37.4% vs 32.3%).48,49
• Induction of ovulation with IUI when associated with mild
to moderate male factor infertility and in those who failed IN-VITRO MATURATION (IVM)
to conceive despite successful induction of ovulation.24
• The clinical pregnancy rate per cycle is 11−20% and • Immature oocytes are retrieved transvaginally from
multiple pregnancy rate is 11−36%.24 antral follicles measuring 2–12 mm in diameter
within unstimulated or minimally stimulated ova-
ries and matured in-vitro for 24–52 hours before
ASSISTED REPRODUCTIVE
fertilization.
TECHNOLOGY (ART) IN PCOS • The pregnancy and live birth rates were lower in IVM
• Anovulation is not an indication for IVF. at 19.6% and 16.5% as compared to 38.3% and 26.2%
Fig. 11.5: Low dose stepdown protocol. Fig. 11.6: Sequential protocol. respectively in regular ART.50
• Indications:24
90 The Infertility Manual Polycystic Ovarian Syndrome 91
• This technique first offered in PCOS for the preven- dose of 400-2000 IU/day or 50,000 IU/week depending on Table 11.13. Diagnostic criteria for PCOS in adolescents.
tion of OHSS cannot be recommended now because the severity of deficiency so as to achieve a serum level of
Parameter ESHRE/ASRM 201262 Endocrine Society 201363
of poor results and as GnRH antagonist and agonist- 25 (OH) D > 20 ng/mL.19
Criteria 1. Clinical or biochemical hyperandrogenisma 1. Clinical or biochemical hyperandrogenisma
trigger protocols reliably prevent OHSS.48 2. Oligo-/anovulationb 2. Persistent oligo-/anovulationb
L-Methylfolate 3. Polycystic ovarian morphologyc
ADJUVANTS: IMPROVEMENT Active form of folic acid helps in DNA synthesis and Limitation Three of three criteria required with exclusion of other Two of two criteria required with exclusion of other
etiologies etiologies
IN METABOLIC FUNCTION reduces homocysteine levels and thereby improves endo
thelial function.19 Note: ASRM, American Society for Reproductive Medicine; ESHRE, European Society for Human Reproduction and Embryology.
a. Increased serum androgens and/or progressive hirsutism.
I. Insulinsensitizing agents: See Table 11.12. b. Oligo-/amenorrhea for at least 2 years, or primary amenorrhea by age 16 years.
Anti-oxidants c. Ovarian volume >10 cm3.
Other Adjuncts Like omega-3 fatty acids, Melatonin, Soy phytoestrogen
(Genistein), Chromium and Zinc.19
Statins PCOS AND PREGNANCY
N-Acetyl cysteine (NAC) • Prior to conception, lifestyle modifications, cessation
Improves lipid profile, decreases systemic inflammation
NAC decreases serum insulin, total cholesterol, LDL and Corticosteriods (C-reactive protein), endothelial inflammation, oxidative of smoking, diet, folic acid supplementation, and con-
homocysteine levels, increase ovulation rate and preg- stress and homocysteine levels.60,61 Category X medica- trol of sugars should be advised.24
Beneficial in CC non-responders mostly Hyperandrogenic
nancy rates and HDL levels.57 As an adjuvant to CC in the tion causing fetal toxicity. Hence effective contraception is • Increased risk of miscarriage (24-36% in PCOS vs
PCOS as it decreases adrenal androgen.17,26 Used as Dexa-
dose: 600 mg twice a day for 6 weeks. essential and not recommended as an adjunct to OI. 15-18% in non PCOS) due to:17,64
methasone 0.25-0.5 mg/day; Prednisone 5 mg/day from
Errors in folliculogenesis, meiotic competence, and
day 2−6 or throughout follicular phase or 10 days.58
Vitamin D PCOS IN ADOLESCENTS oocyte maturation
Hyperandrogenemia
Vitamin D supplementation has beneficial effects on insu-
OCP Pretreatment • The normal physiologic IR in response to growth hor- Hyperinsulinemia, insulin resistance
mone, which peaks during puberty may aggravate the Obesity
lin resistance, hyperandrogenism, follicular maturation, Causes suppression of the HPO axis, reduces LH and
symptoms and phenotypic expression.13 Increased plasminogen activator inhibitor 1
menstrual regularity, and ovulation.19 Given orally at a androgen levels thereby improves folliculogenesis.59
• Rapid weight gain in small for gestational age (SGA) Hyperhomocysteinemia
girls and sustained adiposity in large for gestational Abnormal (elevated as well as decreased) serum
Table 11.12. Insulinsensitizing agents. age (LGA) girls accelerate the prepubertal appearance leptin levels
Metformin Thiazolidinediones Inositol of PCOS.13 ↓ serum and endometrial glycodelin and IGFBP-I
•• Oral biguanide approved for treatment •• Rosiglitazone and Pioglitazone are •• Administration of MI/DCI, in the • Difficulties in diagnosing PCOS in adolescents:12,13 ↓ Uterine vascularity and increased uterine vascu-
of type 2 diabetes mellitus the two prescribed drugs as they physiological plasma ratio (40:1) Relatively high rate of menstrual irregularity and lar resistance
•• Mechanism of action:51,52 are relatively free of liver toxicity; ensures better clinical results56 anovulatory cycles in this period. • Recurrent miscarriage is more common in PCOS
Reduction of hepatic gluconeogenesis though monitoring liver function every •• DCI acts at the periphery and (36-56%) when compared with the general population
Improves peripheral glucose uptake 2 months is recommended55,56 reduces insulin resistance,
Difficulties in interpreting clinical and biochemical
evidence of hyperandrogenism, with acne and hir- (20–23%).65,66
by liver, skeletal muscle and adipose •• Rosiglitazone: Initially 4 mg/ improves glucose metabolism,
tissue day; increased to 8 mg/day improves lipid profile16 sutism being common during adolescence. • Even in singleton pregnancies, there is a higher inci-
Stimulates glycolysis in liver after 8−12 weeks •• MI acts at the ovary: reduces dence of pregnancy complications such as gestational
Uncertainty regarding the significance of PCOM on
– Uses: (a) Adjunct in CC resistant •• Pioglitazone: 15−30 mg once a day Hyperandrogenemia (HA), diabetes (40–50%), gestational hypertensive disorders
ultrasound and has to be differentiated from multi-
cases, (b) Adjunct to OI in PCOS •• Side-effects: Weight gain, fatigue, regularizes cycle with spontane- (such as pre-eclampsia (5%) and neonatal complica-
with glucose intolerance and edema, diarrhea, sinusitis, anemia, ous ovulation, improves ovarian
cystic ovaries (MCO), and ovary volume greater
tions (small-for-gestational-age babies (10–15%) and
Type 2DM, (c) For weight loss in congestive heart failure and in piogl- response and improves oocyte than 10 cm3, being a better marker in adolescents.
admission to neonatal intensive care units).67,68
Obese PCOS along with life style itazone particularly, increased risk of quality16,56 • Diagnostic criteria (Table 11.13).
modifications,53 (d) Adjuvant in
• The spectrum of pregnancy complications, more-so in
bladder cancer •• Dose: 2-4 g/day • Transabdominal pelvic ultrasound, though a routine
IVF cycles may enhance ongoing •• Category C drugs and hence must be
type A and B phenotypes has been related to impaired
ovarian imaging is not indicated. early decidual trophoblastic invasion and/or remodel-
pregnancy rates and reduce the discontinued in pregnancy
incidence of OHSS54 • Serum testosterone levels: High testosterone levels/ ling of spiral vessels and defects in placentation.
•• Dosage: 500-850 mg/day three times/ rapidly progressive hirsutism/virilization to evaluate • Obesity is also associated with increased obstetric
day after confirming normal renal and for malignancy. complications, such as shoulder dystocia and post-
liver function • Screening for metabolic abnormalities as adolescents partum thromboembolism.66
•• Category B drug in pregnancy
•• Side-effects: Dose dependent includes with PCOS independent of body weight have fourfold • Metformin therapy lowers serum insulin and may
nausea/vomiting, bloating, cramps, di- increased risk. have a beneficial effect on miscarriage rates and risk
arrhea flatulence, lactic acidosis (rare, • Management: Involves reproductive, metabolic, and of gestational diabetes though there is no evidence for
fatal), Vit. B12 deficiency cosmetic manifestations of the syndrome. improved live birth rates.21
92 The Infertility Manual Polycystic Ovarian Syndrome 93
PCOS AND MENOPAUSE • Recommended second-line intervention should CC 8. Nam Menke M, Strauss JF. Genetics of polycystic ovarian 24. Thessaloniki ESHRE/ASRM-Sponsored PCOS Consensus
fail to result in pregnancy is either exogenous GTs or syndrome. Clin Obstet Gynecol. 2007;50:188-204. Workshop Group. Consensus on infertility treatment
• Age may improve many manifestations of PCOS, hir- Laparoscopic ovarian drilling.
9. Abbott DH, Barnett DK, Bruns CM, Dumesic DA. Androgen related to polycystic ovary syndrome. Fertil Steril.
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• Induction of ovulation particularly with GTs, care developmental aetiology for polycystic ovary syndrome? 25. Dewailly D, Lujan ME, Marcelle EC, Cedars I, Laven J,
ovarian size and morphology, testosterone levels. must be taken to avoid multi-follicular development Hum Reprod Update. 2005;11:357-74. Norman RJ, et al. Definition and significance of polycystic
• PCOS diagnosis in postmenopausal first formulated by and its adverse consequences—OHSS and multiple 10. Homburg R. Androgen circle of polycystic ovary syndrome. ovarian morphology: a task force report from the Androgen
Endocrine Society (2013), based on previous history of pregnancy. Hum Reprod. 2009;24:1548-55. Excess and Polycystic Ovary Syndrome Society. Hum
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• Chronic low-dose step-up protocol is safe in terms of
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as supportive sign.63 tions of OHSS and multiple pregnancies. 2007;25:352-9. (PCOS): revisiting the threshold values of follicle count on
• PCOS women have a larger cohort of primary follicles • GnRH antagonist protocol with GnRHa trigger for fol- 12. Bremer AA. Polycystic ovary syndrome in the pediatric ultrasound and of the serum AMH level for the definition
than age-matched control women before menopause.21 licular maturation nearly eliminates the risk of OHSS. population. Metab Syndr Relat Disord. 2010;8:375-94. of polycystic ovaries. Human Reprod. 2011;26(11):3123-9.
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• Most reports tend to show normal or increased bone • Women with PCOS should be followed closely dur-
pathophysiology, molecular aspects and clinical implica- concentrations in follicular fluid are associated with poor
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CD001750. 57. Badawy A, State O, Abdelgawad S. N-Acetyl cysteine and • Neuroendocrine Regulation of PRL Secretion • Diagnosis and Management of HPRL
• Actions of Prolactin • Treatment of HPRL
42. Fiedler K, Ezcurra D. Predicting and preventing ovarian clomiphene citrate for induction of ovulation in polycystic
• Other Sources and Functions of PRL • Newer Insights
hyperstimulation syndrome (OHSS): the need for ovary syndrome: a cross-over trial. Acta Obstet Gynecol. • Causes of HPRL • PRL and Ovarian Stimulation in IVF
individualized not standardized treatment. Reprod Biol 2007;86:218-22.
Endocrinol. 2012;10:32. 58. Daly DC, Walters CA, Soto-Albors CE, et al. A randomized
43. Swanton A, Storey L, McVeigh E, Child T. IVF outcome in study of dexamethasone in ovulation induction with
women with PCOS, PCO and normal ovarian morphology. clomiphene citrate. Fertil Steril. 1984;41(6):844-8. INTRODUCTION
Eur J Obstet Gynecol Reprod Biol. 2010;149:68-71. 59. Goenka D, Goenka ML. Oral contraceptive pill pretreatment
44. Humaidan P, Kol S, Papanikolaou EG, on behalf of the ‘The for clomiphene citrate resistant cases followed by repeat • Prolactin (PRL) plays a central role in a variety of
Copenhagen GnRH Agonist Triggering Workshop Group’. clomiphene citrate treatment. J Obstet Gynaecol India. reproductive functions. It is produced by the anterior
GnRH agonist for triggering of final oocyte maturation: 2006;56(2):159-61. pituitary gland. PRL is mainly involved with lactation.
time for a change of practice? Hum Reprod Update. 60. Banaszewska B, Pawelczyk L, Spaczynski RZ, et al. Effects
However, its role in reproductive function is vital and is
2011;17:510-24. of simvastatin and oral contraceptive agent on polycystic
45. Devroey P, Polyzos NP, Blockeel C. An OHSS-Free Clinic ovary syndrome: prospective, randomized, crossover trial. under active interrogation.1,2
by segmentation of IVF treatment. Hum Reprod. 2011;26: J Clin Endocrinol Metab. 2007;92(2):456-61. • Hyperprolactinemia (HPRL) is a common endocrine
2593-7. 61. Kaya C, Cengiz SD, Berker B, et al. Comparative effects disorder with gonadal dysfunction. It presents as amen-
46. Wissing ML, Bjerge MR, Olesen AIG, Hoest T, Mikkelsen of atorvastatin and simvastatin on the plasma total
orrhea, oligomenorrhea, galactorrhea, and infertility.3
AL. Impact of PCOS on embryo cleavage kinetics. Reprod homocysteine levels in women with polycystic ovary
Biomed Online. 2014;28:508-14. syndrome: a prospective randomized study. Fertil Steril.
47. Mulders AG, Laven JS, Imani B, Eijkemans MJ, Fauser BC. 2009;92(2):635-42. PRL BIOSYNTHESIS
62. Fauser BC, Tarlatzis BC, Rebar RW, Legro RS, Balen AH, Fig. 12.1: Structure of PRL.
IVF outcome in anovulatory infertility (WHO group 2) –
including polycystic ovary syndrome – following previous Lobo R, et al. Consensus on women’s health aspects • PRL is a single polypeptide chain of 199-amino acid.3
unsuccessful ovulation induction. Reprod Biomed Online. of polycystic ovary syndrome (PCOS): the Amsterdam • It is encoded by a single gene located on the short arm potent biological form, while usually the larger polymers
2003;7:50-8. ESHRE/ASRM-Sponsored 3rd PCOS Consensus Workshop of chromosome 6. PRL gene belongs to the PRL/growth
48. Ziegler DD, Streuli I, Gayet V, Frydman N, Bajouh O, Group. Fertil Steril. 2012;97:28-38.e25. have reduced receptor affinity and lower bioactivity.3,8
63. Legro RS, Arslanian SA, Ehrmann DA, Hoeger KM, Murad hormone (GH)/HPL group of genes that are structur- • Macroprolactinemia is a common condition reported
Chapron C. Retrieving oocytes from small non-stimulated
follicles in polycystic ovary syndrome (PCOS): in vitro MH, Pasquali R, et al. Diagnosis and treatment of polycystic ally similar to each other. This group of hormones is in 10–46 percent of patients with HPRL and is due to
maturation (IVM) is not indicated in the new GnRH ovary syndrome: an Endocrine Society clinical practice known as group I helix bundle protein hormones.4,5 large molecules of PRL, that is biologically inactive,
antagonist era. Fertil Steril. 2012;98:290-3. guideline. J Clin Endocrinol Metab. 2013;98:4565-92. • Structure of PRL is very similar to the hematopoietic
64. Wang JX, Davies MJ, Norman RJ. Polycystic ovarian but detected by radioimmunoassay as the biologically
49. Heijnen EMEW, Eijkemans MJC, Hughes EG, Laven JSE, cytokines and its receptor. Thus, PRL/GH/HPL family
Macklon NS, Fauser BCJM. A meta-analysis of outcomes syndrome and the risk of spontaneous abortion following active PRL.9,10 This may explain very high PRL levels
of conventional IVF in women with polycystic ovary assisted reproductive technology treatment. Hum Reprod. was also linked to another extended family of proteins sometimes found in women with normal ovulation.
syndrome. Hum Reprod Update. 2006;12(1):13-21. 2001;16:2606-9. known as the hematopoietic cytokines. It is now con- Such cases do not require treatment (Fig. 12.1).
50. Gremeau AS, Andreadis N, Fatum M, Craig J, Turner K, 65. Rai R, Backos M, Rushworth F, Regan L. Polycystic ovaries sidered that PRL is a cytokine with an immune modu-
McVeigh E, et al. In vitro maturation or in vitro fertilization and recurrent miscarriage—a reappraisal. Hum Reprod.
for women with polycystic ovaries? A case–control study of 2000;15:612-5.
latory effect.6,7 PROLACTIN RECEPTORS
194 treatment cycles. Fertil Steril. 2012;98:355-60. 66. Palomba S, Falbo A, Chiossi G, Tolino A, Tucci L, Battista G, • Various hormones can bind to and consequently alter
et al. Early trophoblast invasion and placentation in women the rate of PRL gene expression like estrogen, dopa- • PRL receptor is a transmembrane receptor with close
51. Palomba S, Falbo A, Zullo F, et al. Evidence-based and potential
benefits of metformin in the polycystic ovary syndrome: a with different PCOS phenotypes. Rep BioMed Online. mine (DA), thyroid-releasing hormone (TRH), vasoac- structural similarity to GH receptors. It is composed of
comprehensive review. Endocr Rev. 2009;30(1):1-50. 2014;29:370-81. tive intestinal peptide (VIP).2,3 an extracellular region (that binds PRL), a single trans-
52. Diamanti-Kandarakis E, Economou F, Palimeri S, et al. 67. Boomsma CM, Eijkemans MJ, Hughes EG, Visser GH, membrane region, and a cytoplasmatic region.3,11
Metformin in polycystic ovary syndrome. Ann N Y Acad Fauser BC, Macklon NS. A meta-analysis of pregnancy
• PRL receptors can bind to other two ligands, which are
Sci. 2010;1205:192-8. outcomes in women with polycystic ovary syndrome. Hum PROLACTIN STRUCTURE GH and human placental lactogen.
53. Naderpoor N, Shorakae S, Courten BD, Misso ML, Moran Reprod Update. 2006;12:673-83.
LJ, Helena J, et al. Metformin and lifestyle modification in 68. Kjerulff LE, Sanchez-Ramos L, Duffy D. Pregnancy • PRL is an all-a-helix protein with a wide heterogeneity. • PRL receptors are widely expressed by different cells
polycystic ovary syndrome: systematic review and meta- outcomes in women with polycystic ovary syndrome: a Its 23-kDa nonglycosylated form appears to be the most including the pituitary, breast, liver, pancreas, brain,
analysis. Hum Reprod Update. 2015;21(5):560-74. metaanalysis. Am J Obstet Gynecol. 2011;204:558.e1-6.
96 The Infertility Manual Hyperprolactinemia and Infertility 97
Fig. 12.4: Neuroendocrine regulation of PRL secretion.
of the mammary gland along with stimulation of lac- OTHER SOURCES AND FUNCTIONS OF PRL
togenesis after giving birth.15
• PRL is produced mainly by the pituitary lactotrophs,
• PRL functions along with other hormones such as cor-
which normally comprise about 15–25 percent of the
Fig. 12.2: Circadian rhythm of prolactin versus GH. tisol and insulin. Together these hormones induce the
anterior pituitary.21
transcription of genes that encode for milk proteins.16 • PRL is also secreted by other cells in the body, such as
• During pregnancy, action of PRL is in turn regulated by mammary glands, immune cells, different brain cells,
adrenal cortex, lungs, prostate, epididymus, ovary, and
the sex steroidal hormones such as estrogen and pro- and the decidua of the pregnant uterus.22
lymphocytes.12
gesterone. To get full activity of the prolactin receptor, Following Fig. 12.5 summarises the other sources and
estrogen and progesterone are required. Progester- functions of prolactin.
PRL SECRETION PATTERNS one inhibits the upregulation of the prolactin recep-
tor thus antagonizes the positive action of PRL on its CAUSES OF HPRL
• PRL secretion shows a circadian rhythm. Its concen-
tration is higher during the night and lower circulating receptor.17 High levels of PRL observed during pregnancy and lacta-
level during the day. Levels starts to rise 1 hour after tion. HPRL can also present as a pathological condition at
the sleep onset and continue to rise. Peak values are Fig. 12.3: Hypothalamus GnRH pulses induce secretory pulses of LH. In Males any age. This excess of prolactin may be due to variety of
reached between 5:00 A.M. and 7:00 A.M.3 Most of causes as summarized in (Fig. 12.6).
• PRL is a hormone of sexual gratification. HPRL can be physiological or pathological. A transient
the rise occurs in REM sleep and then falls again before
• The amount of PRL released from the anterior pituitary • Dopamine is normally responsible for sexual arousal elevation in serum PRL can be produced by the venepunc-
the next period of REM sleep. This circadian rhythm is
gland3 is determined by the balance between these and PRL represses the effect of dopamine.18 ture stress. Mildly elevated PRL is frequently seen in PCOS
generated by the suprachiasmatic nuclei of the hypo- • PRL is very important for spermatozoa metabolism,
two opposite signals. patients due to the raised circulating estrogen level.29 In
thalamus (Fig. 12.2).2 that is, glucoseoxidation, fructose utilization, and glyc- patients with primary hypothyroidism, the raised hypo-
• Release of PRL is in a pulsatile pattern. Pulse frequency • PRL also acts as a negative feedback on its own secre-
olysis and is found in high concentrations in semen.19,20 thalamic TRH release constantly stimulates prolactin
ranges between 14 pulses per day (in the late follicular tion, either directly through inhibiting the lactotrophs
phase) to 9 pulses per day (in the late luteal phase). Each or indirectly through stimulating the neuroendocrine
pulse normally lasts for approximately 70 minutes, with dopaminergic neurons.14,15
interpulse interval of approximately 90 minutes.2,13 • Estrogens acts as key regulators of PRL production
• PRL release does not depend entirely on the nursing by enhancing the growth of PRL producing cells and
stimulus but is also affected by other stimuli such as stimulating PRL production either directly through
light, stress, olfaction, and audition.2 activating the PRL gene or indirectly through sup-
pressing DA.2
• Figure 12.4 summarises neuroendocrine regulation of
NEUROENDOCRINE REGULATION PRL secretion.
OF PRL SECRETION
• Hypothalamic gonadotropin-releasing hormone ACTIONS OF PROLACTIN
(GnRH) mediates the stimulatory signal while the inhi
bitory signal is mediated by neurotransmitter dopa
In Females
mine, also known as the prolactin inhibitory factor • PRL exerts many physiological functions, the most
(PIF). The predominant signal is inhibitory (Fig. 12.3).2 prominent of which is inducing lobulo-alveolar growth Fig. 12.5: Other sources and functions of prolactin.
98 The Infertility Manual Hyperprolactinemia and Infertility 99
Fig. 12.7: Role of HPRL in female infertility.
Table 12.2. Effect of HPRL in men.
Effect due to gonadotropins37
Effect at the level of
hypothalamus Effect at the level of testis Effect independent of g
onadotropins37,38
Fig. 12.6: Causes of hyperprolactinemia.
HPRL HPRL Patients with oligospermia or azoospermia with normal
serum levels of gonadotropins show relatively high
↓ ↓ serum levels of PRL thus proving a role of PRL in
Table 12.1. Causes of HPRL and its mechanism.
gametogenesis (independent of gonadotropins)
Dysfunction/disease32 Mechanism ↓Pulsatile release of GnRH Act on PRL receptors (present in
Idiopathic Impaired hypothalamic dopamine secretion Sertoli cells and Leydig cells)
↓
Pituitary tumors: micro/macroprolactinoma, hypothalamic Disruption of dopamine delivery and secretion of PRL
↓
stalk interruption
Spermatogenic arrest
Adenoma
Impaired sperm motility Influences spermatogenesis
Acromegaly PRL secretion from a GH adenoma
Empty Sella syndrome Damage of the pituitary gland ↓ ↓
Primary hypothyroidism Increased hypothalamic TRH
Hypogonadotropic Hypogonadotropic hypogonadism
Polycystic ovary syndrome Raised estrogen concentration hypogonadism and Infertility and infertility
Renal failure Reduced PRL clearance
Drugs Inhibition of dopamine release
- Antidopaminergic drugs
- Anti-psychotics (phenothiazines, haloperidol, butyroph-
a detrimental effect on the reproductive functions both in EFFECT OF HPRL IN MEN
men and women (Fig. 12.7).
enones, risperidone, monoamine, oxidase inhibitors, HPRL can cause primary or secondary hypogonadism and
fluoxetine, sulpiride) is responsible for infertility in about 11% of males with oli-
- Anti-emetics (metoclopramide, domperidone)
- Tricyclic antidepressants EFFECT OF HPRL IN WOMEN gospermia (Table 12.2).3
Opiates Stimulation of opioid hypothalamic receptors • Pulsatile secretion of GnRH is reduced due to excess Sexual Dysfunction
Estrogens Stimulation of lactotrophs production of PRL which in turn reduce the release of
A literature review found about 88% prevalence of sexual
Verapamil Unknown LH and FSH. dysfunction in men with HPRL.39 Most common sexual dys-
• In addition, an excess PRL can directly influence the function was erectile dysfunction (ED) followed by reduced
secretion.30 The occurrence of menopause is also associ- HPRL AND INFERTILITY steroidogeneic activity of the ovary causing menstrual sexual desire, impaired orgasm.39 The mechanism by which
ated with a short-living (weeks–months) PRL secretion.31 irregularities. HPRL causes ED is explained in (Figs. 12.8).
Table 12.1 summarises various causes of HPRL and its PRL has effect on various points of the hypothalamic– • HPRL is a relatively common cause of secondary Nearly half of the ED patients with HPRL show nor-
mechanism. pituitary–gonadal axis. Hence, excess of PRL could have amenorrhea.33 mal serum testosterone level. In addition, in males with
100 The Infertility Manual Hyperprolactinemia and Infertility 101
HPRL in Males Laboratory Evaluation Medical Treatment
• Decreased libido, impotence, erectile dysfunction, As PRL is a dynamic hormone that responds readily to a The first line of treatment for patients with HPRL of any
infertility, gynecomastia, or rarely galactorrhea45 are variety of stimuli, caution must be exercised during diag- cause usually is DA agonist drug.54
the signs and symptoms of HPRL in men caused by nosis. HPRL is usually defined as fasting levels of above Following are the available DA agonists to treat HPRL.
hypogonadotropic hypogonadism. 20 ng/mL in men and above 25 ng/mL in women.49 There • Side effects are more common at the start of drug or
• Hypogonadism in the short term causes decreased is physiological and diurnal variations in levels of PRL and when the dose is increased.
energy and libido are short-term effects of hypogonadism normal levels are between 5 and 25 ng/mL.50 Higher serum • The dose can then be increased gradually. Some patients
Fig. 12.8: Mechanism of ED in HPRL. and in the long term it results in reduced muscle mass, level occurs in afternoon than in the morning, and thus it develop side effects even at the lowest doses. In women,
decreased body hair, and osteoporosis.46 should preferably be measured in the morning, at least drug can be given intravaginally if nausea is trouble-
• Galactorrhea is less common in men than in women 2 hours after waking up. some.57 Nausea is less encountered with cabergoline.
HPRL, serum sex hormone-binding globulin is low. because the glandular breast tissue in men has not
• In case of prolactinoma prolactin level is often more Other drugs that have been studied but are not yet
This in turn increases unbound proportion of testos- been made sensitive to PRL by precedent stimulation
than 250 ng/mL and a level of more than 500 ng/mL is approved include Quinoglide. It is a nonergot DA agonist
terone and attenuates the biological impact of low total of estrogen and progesterone. diagnostic of a macroprolactinoma.
Both sexes may present with neurological symptoms that is given once daily. Long-acting form of bromocrip-
testosterone.40 • PRL levels more than 200 ng/mL may occur with drugs
such as headache, visual disturbances associated with tine is available and can be administered by injection once
In men with HPRL, sexual improvement caused due to such as risperidone and metoclopramide.51
pituitary tumors. a month.58
treatment with PRL lowering agents like bromocriptin cor- • Patients with moderate elevation of PRL should Prolactinomas on the other hand can be managed
relates better with decrease in serum PRL level than with Prolactinomas (even microadenoma) manifests ear- have multiple serum assays in order to minimize the
lier in females compared with males.47 This earlier presen- by observation, medical therapy, irradiation, or surgery
increase in serum testosterone.39 potential for detecting only transient or physiological (Table 12.4).
tation in females is due to greater symptom burden caused increase in PRL.52
by HPRL in them.48 On contrary in males, presentation is
CLINICAL MANIFESTATIONS OF HPRL While making the diagnosis of HPRL is relatively easy,
Surgery
late and is often in the form of visual deterioration or visual
HPRL is often undiagnosed for the absence of specific field defects caused by pressure over optic chiasm by large difficulty lies in identifying the underlying cause. A system- Indications of trans-sphenoidal surgery59
symptoms. prolactinoma (macroprolactinoma). Decreased libido atic evaluation can therefore help in successful treatment. • Patient cannot tolerate medical treatment
may also be a presentation of prolactinoma in male.48 This TSH, BUN, creatinin, and creatinin clearance should be • Treatment with DA agonists fail to lower serum prolac-
checked to test for hypothyroidism and renal insufficiency.
HPRL in Females late and varied presentation can lead to misdiagnosis of tin level or reduce the size of the adenoma
microprolactinoma in male patients.48 • Surgery should be considered to prevent aggressive
Infertility, oligomenorrhea, amenorrhea, galactorrhea, hot Imaging growth of adenoma during pregnancy in women with
flashes, vaginal dryness, headaches, and visual changes giant lactotroph adenoma (>3 cm)
are the clinical manifestations of HPRL in premenopausal DIAGNOSIS AND MANAGEMENT • Imaging of the hypothalamic–pituitary area is indi-
Surgery is usually causes substantial reduction of
women.41 Approximately 10–20% cases of amenorrhea in OF HPRL cated in HPRL without an identified cause.
• PRL secreting prolactinoma often cause high PRL serum PRL level in patients with prolactinomas.59 In
nonpregnant women are due to HPRL.41
• Hypogonadism: HPRL inhibits the release of GnRH
History levels 53 Magnetic resonance imaging (MRI) with gado- macroadenomas, only 20–50 percent of patients had nor-
linium enhancement provides the best visualization of malized prolactin values after surgery, suggesting incom-
thereby causing decreased secretion of luteinizing • History should include history of pregnancy (physi- plete excision and removal of adenoma tissue.60 Another
hormone (LH) and follicle-stimulating hormone (FSH) ologic HPRL). the sellar area.
shortcoming of surgery is the recurrence of the adenoma
resulting in secondary hypogonadism. • History of medications that can cause HPRL (such as Diagnosis of idiopathic HPRL is made in cases with
(50 percent at 4 years) and HPRL (39 percent at 5 years).60 If
• Levels more than 100 ng/mL–Overt hypogonadism estrogen, neuroleptics, antidepressants, risperidone, normal MRI and no other identifiable secondary cause of
serum PRL concentration is 5 ng/mL or less on the first post-
causing amenorrhea, hot flashes, and vaginal metoclopramide, cimetidine, methyldopa, verapamil, HPRL.
operative day, persistent cure is more likely to be expected.
dryness. reserpine).
• Levels of 50–100 ng/mL–Amenorrhea or oligomenor- • Headache, visual symptoms, symptoms of hypothy-
rhea. roidism.
TREATMENT OF HPRL Radiotherapy
• Levels of 20–50 ng/mL–Short luteal phase of the The treatment of HPRL depends on underlying cause. • Radiation treatment is given to prevent regrowth of a
menstrual cycle due to insufficient secretion of
Physical Examination Offending drug should be discontinued in case of drug- residual tumor after trans-sphenoidal debulking of a
progesterone.42 induced HPRL and appropriate therapeutic alternatives very large macroadenoma.
• Visual field examination to test for a chiasmal should be instituted. An associated or underlying primary • Supervoltage radiation by means of external (linear
Long-term negative health effects of HPRL in women
with amenorrhea include osteopenia and osteoporosis.43 syndrome (e.g. bitemporal field loss). hypothyroidism should be corrected by the replacement cobalt or proton beam therapy) and internal (yttrium
• Galactorrhea: The serum PRL concentration is going • Signs of hypothyroidism or hypogonadism. of thyroid hormone. In case alternatives are not available, as pituitary implants) decreases the size of adeno-
to be normal in almost half of the women with galac- • Breast examination for galactorrhea and gynecomastia. then medical therapy with dopamine (DA) agonists may mas. However, PRL levels normalize many years after
torrhea.44 • Chest wall injury. be warranted (Table 12.3). treatment.61
102 The Infertility Manual Hyperprolactinemia and Infertility 103
Table 12.3. Medical management of HPRL. PRL AND OVARIAN STIMULATION IN IVF
Drug→ Bromocriptine Cabergoline Pergolide In the normal menstrual cycle, in the luteal phase, PRL
Class Antiparkinsonian and DA agonist It is a long-acting selective DA Antiparkinsonian and DA level reaches a high level and changes with the E2 level
receptor agonist, exhibiting high agonist
with a small PRL peak after the E2 peak. Wang et al found
affinity for D2 receptors and low Ergot derivative and a D1
affinity for D1, alpha1 and alpha2 and D2 receptor that in the middle menstrual cycle of healthy women, the
adrenergic, and serotonin receptors Agonist level of PRL with biological activity and immune activity
MOA 1. Activates postsynaptic DA By directly stimulating the D2 recep- Acts as an inhibitor to PRL was markedly increased. This increase was not observed
receptors to inhibit PRL tors of the pituitary lactotrophs it secretion and usually results in women with infertility of unknown cause.67 During preg-
secretions inhibits the synthesis and release in a transient elevation in GH nancy and lactation period, there is increase in PRL level
2. Stimulates DA receptors in of PRL concentration and reduced under the stimulation of estrogen. The increased PRL level
the corpus striatum to improve LH concentration
motor functions on the day of hCG administration is associated with the
Pharmakokinetics 1. 90–96% protein bound is 1. The elimination half-life is It is 90%
increase in the number of oocytes obtained thus positively
completely metabolized 63–69 hours protein bound and excreted affecting fertility rate.68 Similar association is noted with
2. 84.6% excreted in the feces 2. Metabolized mainly by the liver 55% by the kidneys and estradiol levels.69 On the contrary, high PRL level after
and 2.5–5.5% in urine 3. Excretion is 60% fecal, 22% ovulation is detrimental to the outcome of IVF.
renal, and 4% unchanged Thus, the patients with an abnormal PRL level should
Side effects (SE) Hypotension, constipation, Nausea is less common than bro- Increased risk of newly be closely monitored in the controlled ovarian stimulation
nausea, vomiting, dizziness, mocriptine and pergolide55 diagnosed cardiac-valve
to improve the clinical pregnancy rate.
headache, and fatigue regurgitation
Contra-indication 1. Postpartum women with Uncontrolled hypertension Uncontrolled hypertension
coronary artery disease or Cardiovascular disorders CONCLUSION
other severe cardiovascular
• Hyperprolactinemia is a frequent finding in young
conditions
2. Uncontrollable hypertension subfertile women.
Dosing 5-mg capsule or 2.5-mg tablet 0.5–0.25 mg oral tablets 0.025–0.6 mg PO once daily
• Spontaneous conception occurs frequently in HPRL
women, although quite often, time to pregnancy
Twice a day and the initial Administered once or twice a week Can be given once a day appears longer than normal.
dose must be one-fourth of the Patients resistant to bromocriptine • DA agonists restores normal ovarian function and is
maintenance dose to decrease may respond to cabergoline.56 the first-line treatment for most patients wishing to
unpleasant SE
conceive.
Monitoring Blood pressure, renal, liver, and Serum PRL level, blood pressure Blood pressure
hematopoietic functions measurements and assessment of measurements
In peptic ulcer patients, signs and liver function PROBABLE QUESTIONS
symptoms for gastrointestinal Dose adjustment is needed in liver
1. Write a short note on prolactin synthesis.
bleeding must be watched failure
Fig. 12.9: Erectile dysfunction in HPRL.
2. Elaborate the causes of hyperprolactinemia.
3. Effect of hyperprolactinemia on fertility in both sex.
Table 12.4. Treatment of prolactinomas.
4. Diagnosis of hyperprolactinemia.
Medical Surgical secretion. Its role is vital in reproductive function regula- 5. What are the treatment modalities available in man-
Treatment of choice is DA agonist Microadenoma: <1 cm Macroadenoma: >1 cm tion and pubertal maturation.64 Kissipeptin is expressed in agement of hyperprolactinemia.
•• To achieve fertility •• No treatment required for asympto- •• F/U MRI at 6 months, 1-y, 2-y, 5-y Arcuate nucleus (ARC) and Anteroventral periventricular
•• Restore ovarian function matic cases intervals
•• Reduce galactorrhea •• F/u MRI at 1-y, 2-y, 5-y intervals •• Symptomatic cases, e.g.: n
eurological or
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C HA PT E R
13
Luteal Phase Defect 107
Table 13.1. Theories regarding pathogenesis of luteal
phase deficiency.7
Luteal Phase Defect Follicular phase factors Luteal phase factors
•• Insufficient FSH •• Disruption of LH surge
production •• Abnormal Large and
Pratibha Malik, Kamini A Rao
•• Insufficient LH production small cells of
•• Abnormal granulosa cells •• Corpus luteum
Chapter Outline •• Deranged follicular •• Insufficient progesterone
• Normal Luteal Phase development secretion
•• Defective steroidogen- •• Decreased progesterone
esis receptors in endometrium
•• Insufficient estrogen •• Defective hCG production
INTRODUCTION when enzymes of steroidogenesis become functional.
priming of by embryo
Progesterone is an indispensable hormone needed to
A normal luteal phase is essential for the establishment •• Endometrium •• Intrinsic endometrial
maintain pregnancy. •• Reduced estrogen levels defects e.g. Progesterone
and continuation of a normal pregnancy. The existence of
• By tradition, the premature onset of menses indicated resistance
a luteal phase defect in a normal ovulatory cycle is under
that there was a luteal phase deficiency of progester-
research. All stimulated cycles do have a degree of luteal
one production, which was shown to be correctable by
phase defect that needs to be corrected to get good results that the luteal function is normal3 (biochemical Fig. 13.1: Mechanism of LPD in IVF cycles. LPD, luteal phase defect, IVF,
in that cycle. Correction of any known cause is the first exogenous progesterone administration.1 in vitro fertilization.
definition).
step followed by adequate supplementation guided by the
The diagnosis of LPD requires abnormal results in at
type of ovarian stimulation protocol. This chapter provides
Incidence least two consecutive cycles.
an evidence based approach to correcting a luteal phase compromised; exogenous hormone replacement in
defect. • Among normal women, the incidence of a luteal phase the luteal phase is thus mandatory.24,25
defect (LPD) is estimated to be around 8.0%.19 Diagnosis
• In antagonist protocols, when GnRH agonist trig-
NORMAL LUTEAL PHASE • In infertile women, there is a large discrepancy in the • According to the recent ASRM guidelines, there is a lack ger is used to induce final oocytematuration, the
reported incidence of LPD ranging from 3.7 to 20%.5 of reliable diagnostic tests for luteal phase insufficiency. production of P4 and E2 is deficient as there is early
• In the menstrual cycle, the luteal phase is the inter-
• Incidence of LPD in gonadotropin stimulated cycle is ASRM suggests that the use of BBT, urinary LH detection luteolysis. The attempts to rescue deficient luteal
val between the occurrence of ovulation till the
higher and varies from 12 to 20%.10 kits, luteal progesterone levels, endometrial biopsy, and phase after agonist trigger is a subject of lot of recent
establishment of a pregnancy or the onset of menses
other diagnostic studies have not been established, and research.
12–14 days later.1
• After the onset of the endogeneous luteinizing hor- Definitions performance of these tests is not r ecommended.6 • Luteal-phase support (LPS) is the term used to signify
mone (LH) surge, the corpus luteum (CL) is produced the supplementation of medications in ART cycles to
According to American Society of Reproductive Medicine Luteal Phase Defect in IVF Cycles support the process of implantation of the embryo in the
from the ovulated dominant follicle. LH released from (ASRM), LPD is perpetuated to be a result of insufficient
• It is well established that all stimulated assisted repro- endometrium.1 LPS in ART cycles markedly improves
the anterior pitutary stimulates the production of both progesterone production to maintain a normal secretory
duction technology (ART) cycles are having abnormal implantation and hence the pregnancy rates.1,24,25
estrogen and progesterone from CL. Progesterone acts endometrium and allow for normal embryo implantation
luteal phases.8 • To correct the LPD in stimulated IVF cycles, exogenous
on the endometrium to produce glandular and stro- and nidation.6 The origin of this condition could be due to
• In in vitro fertilization (IVF) cycles, the supra physi- hCG or progesterone is administered.
mal changes that are essential for the establishment inadequate estrogen priming of endometrium, faulty pro-
ological levels of ovarian steroids cause marked nega-
and continuation of pregnancy to term. If conception gesterone synthesis from the CL, or inability of the endo-
fails, the CL regresses by undergoing apoptosis caus- metrium to react optimally to ovarian steroid hormones
tive feedback effect on the pituitary, leading to the Color Doppler in LPD
suppression of endogenous LH secretion during the
ing fall in progesterone levels that triggers endometrial (Table 13.1). • In the normal menstrual cycle, the resistence index
whole luteal phase. The excessive suppression of LH
shedding and menstruation. • LPD is defined as (RI) of the preovulatory follicle is elevated, which
secretion in all stimulated cycles disrupts CL function
• The CL, which is dependent on pituitary LH for its sur- A luteal phase shorter than 10 days4 (clinical and hence the progesterone secretion (Fig. 13.1). decreases dramatically following ovulation. Luteal
vival, is rescued by human chorionic gonadotropin definition). • Various studies have clearly proven that there occurs RI further decreases progressively in the luteal phase
(hCG) when conception occurs. hCG produced by the A gap of more than 2 days in endometrial endometrial advancement with endometrium show- till the late luteal phase when it again significantly
implanting embryo has luteotropic effect on the CL histological dating compared to the expected day ing evidence of glandular–stromal dyssynchrony in all increases.
and supports its production of progesterone by upreg- of the cycle2 (historical definition) stimulated cycles of IVF resulting in decreased endo- • In LPD, the mean RI of CL during the luteal phase
ulating StAR and vascular endothelial growth factor A midluteal progesterone value of ≥10 ng/mL metrial receptivity. (RI 0.56 ± 0.04) is notably increased compared to nor-
(VEGF) expression. The placenta takes over proges- (≥32 nmol/L) or the sum of three random samples • In all gonadotropin-releasing hormone (GnRH) ago- mal. The mean RI remains high throughout the luteal
terone production at around 8–10 weeks of gestation ≥30 ng/mL and luteal phase at least 12 days denotes nist and antagonist protocols, the luteal function is phase in LPD group.11
108 The Infertility Manual Luteal Phase Defect 109
Table 13.2. Factors Associated with LPD.6 Table 13.3. Routes and doses of progesterone.14,25–27 • As compared to progesterone, dydrogesterone has Intramuscular progesterone use is related to pain,
better bioavailability and is thus patient friendly. It has tenderness, rash, redness, swelling, and formation
First trimester abortion •• Oral route
Short menstrual cycles has a higher affinity for the progesterone receptors and of sterile abscesses at the site of injection.
Dydrogesterone 30 mg daily in divided doses
Breast feeding so can be used at lower oral doses.28,34 Dydrogesterone Transvaginal use of progesterone can lead to dis-
After full term delivery •• Vaginal route
does not bind to androgenic, estrogenic, glucocorti- charge, vaginal irritation, and hence is associated
Infertility -sustained release capsule: 100 mg to 200 mg two to coid, or mineralocorticoid receptors.34
Premenstrual Bleeding
with poor patient compliance.
three times a day
Eating disorders
• Dydrogesterone is known to have immunomodulatory
-vaginal gel 8%, 90 mg per day properties such as decreasing proinflammatory and
Hypothalmic disorders
Excessive physical actvity •• Subcutaneous route 26,27
increasing anti-inflammatory cytokines in early preg- Spontaneous Ovulatory Cycles
Psychological Stress - 25 mg subcutaneous once daily nancy and has excellent safety and tolerability profile • In spontaneous cycles, LPS has no role to augment
High BMI in pregnancy; both for mother and fetus.
•• Intramuscular route fertility.
Polycystic ovary syndrome (PCOS)
Endometriosis - 50 mg once daily • Two recent RCTs have demonstrated that oral dydro- • A recent Cochrane meta-analysis by Haas et al on the
Extremes of reproductive age - 100 mg alternate days gestrone is as effective as vaginal progesterone for LPS use of progestogen for preventing miscarriage showed
Thyroid disorders in women undergoing IUI and IVF cycles.29,30 no evidence to support routine use of progesterone
Elevated Prolactin levels • Lotus I, a phase III RCT, which compares the role of
GnRh trigger for ovulation to prevent miscarriage in early to mid pregnancy (OR
Ovarian induction with or without gonadotropin- releasing Clomiphene Citrate (CC) oral dydrogesterone versus micronized vaginal pro- 0.99, 95 % CI 0.78–1.24).20 The same meta-analysis
agonists in ART gesterone as luteal support in IVF, suggests that oral
• As compared to gonadotropin cycles in which the found a statistically significant reduction (OR 0.39,
American Society for Reproductive Medicine 20156. dydrogesterone in a dose of 30 mg daily is equivalent
luteal LH concentrations decrease, clomiphene 95 % CI 0.21–0.72) in miscarriage rate after progesto-
to MVP 600 mg in the first trimester.33 This could be
citrate (CC) boosts LH levels, even if GnRH antago- gen administration only in women with recurrent
a boon to millions of women undergoing IVF as oral
• In LPD, the endometrium appears thin and non- nists are coadministered.9 When CC is administered, miscarriage, i.e. three or more consecutive miscar-
route is always patient friendly.
echogenic with increased resistance in spiral arteries it causes prolonged occupancy and depletion of estro- riages20 as comparedto placebo.
• The optimum duration of treatment with progesterone
(RI: 0.72 ± 0.06). This may be the underlying mecha- gen receptors in the hypothalamus due to its long • In threatened miscarriage, there is inadequate evi-
remains controversial.
nism of infertility in some cases of “unexplained” half-life (5 days). This leads to increase in endogenous dence to support the routine use of progestogens
• Progesterone supplementation should be started just
infertility.12 FSH and LH secretion resulting to higher estrogen (E) (whether natural or synthetic).21
after oocyte retrieval.
and P levels in the luteal phase. The ability of CC to • It is advisable to continue progesterone supplemen-
Treatment augment CL function has led to its potential use as a tation until placental progesterone production is In IUI Cycles
• Since the diagnosis of LPD is under question, treatment modality for patients with inadequate luteal adequate, around 8–10 weeks of gestation6 although
phase endogenous P4 secretion.5,7 • Two recent systematic review and meta-analysis con-
treatment of LPD is controversial and is usually
strong evidence is lacking.
• In a meta-analysis by Hill et al, it was concluded cluded that in IUI cycles where gonadotropins were
empirical. • A recent meta-analysis by Byung Chul Jee et al sug-
that there is no role of exogenous progesterone sup- used for superovulation, LPS improved the odds of
• According to recent research, LPD has not been proven gested that the addition of E2 to P for LPS does not
port in patients undergoing ovulation induction with clinical pregnancy and live birth rates.13,22
to be an independent entity causing infertility or recur- improve IVF outcomes in GnRH agonist and antago-
CC alone.13 • A very recent RCT by Biberoglu et al established that
rent miscarriage.6 nist cycles.18
300 mg of intravaginal micronized progesterone
• The first approach is the correction of any basic con-
should be the optimum dosage for LPS in IUI cycles
ditions such as obesity, hypothalamic amenorrhea, Progesterone Safety of Progesterone utilizing gonadotropins in follicular phase.23
endocrine disorders such as PCOS thyroid or prolac-
tin disorders (Table 13.2); and the second approach • Supplementation of progesterone can be given orally, • There is robust evidence of safety of progesterone
vaginally, rectally, subcutaneous (SC), or intramuscu- use in pregnancy as there is no statistically signifi-
is the administration of LPS during luteal phase for In IVF Cycles
ART cycles. larly (IM). Patient preference should be the priority for cant difference in the congenital abnormalities seen
• In unstimulated cycles, no treatment for LPD has been prescription (Table 13.3). in the clinical studies between the newborns of the • Vaginal gel is equivalent to all other vaginal pro
shown to improve pregnancy outcomes.6 mothers who received progesterone and those who gesterone forms in terms of clinical pregnancy
• Two main strategies have been suggested: did not. rates.15,24,25
Oral Progesterone • There is no significant difference between the use of
Ovulation induction improves the follicular dynam- • The adverse effects reported with use of various pro-
ics by producing a healthy preovulatory follicle • Dydrogesterone is retro progesterone with excellent gesterone are: vaginal P4 and IM progesterone for luteal LPS in ART
with resultant increase in progesterone production oral bioavailability. It is a biologically active metabo- Oral progesterone may cause bloating, anxiety/ cycles.16,24,25
during the luteal phase. lite of progesterone and has an antiestrogenic effect depression, somnolence, headache, constipation/ • LPS with either hCG or progesterone after ART results
Luteal phase supplementation with progesterone or on the endometrium, achieving the desired secretory diarrhea, fatigue, irritability, breast tenderness, and in an increased pregnancy rate25 and both are equiva-
hCG in IVF cycles. transformation.32 so on. lent in terms of results.17,24,25
110 The Infertility Manual Luteal Phase Defect 111
hCG citrate; IUI, intrauterine insemination; OHSS, ovarian 12. Kupesic S. Assessment of endometrial receptivity by 25. Van der Linden M, Buckingham K, Farquhar C, Kremer
hyper stimulation syndrome; GnRH, gonadotropin-releas- transvaginal Color Doppler and three-dimensional power JAM, Metwally M. Luteal phase support for assisted
• Due to structural and biological similarities with LH, Doppler ultrasonography in patients undergoing in vitro reproduction cycles. Cochrane Database Syst Rev.
ing hormone; FET, frozen-thawed embryo transfer; MVP,
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micronized vaginal progesterone. 13. Hill MJ, Whitcomb BW, Lewis TD, Wu M, Terry N, DeCherney 26. Lockwood G, Griesinger G, Cometti B, 13 European Centers.
triggering final oocyte maturation. hCG stimulates
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the ovaries to boost production of endogenous pro-
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noninferiority randomized controlled study. Fertil Steril.
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Carreras R, Checa MA. Efficacy of luteal phase support intravaginal micronised progesterone as luteal phase
supplementation is needed for LPS, which leads to an endometrial response to normal hormonal stimulation. support in assisted reproductive technology (ART) cycles:
with vaginal progesterone in intrauterine insemination:
improvement of the reproductive outcomes. Intern J Fertil. 1986;31:368-71. results of a randomised study. J Steroid Biochem Mol Biol.
a systematic review and meta-analysis. J Assist Reprod
• All routes of administration of progesterone are similar 6. Practice Committee of the American Society for Reproductive 2005;97:416-20.
Genet. 2014;31:89-100.
Medicine. Current clinical irrelevance of luteal phase 33. Tournaye H, Sukhikh GT, Kahler E, Griesinger G. A
for ART cycles and there is no universal agreement on 23. Biberoglu EH, Tanrikulu F, Erdem M, Erdem A, Biberoglu
deficiency: a committee opinion. Fertil Steril. 2015;103:e27-32. phase III randomized controlled trial comparing the
the optimal doses, timing of initiation, and duration of KO. Luteal phase support in intrauterine insemination
7. Engman L, Luciano AA. Luteal phase deficiency: What we efficacy, safety and tolerability of oral dydrogester-
use.24 cycles: a prospective randomized study of 300 mg
now know, August 2003; OBG Management. one versus micronized vaginal progesterone for luteal
versus 600 mg intravaginal progesterone tablet. Gynecol
• Use of progesterone in LPS in ART cycles should be 8. Duffy DA, Manzi D, Benadiva C, Maier D, Saunders M, support in in vitro fertilization. Hum Reprod. 2017;32(5):
Endocrinol. 2015;19:1-3
preferred over hCG to avoid OHSS. Nulsen J. Impact of leuprolide acetate on luteal phase function 1019-27.
24. Van der Linden M, Buckingham K, Farquhar C, Kremer
in women undergoing controlled ovarian hyperstimulation 34. Schindler AE, Campagnoli C, Druckmann R, Huber J,
JAM, Metwally M. Luteal phase support for assisted
and intrauterine insemination. Fertil Steril. 2006;85:407–11. reproduction cycles. Cochrane Database Syst Rev. Pasqualini JR, Schweppe KW, et al. Classification and
Abbreviations 9. Tavaniotou A, Albano C, Smitz J, Devroey P. Effect of 2011;10:CD009154. pharmacology of progestins. Maturitas. 2008;61:171-80.
clomiphene citrate on follicular and luteal phase luteinizing
ART, assisted reproduction technology; LPD, luteal phase hormone concentrations in in vitro fertilization cycles
deficiency; CL, corpus luteum; ASRM, American Society stimulated with gonadotropins and gonadotropin releasing
of Reproductive Medicine; LUF, luteinized unruptured fol- hormone antagonist. Fertil Steril. 2002;77:733-7.
licle; P4, progesterone; BBT, basal body temperature; hCG, 10. Fatemi HM. The luteal phase after 3 decades of IVF: what
do we know? Reprod Biomed Online. 2009;19(Suppl. 4).
human chorionic gonadotropin; PCOS, polycystic ovarian
www.rbmonline.com/Article/4331 on web 5 June 2009.
syndrome; IVF, in vitro fertilization; RI, resistence index; 11. Tamura H. Changes in blood-flow impedance of the
COS, controlled ovarian stimulation; FSH, follicular stimu- human corpus luteum throughout the luteal phase and
lating hormone; LH, luteinizing hormone; CC, clomiphene during early pregnancy. Fertil Steril. 2008;90:2334–9.
C HA PT E R
14
Obesity 113
be lowered for obesity.2 Overweight is defined as the indi- Effects of Obesity on Natural Fertility
viduals with BMI’s between 25 and 30 (Table 14.2).
Obesity Body weight influences the initiation of puberty in girls as
well as subsequent natural fertility.
TYPES
1. Menstrual cycle abnormalities
Rajitha The distribution of fat is more important than the location • Disturbance of HPO axis is the key pathophysi-
of the fat. ological factor for menstrual disturbances.
Chapter Outline
• Type 1: Excess of body fat without any particular • Excess weight and abdominal fat, especially cen-
• Definition • Obesity and Reproductive Health
• Types • Management distribution pattern—global obesity. tral/visceral fat, increases the risk of having men-
• Type 2: Excess of subcutaneous fat especially on the strual abnormalities.4,5
trunk and abdomen—android obesity—WHR greater • Childhood obesity leads to the risk of developing
INTRODUCTION 19.3% of combined childhood overweight and obesity than 0.8. these menstrual disturbances.6
after 2010.72 • Type 3: Excess of fat in the abdominal viscera— 2. Ovulatory dysfunction
Obesity is a disease of excess adipose tissue mass that Based on data from the 2007 National Family Health abdominal visceral obesity. • Increased insulin in obese women is a stimulus
lead to many comorbidities. The prevalence of obesity as Survey, the percentage of people of different states of India • Type 4: Excess of fat in the gluteofemoral region— for increased ovarian androgen production.15
a worldwide epidemic has risen drastically over the last who are overweight or obese is shown in Table 14.1.73 gynoid obesity, WHR ratio is less than 0.8. Androgens are aromatized to estrogen in the
two decades. In the US, 2/3rd of women and 3/4th of men periphery owing to excess adipose tissue, which
The distribution of adipose tissue in different ana- exerts a negative feedback on HPO axis and affect-
and nearly 50% of the women of reproductive age are over-
weight or obese.1
DEFINITION tomic locations, specifically intra-abdominal and abdomi- ing gonadotropin production.16
nal subcutaneous fat has more significance than sub- • Insulin induces suppression of sex hormone bind-
Fat cells are adapted to store excess energy efficiently Obesity is derived from the Latin word “Obesus,” which cutaneous fat in the buttocks and lower extremities has ing globulin (SHBG), which decreases gonado-
as triglycerides within widely distributed adipose tissue means “one who has become plump through eating”. It is less s ignificance, which may relate to the fact that intra- tropin secretion due to increased production of
and to be released as stored energy whenever needed defined as a state of excess adipose tissue mass.2 abdominal adipocytes are more lipolytically active than estrogen from the peripheral conversion of andro-
as free fatty acids for use at other sites.2 Excess free fatty In simple daily practice, body mass index (BMI), those from other deposits. gens by adipose aromatase.7-9
acids have a toxic effect on reproductive tissues leading waist to hip ratio (WHR), and waist circumference are the
• Increased adipose tissue leads to production
to cellular damages and chronic low-grade inflamma- parameters used to measure obesity. Other parameters OBESITY AND REPRODUCTIVE HEALTH of adipokines, which directly inhibits ovarian
tory condition.3 This physiologic system, orchestrated used to measure total body fat are anthropometry (skin
function.10,11
through endocrine and neural pathway, permits humans fold thickness), densitometry (under water weighing), Effects on HPO Axis • There is a decrease in early follicular LH pulse
to balance starvation. But in the presence of nutrition computed tomography (CT) or magnetic resonance imag- amplitude, but not frequency in obese women.
Obese women have increased levels of leptin, a cell-
abundance, sedentary lifestyle genetic factor increases ing (MRI), and electric impedance, but their use is limited Women with prolonged folliculogenesis and
signaling protein, produced in adipose tissue and termed
adipose tissue energy stores and leads to adverse health to research purposes. diminished luteal progesterone levels12-14
as adipokine, which may lead to chronic downregulation
conditions. BMI of 30 is most commonly used as a threshold for decreases spontaneous conception.
of its receptor in the brain.
In 1934, Irving Stein and Michael Leventhal described obesity for both men and women. Large scale epidemio- Jain et al found that the amplitude of LH pulsatility was
logic studies suggest that all comorbidities begin to rise significantly decreased, again pointing to a central defect
Evidences
the relation between obesity and infertility, who later
when BMI is greater or equal to 25, suggesting the cutoff to that may be unique to this disease in eumenorrheic obese A large Dutch study by Vander Steegetal of more than 3,000
described the triad of obesity, hirsutism, and infertility.
A minimum fat mass of 22% of total body weight is women.18 women with normal cycles, in which with each BMI point
required for the initiation and maintenance of reproduc- greater than 29 kg/m2, the probability of spontaneous con-
Table 14.1. Prevalence of overweight or obesity in ception declined linearly.17
tive function in both men and women. Obesity impairs Table 14.2. Categories of obesity by body mass index
India and different states.
reproductive potential in the couple, leading to infertil- (BMI) (WHO, 2004).
Category Female (%) Male (%)
ity and subsequent complications in pregnancy and the Category BMI (kg/m2) Effects of Obesity on Assisted
India 16 12%
adverse effects of it on their offspring. Underweight <18.5 Reproduction
Punjab 37.5 30.3
Table 14.1 shows the prevalence of obesity in India. Normal 18.5–24.9
Kerala 34.3 24 1. Effects on the oocyte
The prevalence data from 52 studies conducted in 16 of the Overweight 25.0–29.9
Goa 27 20.8 • Obesity leads to an altered follicular environment,
28 states in India concluded that the prevalence of child- Obesity, grade I 30.0–34.9 with higher level of insulin, triglycerides, and
Tamil Nadu 24.4 19.8
hood and adolescent obesity was higher in north India Obesity, grade II 35.0–39.9 markers of inflammation such as C-RP and lactate
than in south India. There is a significant increase from Andhra Pradesh 22.7 17.6
Obesity, grade III 40.0 in follicular fluid in women undergoing in vitro
the earlier prevalence of 16.3% reported in 2001–2005 to Karnataka 17.3 14 Practice Committee. Obesity and reproduction. Fertil Steril. 2015. fertilization (IVF).19
114 The Infertility Manual Obesity 115
• Obesity is associated with higher dose of gonado- multiple pregnancy rates are unaffected30 in obese
tropin stimulation and longer treatment courses women (BMI >30 kg/m2).
needed for follicular development20,21 and lower 2. Effects on the embryo
oocyte yield and has a higher chance of cycle can- • In vitro leptin has a stimulating effect on human
cellation.22 trophoblastic stem cell growth, and its inhabi-
• Machtinger et al studied the oocyte that failed to tation decreases proliferation and dramatically
fertilize in IVF cycles of morbidly obese women, increases apoptosis.31
described disarrayed meiotic spindles with mis- • Elevated leptin levels in obese women may
aligned metaphase chromosomes.23 decrease the sensitivity of the trophoblastic to its
• Obesity also appears to alter mitochondrial func- effects.
tion and distribution in the oocyte, with clumping
Leary et al showed an abnormal embryogenesis with
throughout ooplasm compared with controls.24
• In a study of high-fat diet fed mice, there was reduced blastocyst survival from women with BMI greater
an increase in the expression of ER stress mark- than or equal to 25 kg/m2. In addition, there was a higher
ers ATF4 and GRP78 and increase granulosa cell triglyceride content, lower glucose consumption, and
apoptosis, thus corelating with increased activat- altered amino acid metabolism, fewer cells in the trophec-
ing transcription factor levels in the follicular fluid toderm in the embryos that reached blastocyst stage com-
of the obese women undergoing IVF.25 Thus show- pared with controls (BMI <24.9 kg/m2).32 Fig. 14.1: Pathogenesis of obesity. LH, luteinising hormone; IR, insulin resistance.
ing an evidence of endoplasmic reticulum (ER)
3. Effects on the endometrium
stress in obese state.
• The diet-induced obese (DIO) mouse study Pathophysiology
• Excess fatty acids obtained from diet can be stored A Central Pathway Through which Leptin Acts
showed that endometrial decidualization that
as triglycerides in adipocytes, when this capac-
is the necessary step for uterine receptivity is Leptin, word is derived from the greek word leptos, to Regulate Appetite and Body Weight
ity is exceeded with continued excess diet, fatty meaning ”thin”. Leptin is discovered in 1994, consists of a
impaired, leading to decrease in implantation • Leptin signals through proopiomelanocortin (POMC)
acids accumulate in other tissues and exerts toxic 167 amino acid peptide. Lep gene is located on chromo-
sites and decrease in the response to hormonal neurons in the hypothalamus to induce increased pro-
effects, termed as lipotoxicity.26
stimulation in the endometrial stromal cells.33 some 7q31.3 and leptin receptor on chromosome: duction of α-MSH, requiring the processing enzyme
• Lipotoxicity plays a role in the development of
• Similar findings were found in a human endome- 1. Functions as a satiety factor PC-1 (proenzymeconvertase 1).
insulin resistance (IR) and chronic low-grade
trial stromal cell line triggered to undergo decidu- • α-MSH acts as an agonist on melanocortin-4 receptors
inflammatory state with high CRP in obese 2. Synthesized in white fat tissue
alization. to inhibit appetite, and the neuropeptide AgRp acts as
women.28 3. Increased blood glucose and insulin levels stimulates
• Abnormal morphology of cumulus–oocyte com- • Decidualization and implantation defects may an antagonist of this receptor.
leptin synthesis
plexes are seen in obese women with elevated negatively affect the placentation process, leading • Leptin is associated with a decrease in the neuropep-
4. Increased leptin level increase metabolic acti -
levels of free fatty acids in the follicular fluid in to pregnancy complications such as stillbirth and tide Y (NPY), a 36 amino acid polpeptide.
vity and reduce food intake and enhances fat meta-
patients undergoing IVF.27 pregnancy-induced hypertension. • Fasting and exercise decrease leptin secretion and
bolism
• High serum leptin levels correlates with high level • Obesity causes implantation failure by affect- increase NPY in the arcuatenucleus. NPY neurons
5. Overweight subjects are resistant to leptin
of leptin in follicular fluid, which affects steroi- ing the preimplantation embryo and physiologi- stimulates food intake, decrease heat production by
6. Leptin is also secreted by the granulosa and cumulus
dogenic pathway in granulosa cells, decreasing cal cross talk between the endometrium and the inhibiting symphathetic nervous activity, and increase
embryo, which is critical for normal implantation. cells and present in follicular fluid
estrogen and progesterone production, which insulin and cortisol production.
• A meta-analysis by Metwally et al in 2008 showed an 7. Identification of ob gene mutation in genetically
may have adverse effects on endometrial receptiv-
obese mice represented major breakthrough in the
ity (ER) and endometrial implantation. A system- increased risk of miscarriage at less than 20 weeks,
field. The product of ob gene is the peptide leptin.
Effects of Leptin in Obesity
atic review of 27 IVF studies showed that there is a with an odds ratio of 1.67 in women with a BMI
10% lower LBR than normal weight women (odds greater than or equal to 25 kg/m2 of both sponta- Ob/ob mouse is homozygous for a mutation of the • Dysregulation of gonadotropin-releasing hormone
ratio (OR) 0.90; 95% confidence interval (CI), neous and assisted reproductive conception.34 ob gene on chromosome 6. (GnRH) secretion
0.82–1.0) in overweight women (BMI >25 kg/m2) • Leptin stimulates proliferation and apoptotic cell 8. The db/dbmouse,is homozygous for a mutation of db • Altered ovarian steroidogenesis
undergoing IVF.29 pathways in vitro and thus has a regulatory role in gene, which encodes the leptin receptor gene and is • Dysregulation of folliculogenesis
• Sallam et al conducted a meta-analysis of 26 IVF remodeling of the human endometrial epithelium.35 located on chromosome 4 • Dysregulation of perifollicular blood flow
studies, which showed that there is a significant • Leptin also modulates ER, so chronic dysregu- 9. Mutations in several other obesity genes cause severe Increase in large fat cells in obese women leads to
reduction in live birth, CPR, and higher cancella- lation of leptin pathway may negatively affect obesity in humans and mice, which is rare (Fig. 14.1 increased production of leptin level, and this high level
tion and miscarriage rates, but implantation and implantation. and Table 14.3). of leptin suggest resistance to action of leptin. Compared
116 The Infertility Manual Obesity 117
Table 14.3. Obesity genes in humans and mice2 Thermal mechanism modification is the first line of management for the ini-
tial 10% of weight loss goal of over 6 months and may also
Gene Gene product Mechanism of obesity In humans In mice • Obesity is associated with increased fat deposits in
include pharmacotherapy or surgery, depending on BMI
Lep (ob) Leptin, a fat derived hormone Mutation prevents leptin from delivering Yes Yes the abdominal area and upper thighs, with increased
risk category (Table 14.4).2
satiety signal; brain perceives starvation waist to hip circumference and also has a characteris-
LepR(db) Leptin receptor Same as above Yes Yes tic scrotal fat deposition.49
• As scrotum remains in closer contact with surrounding Lifestyle Modification
POMC Propiomelanocortin, a precursor of Mutation prevents synthesis of MSH, a Yes Yes
several hormones and neuropeptides satiety signal tissue than in normal weight men there is an increase To reduce pregnancy-related complications and mor-
in intratesticular temperature, which alters spermato- bidity from anaesthesia-related surgical procedures,
MC4R Type 4 receptor for MSH Mutation prevents reception of satiety signal Yes Yes
from MSH
genesis. such as oocyte retrieval, obese women should consider
Genetic mechanism a weight loss (BMI <35 kg/m2). Diet modification, physi-
AgRP Agouti-related peptide, a neuropep- Antagonises α-MSH action at MC4 No Yes
tide expressed in the hypothalamus receptors, a key hypothalamic receptor that cal activity, behavioral and stress management strategies
inhibits eating
• Genetics of obesity is complex. Obesity is probably are the best lifestyle modification programs for weight
due to interaction between multiple genes and several management.61,62
PC-1 Pro hormone convertase 1, Mutation prevents synthesis of Yes No
environmental factors including diet and activity level In anovulatory women, weight loss improves the rates
a processing enzyme neuropeptide, probably MSH
• Klinefelter, PraderWilli, Laurence Moon Biedel syn- of pregnancy. Moderate short term weight loss (approx
Fat Carboxypeptidase E, a processing Same as above No Yes dromes are the genetic or chromosomal conditions,
enzyme
3.1 kg decrease over 140 days) before starting IVF is associ-
which can result in obesity and male infertility. ated with a higher number and percentage of metaphase
TrkB TrkB, a neurotrophin receptor Hyperphagia due to uncharacterized Yes Yes
In humans, with severe early onset obesity, delayed 2 oocytes unrelated to pregnancy outcome.63 There is an
hypothalamic defect
puberty and hypogonadism are seen in leptin deficiency improvement in total sperm count, sperm morphology,
due to mutation in the leptin gene and leptin resistance and increase SHBG and total testosterone with weight
to postmenopausal women, leptin levels are higher in reproduction. These discrepancies are due to the due to mutation in leptin receptor gene.50 reduction in obese men.43 However, to date, there is no
females and in premenopausal women than in males. differences in data acquisition, study population,
strong evidence that preconception weight loss in women
Obesity and male sexual dysfunction
patient lifestyle, and comorbidities. improves IVF-related pregnency outcome,61 and data is
• Obesity is associated with a 1.3-fold relative risk of less clear in men.
Ghrelin • Due to obesity male fertility is adversely affected
through endocrinological, thermal, and genetic mech- erectile dysfunction.52 Weight loss of 7% of total body weight and increased
It is a complex hormone, a 28-amino acid peptide discov- anisms. • Decrease in testosterone levels and elevated levels of physical activity of at least 150 minutes per week of moder-
ered in 1999. several proinflammatory cytokines in obese men lead ate activity such as walking is the current recommendation
Endocrinological mechanism to erectile dysfunction.53 for lifestyle modification of obesity.64,65 Calorie restriction
• Ghrelin stimulates the release of growth hormone. It
is secreted mainly in the stomach, intestine, pituitary, • In obese men, more androgens are converted to estro- • Markers of inflammation are positively associated with of 500–1,000 kcal/day from usual dietary intake should
hypothalamus, kidney, ovary, and testis. gens via aromatization of peripheral fat.36 endothelial dysfunction through disturbed nitric oxide lead to 1–2 pound weight loss per week, with a low calo-
• Stimulates food intake via the NPY pathway. • Gonadotropins concentration may be suppressed by pathway.54 rie diet of 1,000–1,200 kcal/day, achieves a average 10%
• The circulating level of ghrelin is lower in obesity, it is increased negative feedback of estrogens37; conse- decrease in total body weight over 6 months. About 60–80%
reduced with food intake and increased with fasting. quently, serum total testosterone levels are reduced,38 MANAGEMENT may regain weight in 3 years, 75–100% may regain weight
• Ghrelin exerts inhibitory effects on luteal function along with decreased formation of inactive 2-hydroxy in 5 years when lifestyle changes are not maintained.
(Tropea et al., 2007) estogens. The Goal of Therapy
• The biosynthesis of estradiol and progesterone in • Reduced gonadotropin secretion in obese men is due The primary goal of therapy is to improve obesity-related
Medical Treatment
granulosa lutein cells to increased insulin levels leading to suppression of comorbid conditions and reduce the risk of future devel- Patients with a BMI greater than 30 kg/m2 or a BMI greater
SHBG, increased androgen availability for estradiol opment of pregnancy-related complications. Lifestyle than 27 kg/m2 with a persistent comorbidity and for whom
production by adipose aromatase.39-43,51
Effect of Obesity on Male Reproductive System
• There is a decrease in total and bioavailable testoster-
• Incidence of oligozoospermia and asthenozoospermia one levels and inhibin-B concentration45 combined Table 14.4. Guide to select treatment.71
is increased in obese men. with diminished LH pulse amplitude.44 BMI category
• There are various evidences as to show whether • Obese men have a decreased Leydig cell testosterone Treatment 25–26.9 27–29.9 30–35 35–39.9 ≥40
male obesity alters sperm function,41 decreased secretion, with testosterone levels negatively corre-
Diet, exercise, behavior therapy With comorbidities With comorbidities + + +
sperm mitochondrial activity,55,56 increased sperm lated with fasting insulin and leptin levels.46-48
DNA damage,55-59 induced seminal oxidative stress,60 • Obese men have a negative effect on the GnRH secre- Pharmacotherapy With comorbidities + + +
impairs blastocysts development, reduces pregnancy tion by the hypothalamus due to increase in endor-
phin levels. Surgery With comorbidities +
outcome, or increases miscarriage following assisted
118 The Infertility Manual Obesity 119
dietary and physical activity therapy has not been success- surgeries. Surgical procedures are further divided as restric- Table 14.6. Surgical options in obesity.66 4. Describe the role of leptin in reproductive function.
ful is the criteria for the use of pharmacotherapy by the US tive or combined restrictive/malabsorptive procedures. 5. Write a note on gene, environment and lifestyle, and
Restrictive procedures Combined procedures
National Institutes of Health or the European union. Restrictive procedures such as sleeve gastrectomy (SG) human reproduction.
Vertical band gastroplasty Roux-en-Y gastric bypass
The role of pharmacotherapy for obesity is and laparoscopic adjustable gastric band (LAGB) create a 6. Impact of male obesity on infertility.
Adjustable gastric banding Sleeve gastrectomy with
small gastric pouch with staples or a band that fills rapidly 7. Impact of female obesity on infertility.
1. Suppression of appetite via centrally active medica- duodenal switch
to induce early satiety. 8. Semen parameters and hormonal profile in obese fer-
tions that alter monoamine neurotransmitters. Sleeve gastrectomy Implantable gastric stimulation
Bypass of a large section of the small bowel in malab- tile and infertile males.
2. Reduce the absorption of selective macronutrients Intragastric balloon
sorptive procedures resulting in restricting food intake and 9. Contribution of environmental factors to the risk of
from the gastrointestinal tract, such as fat (Table 14.5). Gastric plication
causing malabsorption. Laparoscopic adjustable banding male and female infertility.
Centrally Acting Anorexiant Medications of the stomach along with Roux-en-Y and other forms of
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14. Grenman S, Ronnemaa€ T, Irjala K, Kaihola HL, Gronroos€ 2011;95:1970-4. Endocrinol Metab. 1993;76:1140-6. 2010;93:2222-31.
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abdominal fat cell size and effect of weight reduction. J perspectives. Cardiol Res Pract. 2014;2014:605810. hormones and semen quality in fertile Australian men: damage in infertile patients. Asian J Androl. 2013;15:622-5.
Clin Endocrinol Metab. 1986;63:1257-61. 29. Rittenberg V, Seshadri S, Sunkara SK, Sobaleva S, Oteng- inverse relationship between obesity and sperm output. 60. Tunc O, Bakos HW, Tremellen K. Impact of body mass index
15. Rachon D, Teede H. Ovarian function and obesity— Ntim E, El-Toukhy T. Effect of body mass index on IVF Hum Reprod. 2009;24:1561-8. on seminal oxidative stress. Andrologia. 2011;43:121-8.
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16. Jungheim ES, Moley KH. Current knowledge of obesity’s 30. Sallam HN, Moeity F, Abdel-Baki TN. Effect of obesity with a decrease in leydig cell testosterone secretion in outcomes? A systematic review. Obes Rev. 2014;15:839-50.
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for future research. Am J Obstet Gynecol. 2010;203:525-30. studies. Fertil Steril. 2011;S81 47. Isidori AM, Caprio M, Strollo F, Moretti C, Frajese G, infertility. In: Tarlatzis BC, Bulun SE (Eds). Transforming
17. Van der Steeg JW, Steures P, Eijkemans MJ, 31. Magarinos MP, Sanchez-Margalet V, Kotler M, Calvo JC, Isidori A, et al. Leptin and androgens in male obesity: Reproductive Medicine World-wide. Birmingham:
Habbema JD, Hompes PG, Burggraaff JM, et al. Obesity Varone CL. Leptin promotes cell proliferation and survival evidence for leptin contribution to reduced androgen American Society for Reproductive Medicine; 2013.
affects spontaneous pregnancy chances in subfertile, of trophoblastic cells. Biol Reprod. 2007;76:203-10. levels. J Clin Endocrinol Metab. 1999;84:3673-80. p. 66-73. Proceedings of the The International Federation
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18. Jain A, Polotsky AJ, Rochester D, Berga SL, Loucks T, overweight and obese women display phenotypic and Abd El-Azeem HG, et al. Semen parameters and hormonal Sterility and The 69th Annual Meeting of the American
Zeitlian G, et al. Pulsatile luteinizing hormone amplitude metabolic abnormalities. Hum Reprod. 2015;30:122-32. profile in obese fertile and infertile males. Fertil Steril. Society for Reproductive Medicine; 2013 Oct 12-17; Boston.
and progesterone metabolite excretion are reduced in 33. Rhee JS, Saben JL, Mayer AL, Schulte MB, Asghar Z, 2010;94:581-4. 63. Chavarro JE, Ehrlich S, Colaci DS, Wright DL, Toth TL, Petrozza
obese women. J Clin Endocrinol Metab. 2007;92:2468-73. Stephens C, et al. Diet- induced obesity impairs endometrial 49. Ivel lR. Lifestyle impact and the biology of the human JC, et al. Body mass index and short-term weight change
19. Robker RL, Akison LK, Bennett BD, Thrupp PN, Chura LR, stromal cell decidualization: a potential role for impaired in relation to treatment outcomes in women undergoing
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Russell DL, et al. Obese women exhibit differences in autophagy. Hum Reprod. 2016;31:1315-26. assisted reproduction. Fertil Steril. 2012;98:109-16.
50. Farooqi IS, Wangensteen T, Collins S, Kimber W, Matarese G,
ovarian metabolites, hormones, and gene expression 34. Metwally M, Ong KJ, Ledger WL, Li TC. Does high 64. Wyatt HR. Update on treatment strategies for obesity. J Clin
Keogh JM, et al. Clinical and molecular genetic spectrum
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Med. 2007;356:237-47.
20. Souter I, Baltagi LM, Kuleta D, Meeker JD, Petrozza JC. the evidence. Fertil Steril. 2008;90:714-26. in diabetes—2013. Diabetes Care. 2013;36:S11-66.
51. Hammound AO, Peterson M, Gibson M, Meikle AW,
Women, weight, and fertility: the effect of body mass 35. Tanaka T, Umesaki N. Leptin regulates the proliferation 66. Practice Committee of American Society for Reproductive
Carrell DT. Impact of male obesity on infertility. Fertil
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insemination cycles. Fertil Steril. 2011;95:1042-7. J Mol Med. 2008;22:683-9. bulletin. Fertil Steril. 2008;90:S21-9.
52. Bacon CG, Mittleman MA, Kawachi I, Giovannucci E,
21. Fedorcsak P, Dale PO, Storeng R, Ertzeid G, Bjercke S, 36. Jarow JP, Kirkland J, Koritnik DR, Cefalu WT. Effect of 67. Dixon JB, le Roux CW, Rubino F, Zimmet P. Bariatric
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2004;19:2523-8. 37. Bray GA. Obesity and reproduction. Hum Reprod. up study. Ann Intern Med. 2003;139:161-8. effect of bariatric surgery on type 2 diabetes mellitus. Ann
22. Pinborg A, Gaarslev C, Hougaard CO, Nyboe Andersen A, 1997;12:26-32. 53. O’Brien JH, Lazarou S, Deane L, Jarvi K, Zini A. Erectile Intern Med. 2009;150:94-103.
Andersen PK, Boivin J, et al. Influence of female bodyweight 38. Oliva A, Spira A,Multigner L. Contribution of environmental dysfunction and andropause symptoms in infertile men. 69. Tsur A, Orvieto R, Haas J, Kedem A, Machtinger R. Does
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study of 487 infertile couples. Reprod Biomed Online. 2001;16:1768-76. 54. Seftel A. Male hypogonadism Part 2: etiology, oocyte yield, or embryo quality? J Ovarian Res. 2014;7:116.
2011;23:490-9. 39. Pasquali R. Obesity and androgens: facts and perspectives. pathophysiology, and diagnosis. Int J Impot Res. 70. Reis LO, Zani EL, Saad RD, Chaim EA, de Oliveira LC, Fregonesi
23. Machtinger R, Combelles CM, Missmer SA, Correia KF, Fertil Steril. 2006;85:1319-40. 2006;18:223-8. A. Bariatric surgery doesn’t interfere with sperm quality—A
Fox JH, Racowsky C. The association between severe 40. MacDonald AA, Herbison GP, Showell M, Farquhar CM. 55. Fariello RM, Pariz JR, Spaine DM, Cedenho AP, Bertolla RP, preliminary long term study. Reprod Sci. 2012;19:1057-62.
obesity and characteristics of failed fertilized oocytes. The impact of body mass index on semen parameters and Fraietta R. Association between obesity and alteration of 71. National Heart, Lung, and Blood Institute, North American
Hum Reprod. 2012;27:3198-207. reproductive hormones in human males: a systematic review sperm DNA integrity and mitochondrial activity. BJU Int. Association for the study of obesity: Practice Guide:
24. Igosheva N, Abramov AY, Poston L, Eckert JJ, Fleming TP, with meta-analysis. Hum Reprod Update. 2010;16:293-311. 2012;110:863-7. Identification, Evaluation, and Treatment of Overweight
Duchen MR, et al. Maternal diet-induced obesity alters 41. Palmer NO, Bakos HW, Fullston T, Lane M. Impact of 56. La Vignera S, Condorelli RA, Vicari E, Calogero AE. Negative and Obesity in Adults. Bethesda, MD: National Institutes
mitochondrial activity and redox status in mouse oocytes obesity on male fertility, sperm function and molecular effect of increased body weight on sperm conventional of Health, pub number 00-4084; Oct 2000.
and zygotes. PLoS One. 2010;5:e10074. composition. Spermatogenesis. 2012;2:253-63. and nonconventional flow cytometric sperm parameters. 72. Ranjani H, Mehreen TS, Pradeepa R, Anjana RM, Garg R,
25. Wu LL, Dunning KR, Yang X, Russell DL, Lane M, 42. Teerds KJ, de Rooij DG, Keijer J. Functional relationship J Androl. 2012;33:53-8. Anand K, et al. Epidemiology of childhood overweight &
Norman RJ, et al. High-fat diet causes lipotoxicity responses between obesity and male reproduction: from humans to 57. Rybar R, Kopecka V, Prinosilova P, Markova P, Rubes J. obesity in India: a systematic review. Indian J Med Res.
in cumulus-oocyte complexes and decreased fertilization animal models. Hum Reprod Update. 2011;17:667-83. Male obesity and age in relationship to semen parameters 2016;143(2):160-74.
rates. Endocrinology. 2010;151:5438-45. 43. Hakonsen LB, Thulstrup AM, Aggerholm AS, Olsen J, and sperm chromatin integrity. Andrologia. 2011;43: 73. National Family Health Survey, 2005-06. Mumbai:
26. Sorensen TI, Virtue S, Vidal-Puig A. Obesity as a clinical Bonde JP, Andersen CY, et al. Does weight loss improve 286-91. International Institute for Population Sciences; 2007.
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Acta 2010;1801:400-4. 2011;8:24.
SE C T I O N
3
Male Factor Infertility
C HA PTE R
Semen Analysis 15 Veronica Irene Yuel
Chapter Outline
• Semen • Testing for Immunoassays
• Steps • Other Analysis Parameters
Infertility is on the rise and the male component seems to bulbourethral (Cowper’s) glands and epididymides are also
be paralleling the female counterpart. Therefore, semen there.
analysis remains the mainstay of male fertility assessment.
Accurate prediction of fertility status of male partner needs Sample Collection (WHO 2010)
a standardized semen analysis with the latest and pre-
• Collection of semen sample should be done in a sepa-
cise methods of reference values, so well covered in this
rate and clean room near the laboratory. This limits the
chapter.
exposure of the semen to fluctuations in temperature
and the time between collection and analysis.
INTRODUCTION • Abstinence: A minimum of 2 days and a maximum of
Infertility impacts one of every six couples attempting 7 days of abstinence from sexual activity are a dvocated.
pregnancy or may be even earlier1 out of which, the male For additional samples, if needed, the abstinence
partner contributes in 30–50% of cases and is the sole fac- period should remain constant every time.
tor in up to 20%.2 Semen analysis still remains “THE” test • The man should be given clear written and spo-
which defines the fertility status of a male and parameters ken instructions regarding the method of collection
include sperm concentration, motility, and morphology3; emphasizing that the semen sample should be com-
this, in turn, helps one in monitoring spermatogenesis dur- plete and any loss of any volume of the sample must
ing fertility treatments. However, this test has been shown be reported.
to be ineffective in reliably predicting the fertility status of • Method of collection: Mostly by masturbation and
men. This is mainly due to the varied reference ranges of ejaculated into a clean, wide-mouthed container made
the semen parameters and methods of analysis and prepa- of glass or plastic, non-toxic container labeled with the
ration. This chapter has been specially designed keeping in man’s name, identification number, and the date and
mind the latest and precise methods of reference values. time of collection.
• The container should be then kept between 20 and
37°C.
SEMEN
• Sample collection at home: Sample should be c ollected
Semen has two major components: after all the above precautions and brought to the hos-
• Total number of spermatozoa: This is the total sperm pital laboratory as early as possible maximum within
produced by the testis and hence a marker of testicu- 1 hour of collection.
lar function. Presence of sperms in the testis also is an • Collection of semen in condom: For men collecting
indirect test indicating the patency of ducts. samples by coitus interruptus, special non-toxic con-
• Total fluid volume: Produced by the various accessory doms should be used. Ordinary latex condoms should
glands and is a marker of the secretory activity of the not be used as they are spermicidal.
glands.
Majority of the semen volume, around 90%, comprises
STEPS
secretions from the accessory organs,4 mainly the pros- • Place the sample container on a table or in an incuba-
tate and seminal vesicles. Small contributions from the tor at 37°C to allow liquefaction
126 The Infertility Manual Semen Analysis 127
• Assess liquefaction by a pipette and appearance of the Semen Volume Initial Microscopic Examination in the middle of the row. If 200 spermatozoa are not
semen observed in the five rows of the central grid, continue
• Measure semen volume with a volume pipette Semen volume can be measured directly as follows: • A wet preparation of the homogenous semen sample counting in the rows of the two adjacent grids.
• Measure semen pH (if required) with a pH meter • Collect the sample directly into commercially avail- is used • Assess 200 spermatozoa.
• Prepare a wet preparation for assessing microscopic able modified graduated glass cylinder. • A drop of around 10 µL is placed on a glass slide, which • Tally the number of spermatozoa and rows with a
appearance, sperm motility, and the dilution required • Volume can thus be directly read from the graduations is then covered with a coverslip; a phase-contrast laboratory counter.
for assessing sperm number on the cylinder up to 0.1 mL accuracy. microscope is recommended for the examination • Compare replicate counts to see if they are acceptably
• Assess sperm number • Scan the preparation at a total magnification of 100× close.
Alternatively, semen volume can also be measured (i.e. a combination of a 10× objective lens with a 10×
• Assess sperm vitality (if the percentage of motile cells • Calculate the concentration in spermatozoa per
by weighing the sample in the container in which it is ocular lens). This will detect any mucus strand forma-
is low) mL by the formula number (N) divided by the vol-
collected as follows: tion; sperm aggregation or agglutination; cells other
• Make semen smears for assessing sperm morphology ume in which they were found, i.e. the volume of the
• Make semen dilutions for assessing sperm concentra- • Collect the sample in a pre-weighed, clean, and than spermatozoa, e.g. epithelial cells, “round cells,” total number (n) of rows examined for the replicates,
tion disposable container and isolated sperm heads or tails multiplied by the dilution factor. That is, C = (N/n) ×
• Perform the mixed agglutination reaction (MAR) test • Weigh the container with the sample in it • The preparation should then be observed at a higher (1/20) × dilution factor.
• Assess peroxidase-positive cells (if round cells are • Subtract the weight of the container magnification, i.e. 200× or 400× (i.e. a combination of a • Calculate the total number of spermatozoa per
present) • Semen volume can be then calculated from the weight of 20× or a 40× objective with a 10× ocular), which helps ejaculate which is obtained by multiplying the sperm
• Prepare spermatozoa for the immunobead test the sample, assuming the density of semen to be 1 g/mL5 in the assessment of sperm motility and determina- concentration by the volume of the whole ejaculate.
• Fixing, staining, and assessing smears for sperm (semen density varies between 1.043 and 1.102 g/mL.6 tion of the dilution required for accurate assessment
of sperm number. The lower reference limit for sperm concentration is
morphology Semen volume may be low in conditions such as the 15 × 106 spermatozoa per mL (WHO 2010).
• Assaying accessory gland markers (if required). following: Sperm Count The lower reference limit for total sperm number is
• Obstruction of the ejaculatory duct or congenital bilat- 39 × 106 spermatozoa per ejaculate (WHO 2010).
The total number of sperm in an ejaculate represents
Macroscopic Examination of Semen eral absence of the vas deferens (CBAVD)7,8; sperm count.
Clinical significance: There is a direct correlation of
• Collection problems either erectile dysfunction or loss sperm numbers with fertility potential in terms of both preg-
Liquefaction of a fraction of the ejaculate;
The terms “total sperm number” and “sperm concen-
nancy rates and prediction of pregnancy.7,10,11 Also, the total
tration” are two separate identities. Sperm concentration
Soon after the sample is collected, semen is in semi-solid • Partial retrograde ejaculation; sperm numbers are a direct measure of testicular function.12
refers to the number of spermatozoa per unit volume
coagulum form. It has to be liquefied into a homogenous • Androgen deficiency. Azoospermia: Complete absence of spermatozoa
of semen. It is a function of the number of spermatozoa
form for proper analysis. Within a few minutes, i.e. 15–30 at in an ejaculate is termed azoospermia. A semen sample
Semen volume may be high when there is an active emitted and the volume of fluid diluting them, while total
room temperature, it usually liquefies. If it does not liquefy should be termed “azoospermia” if no spermatozoa are
exudation due to inflammation of the accessory organs. sperm number refers to the total number of spermatozoa
within 30 minutes, wait for another 30 minutes. If still liq- found in the sediment of a centrifuged sample.13 However,
Lower reference limit—the lower reference limit for in the entire ejaculate. This is obtained by multiplying the
uefaction has not occurred, continuous gentle shaking of centrifugation at 3,000 g for 15 minutes does not pellet all
semen volume is 1.5 mL8 (WHO 2010). sperm concentration by the semen volume.
the sample container on a two-dimensional shaker, either spermatozoa from a sample14; and after centrifugation,
Determination of sperm number consists of the
at room temperature or in an incubator at 37°C, produces motility, and concentration may also not be correct.15
Semen pH following:
a homogeneous sample.
pH is usually measured after the semen sample has lique- • Mix the sample well and examine a drop of this lique- Sperm Motility
fied and within a maximum of 60 minutes. pH paper in the fied semen on a glass slide under a coverslip. This will
Semen Viscosity determine the appropriate dilution.
Sperm motility in a semen sample should be assessed once
range of 6.0–10.0 should be used for measuring the pH.
liquefaction of the sample is complete preferably within
• The viscosity of a semen sample is assessed by intro- • Mix the semen and prepare dilutions with fixative.
• Stir the semen sample to make it homogenous 30 minutes of ejaculation. The steps include the following:
ducing a glass rod into the sample and observing the • Load the hemocytometer chamber with spermatozoa
• Put a drop of semen evenly onto the pH paper
length of the thread that forms upon withdrawal of and allow them to settle. • Mix the semen sample well
• Wait for the color of the semen drop area to change
the rod. It is recorded as abnormal when the length of • Assess the samples after 15 minutes. • Remove an aliquot of semen immediately once mixed
uniformly
the thread exceeds 2 cm. • Examine the hemocytometer with phase-contrast thoroughly
• Match the color with the pH paper selected to read the
• Alternatively, viscosity can also be estimated by pipet- microscope at 200× or 400×. • Remix the semen sample before removing a replicate
pH and draw your inference.
ting the sample into a (approximately 1.5 mm diameter, • Count 200 spermatozoa in each replicate. aliquot
wide bore) disposable pipette, allowing the semen to Reference value: According to the WHO 2010 criteria, • First assess the central grid of one side of the improved • For each aliquot, make a wet preparation around
drop by gravity and observing the length of any thread. pH of 7.2 is the reference value and pH less than 7.0 with Neubauer chamber, row by row. 20 µm deep
A normal sample flows out of the pipette in small dis- low volume and low sperm count may be associated with • Count at least 200 spermatozoa have been observed • Wait for the sample to stop drifting
crete drops. A viscous semen sample will form a thread ejaculatory duct obstruction or CBAVD.9 However, a high and a complete row has been examined. Counting • Examine the slide with phase-contrast optics at 200× or
more than 2 cm long. pH may be there naturally and is not of much significance. must be done by complete rows ensuring not to stop 400× magnification
128 The Infertility Manual Semen Analysis 129
• Assess 200 spermatozoa per aliquot for the percentage Hypo-osmotic swelling swelling test—this test in the ejaculate by the percentage of normal forms. A • Calculate the percentage of motile spermatozoa with
of different motile categories. is based on the principle that only cells with intact direct correlation has been established between various particles attached
membranes, i.e. live cells will swell in hypotonic solutions, percentages of normal sperm morphology and the preg- • Record the class (IgG or IgA) and the site of binding of
Grading of sperm motility (WHO 2010): Grading of
signifying that sperms that are immotile but living which nancy rates both in vivo and on vitro.19 the latex particles to the spermatozoa (head, midpiece,
motility needs to be simple that distinguishes sperma-
tozoa with progressive motility (PR) or non-progressive swell when dipped in a hypertonic solution and thus can principal piece).
motility (NP) from those that are immotile. The motility of be identified from a dead sperm as follows: Presence of Non-sperm Cells
Reference value: More than 50% motile spermatozoa
each spermatozoon is graded as follows: • Thaw the frozen swelling solution and stir it well. This includes counting of cells other than the sperm and with adherent particles is significant (WHO 2010).
• PR: Spermatozoa moving actively, either linearly or in • Warm 1 mL of swelling solution. is suggestive of testicular damage and inflammation Passage of the sperm through the cervical mucus bar-
circles, regardless of speed. • Mix the semen sample well. of accessory glands or efferent ducts. Non-sperm cells rier is significantly impaired when 50% or more of the
• NP: Patterns of movement but no forward progression. • Pipette out 100 µL of semen, add to the swelling solu- include the following: round cells, epithelial cells, iso- motile spermatozoa have antibody bound to them, thus
• Immotility (IM): No movement. tion, and mix it gently. lated sperm head or tail, and leukocytes. The prevalence affecting in vivo fertilization. However, if the particles are
• Incubate at 37°C for 5 minutes, then transfer a 10-µL ali- of round cells as to the number of spermatozoa can be found bound to the tail tip, fertilization is not affected and
Reference limit: The lower reference limit for total assessed from slides or during the estimation of peroxi-
otility (PR + NP) is 40% (WHO 2010). The lower reference
m quot to a clean slide, and cover with a 22 mm × 22 mm is a common finding among fertile men.20
coverslip. dase-positive cells.
limit for PR is 32% (WHO 2010). 1. The immunobead assay: This is a more accu-
Clinical significance: Sperm motility has been directly • Examine the slide with phase-contrast microscope at
200× or 400× magnification.
TESTING FOR IMMUNOASSAYS rate method of detecting antisperm antibodies on
associated with fertility and pregnancy rates.11,16 the sperm, though more elaborate and time con-
• Tally the number of unswollen (dead) and swollen Adherance of sperm to each other in an ejaculate indi-
suming.
Sperm Vitality (live) cells. cates the presence of antisperm antibody in the sample.
2. In the direct immunobead test, beads coated with
• Evaluate 200 spermatozoa and count the average per- Sperms may adhere head to head, head to tail, or tail to
Sperm vitality is a useful tool for assessing the membrane covalently bound rabbit antihuman immunoglobu-
centage of vital spermatozoa. tail. Also, sperm antibodies may be there in the semen
integrity of the sperm cell. It, thus, is an efficient means of lins against IgG or IgA are mixed directly with washed
without the presence of any agglutination of sperms.
differentiating between a live but immotile sperm and a Lower reference limit: The lower reference limit for spermatozoa. The binding of beads with antihuman
Two classes of anti-sperm antibodies are found—
dead sperm. Test used for assessing the membrane stabil- vitality (membrane-intact spermatozoa) is 58% (WHO IgG or IgA to motile spermatozoa indicates the pres-
ity are dye exclusion test and hypo-osmotic swelling test. immunoglobulin G (IgG) and immunoglobulin A (IgA).
2010). ence of IgG or IgA antibodies on the surface of the
The dye exclusion method is based on the principle IgM being a heavier molecule due to its size is not found
Clinical significance: The presence vital but immotile in the semen. Two types of immunoassays are there to spermatozoa.
that damaged plasma membranes allow entry of mem- cells are indicative of structural defects in the flagellum17;
brane-impermeant stains. This includes eosin–nigrosin detect these antibodies—immunobead assay and sperm Reference value: 50% motile spermatozoa with adher-
a high percentage of immotile and non-viable cells suggest MAR test.
test, which forms the basis of this test. ent particles. 50% or more of the motile spermatozoa with
epididymal pathology.18
• MAR test—the mixed agglutination reaction (MAR) adherent particles is diagnostic of immunological infertil-
Procedure test is a sensitive, easy, and cost-effective screening test ity (WHO 2010).
Sperm Morphology
• Mix the semen sample well. In the MAR test, a “bridging” antibody (anti-IgG or
• Remove a 50-µL aliquot of semen and mix with an Determination of sperm morphology comprises the fol- anti-IgA) is used to bring the antibody-coated beads
equal volume of eosin–nigrosin suspension and wait lowing steps: into contact with unwashed spermatozoa in semen OTHER ANALYSIS PARAMETERS
for 30 seconds. bearing surface IgG or IgA Certain other procedures are there which may not be a
• Prepare a smear of semen on a slide. Air-dry, fix, and
• Make a smear on a glass slide and allow it to dry in air. • The direct IgG and IgA MAR tests are performed by part of routine semen analysis but definitely are of impor-
stain the slide.
• Examine immediately after drying, or later after mixing fresh, untreated semen separately with latex tance in diagnostic and research purposes. A detailed
• The use of the Papanicolaou, Shorr, or Diff-Quik stain
mounting with a permanent non-aqueous mounting particles (beads) or treated red blood cells coated with description of these procedures is beyond the scope of this
(commercially available) is recommended.
medium. human IgG or IgA
• Examine the slide with brightfield optics at 1,000× chapter, hence only enumerating those tests.
• Examine the slide with brightfield optics at 1,000× • The suspensions are added with a monospecific anti-
magnification and oil immersion. magnification with oil immersion. 1. Multiple sperm defect indices—morphologically
human IgG or antihuman IgA. The formation of mixed
• Tally the number of stained (dead) or unstained (vital) • Assess approximately 200 spermatozoa for the percent- abnormal spermatozoa defects, an assessment of the
agglutinates between particles and motile spermato-
cells. age of normal forms or of normal and abnormal forms. morphological abnormalities may be more useful
zoa indicates the presence of IgG or IgA antibodies on
• Evaluate 200 spermatozoa. Calculate the percentages The lower reference limit for normal forms is 4% (WHO the spermatozoa than a simple evaluation of the semen. Three indices
of vital cells from the slide. 2010). • Score only motile spermatozoa and determine the per- are as follows:
Lower reference limit: The lower reference limit for The total number of morphologically normal sperma- centage of motile spermatozoa that have two or more • Multiple anomalies index (MAI)21
vitality (membrane-intact spermatozoa) is 58% (WHO tozoa in the ejaculate is of biological significance. This is latex particles attached • Teratozoospermia index (TZI)22
2010). obtained by multiplying the total number of spermatozoa • Assess 200 motile spermatozoa • Sperm deformity index (SDI).23
130 The Infertility Manual Semen Analysis 131
These indices have been correlated with fertility in vivo 7. van der Steeg JW, Steures P, Eijkemans MJ, F Habbema JD, 21. Jouannet P, Ducot B, Feneux D, Spira A. Male factors and oxygen species production, sperm morphological defects,
(MAI and TZI)24 and in vitro (SDI),23 and may be useful in Hompes PG, Kremer JA, et al. Role of semen analysis in the likelihood of pregnancy in infertile couples. I. Study of and the sperm deformity index. Fertil Steril. 2004;81:349-54.
sub-fertile couples. Fertil Steril. 2011;95:1013-9. sperm characteristics. Int J Androl. 1988;11:379-94. 24. Slama R, Eustache F, Ducot B, Jensen TK, Jørgensen N,
assessments of certain other conditions.25
8. Daudin M, et al. Congenital bilateral absence of the vas 22. Menkveld R, Wong WY, Lombard CJ, Wetzels AM, Thomas Horte A, et al. Time to pregnancy and semen parameters:
2. Panleukocyte (CD45) immunocytochemical deferens: clinical characteristics, biological parameters, CM, Merkus HM, et al. Semen parameters, including WHO a cross-sectional study among fertile couples from four
staining—this is detection of the peroxidase negative cystic fibrosis transmembrane conductance regulator gene and strict criteria morphology, in a fertile and subfertile European cities. Hum Reprod. 2002;17:503-15.
polymorphonuclear leukocytes, which can only be mutations, and implications for genetic counseling. Fertil population: an effort towards standardization of in-vivo 25. Auger J, et al. Sperm morphological defects related to
detected by immunocytochemical means. This stain- Steril. 2000;74:1164-74. thresholds. Hum Reprod. 2001;16:1165-71. environment, lifestyle and medical history of 1001 male
ing helps in differentiating between leukocytes and 9. Weiske WH, Sälzler N, Schroeder-Printzen I, Weidner W. 23. Aziz N, Saleh RA, Sharma RK, Lewis-Jones I, Esfandiari N, partners of pregnant women from four European cities.
Clinical findings in congenital absence of the vasa deferentia. Thomas AJ Jr, et al. Novel association between sperm reactive Hum Reprod. 2001;16:2710-7.
germ cells.
Andrologia. 2000;32:13-8.
3. Computer-aided semen analysis (CASA) 10. Slama R, et al. Time to pregnancy and semen parameters:
4. Measurement of reactive oxygen species a cross-sectional study among fertile couples from four
5. Assessment of acrosome reaction European cities. Hum Reprod. 2000;17:503-15.
6. Assessment of sperm chromatin. 11. Larsen L, Scheike T, Jensen TK, Bonde JP, Ernst E,
Hjollund NH, et al. Computer-assisted semen analysis
parameters as predictors for fertility of men from the
PROBABLE QUESTIONS general population. The Danish First Pregnancy Planner
Study Team. Hum Reprod. 2002;15:1562-7.
1. Elaborate WHO 2010 guidelines on semen analysis 12. Andersen AG, Jensen TK, Carlsen E, Jørgensen N,
and how are they different from previous guidelines? Andersson AM, Krarup T, et al. High frequency of sub-
2. What is the composition of semen? optimal semen quality in an unselected population of
young men. Hum Reprod. 2000;15:366-72.
3. Write a note on semen collection.
13. Eliasson R. Basic semen analysis. In: Matson P (Ed).
4. Enumerate tests for detection of antisperm antibodies. Current Topics in Andrology. Perth: Ladybrook Publishing;
5. Discuss the macroscopic examination of semen. 2003. pp. 35-89.
6. Elaborate HOS test and its clinical significance. 14. Corea M, Campagnone J, Sigman M. The diagnosis of
7. Define leucocytospermia and test to differentiate azoospermia depends on the force of centrifugation. Fertil
round cells on microscopic examination. Steril. 2005;83:920-2.
15. Cooper TG, Hellenkemper B, Jonckheere J, Callewaert N,
Grootenhuis AJ, Kersemaekers WM, et al. Azoospermia:
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Health Stat. 2005;23:25. 17. Chemes HE, Rawe YV. Sperm pathology: a step beyond
2. Thonneau P, Marchand S, Tallec A, Ferial ML, Ducot B, descriptive morphology. Origin, characterization and
Lansac J, et al. Incidence and main causes of infertility in fertility potential of abnormal sperm phenotypes in
a resident population (1,850,000) of three French regions infertile men. Hum Reprod Update. 2003;9:405-28.
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3. Deveneau NE, Sinno O, Krause M, Eastwood D, Sandlow JI, producing ejaculates characterized by high levels of
Robb P, et al. Fertil Steril. 2014;102(6):0015-282. dead sperm and altered seminal plasma factors consistent
4. Elzanaty S, Richthoff J, Malm J, Giwercman A. The impact with epididymal necrospermia. Fertil Steril. 2004;81:
of epididymal and accessory sex gland function on sperm 1148-50.
motility. Hum Reprod. 2002;17:2904-11. 19. Garrett C, Liu DY, Clarke GN, Rushford DD, Baker HW.
5. Auger J, Kunstmann JM, Czyglik F, Jouannet P. Decline in Automated semen analysis: “zona pellucida preferred”
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6. Cooper TG, Brazil C, Swan SH, Overstreet JW. Ejaculate 2003;18:1643-9.
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C HA PT E R
16
Evaluation of Sperm Damage 133
Table 16.1. Factors contributing to sperm damage.6,20,33–36 1. ROS generated by leucocytes during male reproduc-
tive tract infections24
Evaluation of Sperm Damage Intrinsic factors
•• Protamine
Extrinsic factors
•• Elevated testicular temperature
2. ROS due to electromagnetic radiation including heat25
deficiency (e.g. use of hot baths, saunas, or radio frequency radiation in the mobile phone
•• Mutations affecting down-filled blankets, laptop range26
Chandrika Kulkarni
deoxyribonucleic computers, and prolonged peri- 3. ROS produced by redox cycling metabolites or xeno-
acid (DNA) com- ods of driving) biotics such as catechol estrogens or quinones27
Chapter Outline paction •• Cancer and radiation exposure
• Sperm Chromatin Structure • Tests for Assessment of DNA Damage •• Gonadotoxins
4. Advanced age—the DNA integrity of gametes pro-
• Mechanisms of Sperm Damage • Treatment •• Varicocele duced at older age is decreased.28
• Effects of DNA Damage • Selective Isolation •• Other causes: 5. Cigarette smoking—smokers have a high sperm DNA
• Indications for Testing
High fever and infections, air damage compared to non-smokers.29,30 Also, progeny
pollution, cigarette smoking, of smokers have higher risk of childhood cancer.31,32
alcohol, and advanced age
INTRODUCTION the strong disulfide crosslinks between the cysteine
molecules.11 EFFECTS OF DNA DAMAGE (TABLE 16.1)
• Sperm deoxyribonucleic acid (DNA) damage results in • The compact structure of sperm nucleus probably
decreased fertility and recurrent miscarriage. • These breaks serve the purpose of giving the DNA tem- • Sperm DNA damage is associated with increased infer-
protects its genome during movement through the porary respite from the stress of torsion. tility rates and also poor fertilization rates and altered
• Sperm DNA damage is due to various external and
genital tract.12 • Thereafter, these DNA breaks are repaired by the embryo cleavage during in vitro fertilization (IVF).22,37–39
internal factors.
• The sperm DNA, as already stated, predominantly • Sperm DNA damage may be overcome by the oocyte’s
• Treatment of amenable factors and selective sperm internal mechanisms (topoisomerase II) of the sperm
contains protamines, which form 85% of the nuclear capacity to repair. This repair could be successful,
isolation improves outcome. nucleus.
proteins while the rest 15% is made up of histones.13 unsuccessful, or partially successful leading to a nor-
• Semen analysis is a preliminary test used for the • In case of non-repair of the DNA structure, there
• The histones are associated with genes at the nuclear
assessment of male infertility. may be persistent DNA fragments in mature sperma- mal pregnancy, failed fertilization/implantation, or
periphery and telomeres. These genes may be involved
• However, the information derived from semen analy- tozoa. miscarriage respectively.40
in fertilization and early embryonic development.14
ses may not always be accurate.1 • Higher sperm DNA damage is associated with lower
• The compact chromatin structure is disturbed when
• Conventional semen analysis may not indicate the Abortive Apoptosis21 live birth rates following IVF.5,41
there are increased histones (>15%). This makes the
fertilization potential of the sperm.2,3 • A poor DNA sperm integrity is associated with signifi-
nuclear DNA vulnerable to external stresses.12 • Apoptosis (programmed cell death) is a mechanism
• Men with normal semen profiles are not necessarily cant increase in miscarriage rate,42 childhood cancers
• There is an increased sperm histone to protamine ratio for elimination of inappropriate sperms.
fertile, which can be due to abnormal sperm DNA.4 such as leukemia and conditions such as autism.31,43,44
in infertile men.15 • The ability of sperms to undergo complete apoptosis
• Sperm DNA damage contributes to around 80% of • However, according to ASRM Committee report 2013,
• The human spermatozoan is more complicated in that gradually decreases during later stages of spermato-
couples diagnosed with unexplained infertility.5 “existing data do not support a consistent relationship
it has two types of protamines (P1, P2) when compared genesis.
• A normal sperm DNA is required for the development between abnormal DNA integrity and reproductive
to other mammals which have only one type (P1).16,17 • Sperms may sometimes undergo a form of apoptosis
of embryo as well as the correct transmission of hered- outcomes.”45
• An altered ratio of the two protamines (P1:P2) is also wherein the DNA in the nucleus gets fragmented but
itary information.6,7
associated with fertility problems.18,19 they can still differentiate in mature forms with ability INDICATIONS FOR TESTING
SPERM CHROMATIN STRUCTURE to fertilize.
MECHANISMS OF SPERM DAMAGE Prolonged idiopathic infertility
• The sperm chromatin is tightly bound unlike the chro- Oxidative Stress
matin in somatic cells.8,9 Low fertilization rates or bad quality embryos in in vitro
• Reactive oxygen species (ROS) may be the main rea- fertilization (IVF)
• The predominant nuclear proteins in sperms are pro-
son for sperm DNA damage during transit through Implantation failure following IVF
tamines and histones in somatic cells.10
epididymis.20 Repeated abortions
• The histones in the sperm chromatin are initially
• ROS leads to stimulation of various protease enzymes Prolonged exposure to toxic environmental conditions
replaced by transitional proteins and later by prota- affecting fertility
mines during spermatogenesis.10 involved in the cleavage of DNA such as caspases and
Conventional seminal parameters found below the
• Highly organized loops called toroids are formed nucleases.22 reference ranges
from tightly bound DNA strands and protamine mole- Defective sperm condensation20 • ROS-led oxidative damage causes impaired develop-
Advanced male partner age
cules.10 ment of the embryo.23
• In the elongating spermatids, double-stranded (ds) Varicocele patients
• The sperm nucleus is compact and transcriptionally DNA breaks are introduced during the replacement of These are some of the many reasons for the production of Cancer patients
inert because of the smaller size of protamines and histones with protamines. oxidative stress in the male genital tract: (Courtesy: Evgeni et al.11)
134 The Infertility Manual Evaluation of Sperm Damage 135
TESTS FOR ASSESSMENT Tests for Sperm Chromatin Chromomycin A3 (CMA3)49 • DNA fragmentation index (% DFI) is calculated using
the ratio of red fluorescence to the total fluorescence
OF DNA D
AMAGE46 Packaging Defects • CMA3 is a guanine cytosine-specific fluoroch- (red + green).
rome. • DFI of greater than or equal to 25–27% has poor
• Tests for sperm chromatin packaging defects are as f ollows: Aniline Blue Staining • CMA3 competes with protamines for association with
Aniline blue staining prognosis.54
Toluidine blue staining • The histone rich immature sperms take up the DNA.
Chromomycin A3 (CMA3)
blue stain with aniline dye, whereas mature • The higher intensity of CMA3 staining indicates lesser Acridine Orange Test (AOT)
s permatozoa which are protamine rich do not take protamine content of sperm nucleus.
• Tests for sperm DNA integrity are as follows: • It is a simplified form of SCSA.
up any stain. 47
Terminal deoxynucleotidyltransferase (Tdt)-mediated • Fluorescing spermatozoa are counted using the micro-
deoxyuridine triphosphate (dUTP) nick end
Tests for Sperm DNA Integrity scope.
labeling (TUNEL) Toluidine Blue Staining48 • Sperm samples are first treated with citric acid and
COMET (Single-Cell Gel Electrophoresis)
COMET • Toluidine blue easily binds to the phosphate residues later stained with acridine orange.55
Sperm chromatin structure assay (SCSA) of sperm DNA in nuclei with impaired DNA. • Principle—fragmented DNA moves rapidly toward the • As with SCSA, sperms with dsDNA are green and
Acridine orange test (AOT) • There is a metachromatic shift from light blue to anode of an electrophoretic field in agarose gel.46 sperms with ssDNA are red under fluorescent micro-
Sperm chromatin dispersion (SCD) test. purple-violet color. • The comet head is formed by the intact mature DNA, scope.55
while the fragmented DNA represents the tail. • The limitations of this test are that the fluorescence
• The larger the amount of DNA, the greater the fluores- fades rapidly and color is sometimes indistinct.56
Method Principle Advantage Disadvantage cent intensity.50
COMET (single-cell gel Faster rate of migration •• Very sensitive technique •• Decreased accuracy • The neutral comet assay detects double-stranded (ds) Sperm Chromatin Dispersion—SCD Test
electrophoresis) of smaller fragmented •• Requires only a few number •• Lack of standardization DNA and is much more sensitive to identify DNA dam- (Halo Sperm Assay)
deoxyribonucleic acid of cells
age related to infertility.
(DNA) toward anode in •• Degree of DNA damage • Principle—intact DNA loops around the nucleus when
an electrophoretic field in individual cell can be • The alkaline comet assays detects single-stranded (ss)
embedded in agarose give a characteristic ‘halo’ appear-
analyzed DNA breaks.
ance due to the release of chromatin from proteins.
• The advantage of comet assay is that it requires only a
TUNEL Quantifies the incor- •• Can simultaneously detect •• Degree of DNA damage within a • It can be easily analyzed using fluorescence or bright
Terminal deoxynucle- poration of dUTP at single- and double-strand cell cannot be quantified few number of cells and the degree of DNA damage in field microscopy.
otidyltransferase (Tdt)- double-stranded (ds) breaks unlike comet assay •• Only reveals the number of cells individual cell can be analyzed.46 • Sperm DNA fragmentation as reported by the SCD test
mediated deoxyuridine DNA breaks in a reaction within a population with DNA • Decreased accuracy and lack of standardization pre-
triphosphate (dUTP) nick catalyzed by the enzyme damage are negatively correlated with fertilization rate and
cludes its routine usage.46 embryo quality in IVF/intracytoplasmic sperm injec-
end labeling TdT
tion (ICSI) but not with live birth rates.57
Sperm chromatin DNA denaturation follow- •• SCSA gives DNA fragmen- •• Requires expensive flow cytom- TUNEL
structure assay (SCSA) ing heat or acid treatment tation index (% DFI) eter
is determined by measur- •• Extensively used parameter •• Requires highly skilled person- • Principle—terminal deoxynucleotidyl transferase TREATMENT
ing the metachromatic •• Simple and faster method nel (TdT)-mediated deoxyuridine triphosphate (dUTP)
shift from green fluores- of analysis nick end labeling (TUNEL) quantifies the incorpora-
Modifiable Factors in the Treatment
cence (acridine orange tion of dUTP at dsDNA breaks in a reaction catalyzed of Oxidative Stress Causing DNA
intercalated into dsDNA)
to red fluorescence (sin-
by the enzyme TdT.47 Fragmentation
gle stranded DNA) • dUTP can be detected by flow cytometry, fluorescence
microscopy, or light microscope. Medical Life style Dietary
Acridine orange test Microscopic method of •• Simple method •• Indistinct color factor change change Supplement
(AOT) SCSA which relies on •• Does not require expensive •• Rapidly fading fluorescence
• TUNEL assay detects both ssDNA and dsDNA.51
visual interpretation of flow cytometry • TUNEL value of greater than or equal to 36% is associ- •• Treat- •• Smoking •• Healthy diet •• Vitamin C
fluorescing spermatozoa ated with poorer outcomes.52,53 ment of cessation •• Increase •• Vitamin E
leucocyto- •• Avoid testicu- in fruits/ •• Mixed
Sperm chromatin Sperm with intact DNA •• Easy method •• Low number of Spermatozoa spermia lar heat vegetables antioxidants
dispersion produce a dispersion •• Commercial kits available analyzed Sperm Chromatin Structure Assay (SCSA) •• Surgical •• Avoid and
SCD test halo as a result of the •• Observer variation repair of testicular sources of
• Principle—acridine orange (AO) dye fluoresces green
chromatin released from varicocele mobile phone antioxidants
proteins with dsDNA and red with ssDNA after denaturation of radiation •• Weight loss
Analyzed using fluo- DNA with heat or acid46 •• Avoid heavy/
rescence or bright field • Flow cytometer is used to measure the red–green toxic metals
microscopy (Courtesy: Wright et al.58)
fluorescence.
136 The Infertility Manual Evaluation of Sperm Damage 137
Cause-Specific Approach • Clinical pregnancy rates with ICSI are higher than that in male infertility and comparisons with other techniques. vulnerability: from basic science to clinical applications—a
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Reprod. 2008;23:2663-8. 2004;81:965-72. 68. Niu ZH, Shi HJ, Zhang HQ, Zhang AJ, Sun YJ, Feng Y. Sperm
73. Zini A, Blumenfeld A, Libman J, Willis J. Beneficial effect
39. Lewis SEM, Simon L. Clinical implications of Sperm DNA 54. Henkel R, Kierspel E, Hajimohammad M, Stalf T, chromatin structure assay results after swim-up are related
Hoogendijk C, Mehnert C, et al. DNA fragmentation only to embryo quality but not to fertilization and pregnancy of microsurgical varicocelectomy on human sperm DNA
damage. Hum Fertil. 2010;13:201-7. integrity. Hum Reprod. 2005;20:1018-21.
40. Broer SL, Mol B, Dolleman M, Fauser BC, Broekmans FJ. of spermatozoa and assisted reproduction technology. rates following IVF. Asian J Androl. 2011;13:862-6.
Reprod Biomed Online. 2003;7:477-84. 69. Chi HJ, Chung DY, Choi SY, Kim JH, Kim GY, Lee JS, et al. 74. Werthman P, Wixon R, Kasperson K, Evenson DP. Signi-
The role of anti-Mullerian hormone assessment in assisted ficant decrease in sperm deoxyribonucleic acid frag-
55. Spano M, Cordelli E, Leter G, Lombardo F, Lenzi A, Integrity of human sperm DNA assessed by the neutral
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Gandini L. Nuclear chromatin variations in human comet assay and its relationship to semen parameters and
Gynecol. 2010;22:193-201. 1800-4.
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41. Frydman N, Prisant N, Hesters L, Frydman R, Tachdjian G,
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Cohen-Bacrie P, et al. Adequate ovarian follicular status
assay. Mol Hum Reprod. 1999;5:29-37. 70. Dar S, Grover SA, Moskovtsev SI, Swanson S, Baratz A, The assessment of oxidative stress in infertile patients with
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Ayhan A. A logistic regression model including DNA
Steril. 2008;89:92-7. status and morphology of spermatozoa for prediction of
42. Robinson L, Gallos ID, Conner SJ, Rajkhowa M, Miller D, fertilization in vitro. Hum Reprod. 1998;13:1235-9.
Lewis S, et al. The effect of sperm DNA fragmentation on 57. Velez de la Calle JF, Muller A, Walschaerts M, Clavere JL,
miscarriage rates: a systematic review and meta-analysis. Jimenez C, Wittemer C, et al. Sperm deoxyribonucleic
Hum Reprod. 2012;27(10):2908-17. acid fragmentation as assessed by the sperm chromatin
43. Aitken RJ, De Iuliis GN. Value of DNA integrity assays dispersion test in assisted reproductive technology
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81-92. Fertil Steril. 2008;90:1792-9.
44. Sorahan T, Prior P, Lancashire RJ, Faux SP, Hulten MA, Peck 58. Wright C, Milne S, Leeson H. Sperm DNA damage
IM, et al. Childhood cancer and parental use of tobacco: caused by oxidative stress: modifiable clinical, lifestyle
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CH AP T E R
Current Trends in the
17
Current Trends in the Management of Azoospermia and Oligoasthenospermia 141
Management of Azoospermia and Evaluation of Other Causes • Following TURED improves semen parameters in
patients with ductal obstruction.
of Azoospermia
Oligoasthenospermia • Congenital absence of vasdeferens—genetic coun-
• Men with normal sized testes and low ejaculate volume
may have ejaculatory duct obstruction or dysfunction.
seling of both the partners with testing for cystic fibro- • Vasography helps in the identification of vas deferens
Soumya Davuluri
sis transmembrane conductance regulator mutations or ejaculatory ducts.
(point mutations and 5T allele),2,3 imaging for renal • Normal testicular biopsy also indicates obstruction in
Chapter Outline
abnormalities and transrectal ultrasound.4 the reproductive system.
• Azoospermia • Nonobstructive Azoospermia
• ICSI • Genetic sequencing to be considered in cases where • Seminal pH and fructose along with seminal vesicle
wife is a carrier and husband is negative for CBAVD aspiration and seminal vesiculography performed
(Flowchart 17.1). under transrectal ultrasound guidance help in the
INTRODUCTION fertility, infections of the genital tract, gonadotoxins diagnosis of ejaculatory duct obstruction
such as prior chemotherapy or radiation, recent fever • The site of obstruction can be located by seminal vesic-
Azoospermia accounts for 1% among 10–15% of infertile
or heat exposure and current medication (cimetidine,
Bilateral Testicular Atrophy ulography.6,5
men. This can be classified in to pretesticular (2%), testicu- calcium channel blockers), family history of birth • Can be caused either by ischemia to the testes, expo-
lar failure or nonobstructive azoospermia (49–93%), post defects and mental retardation, reproductive failure or sure to mumps/HIV virus, or due to injury to the groin. Treatment of Obstructive Azoospermia
testicular (7–51%), obstructive or ejaculatory dysfunction. cystic fibrosis. Usually presents with low serum testosterone levels
Pregnancy rates are as close to 50% in women under the Clinical management: Correction of the cause.
and low semen volume and testicular atrophy.
age of 35 years and are independent of the site of retrieval Surgical management is done in two ways:
Physical Examination
of sperm. Oligoasthenospermia is the most common iden- (1) Correction of the obstruction and (2) surgical retrieval
tifiable anomaly that can be treated in most of the cases. • Secondary sexual characters—inspection of the body- Ductal Obstruction of sperms followed by in vitro fertilization (IVF)/intracyto-
Specific treatment is possible with the aim to improve the habitus, hair distribution and gynaecomastia, and a • Normal ejaculate volume and azoospermia may indi- plasmic sperm injection (ICSI).
sperm concentration and the fertility potential of sperms. eunuchoid strature. cate ductal obstruction or abnormal spermatogenesis.5 • Surgical management varies with the site of the obstruc-
This twofold approach is helpful in both natural and • Genital examination (inspection and palpation)— • Transrectal ultrasound is helpful in the diagnosis of tion. Obstruction in the vas deferens and epidydimis is
assisted reproductive techniques. penile curvature and hypospadiasis. ductal obstruction. treated by microsurgical reconstruction.
Testes size (normal testicular volume >12 mL), consist-
AZOOSPERMIA ency, and presence of testicular mass.
Flowchart 17.1: Genetic testing algorithm in men with azoospermia.*
Introduction Presence and consistency of vas deferens
Consistency of epidydimis
When no spermatozoa can be detected on high-power Presence of varicocele
microscopic examination of a pellet after centrifuga-
Presence of mass upon per rectal examination
tion for 15 minutes at 3,000×g or greater of the seminal
• Hormonal evaluation includes follicle-stimulating
fluid on at least two separate occasions is defined as
azoospermia.1 hormone (FSH), luteinizing hormone (LH), inhibin B,
These men can further be classified as obstructive and serum testosterone (Table 17.1).
and nonobstructive azoospermia. Nonobstructive azoo-
spermia (50–60%) is more common than the obstructive
Table 17.1. Hormonal profile in Azoospermia.*
azoospermia (OA) (15–20%).
Testos- Testis
OA is defined as the absence of spermatozoa in the
Etiology Subtype FSH LH terone volume
ejaculate despite normal spermatogenesis.
Obstructive
azoospermia
⇔ ⇔ ⇔ ⇔
Management Nonob- Primary
Evaluation should start after confirming azoospermia on structive testicular ⇑ ⇑ ⇓ ⇓
azoospermia failure
atleast two semen analyses.1
Hypogon-
• History: Should include history of childhood illness adotropic
⇓ ⇓ ⇓ ⇓
such as viral orchitis or cryptorchidism, genital trauma hypogonadism
or prior pelvic or inguinal surgeries, history of prior *CUA guideline: The work up of azoospermic males. *Wosnitzer MS. Genetic evaluation of male infertility. Transl Androl Urol. 2014.
142 The Infertility Manual Current Trends in the Management of Azoospermia and Oligoasthenospermia 143
• Ejaculatory duct obstruction is treated by trans- can be performed for congenital, infectious, post- the extensive inflammatory reaction cannot be repaired Treatment
urethral resection of ejaculatory ducts (TURED). vasectomy or idiopathic epididymal obstruction. by surgical reconstruction in tuberculosis, VE and/or
• The female partner should be evaluated prior to per- complex repairs requiring trans-septal, inguinal, or pelvic • Gonadotropins: Helps to induce sperms in the ejacu-
forming microsurgery in the male. approaches are frequently required for success along with late and also increases the success rates of TESE. The
Transurethral Resection of Ejaculatory rationale is that high levels of exogenous gonado-
• The micro surgical vaso-vasostomy is the gold stand- simultaneous sperm retrieval and cryopreservation.7
ard for vasal reconstruction, as the precision in Ducts (TURED) Vaso-epididymostomy or other complex repairs may tropins will decrease the endogenous gonadotropin
mucosal to mucosal anastomosis results in superior • Treatment of choice in the management of ejacula- be required if vasectomy is not the cause of the obstruc- secretion, which helps in improving the functioning of
outcomes. tory duct obstruction. Complications of TURED occur tion or if the surgery is a repeat reconstruction. Microsur- the FSH and LH receptors in the Leydig and the Ser-
• Success of vasectomy reversal is influenced by the in 20% of the patients including hematuria, hemato- gical repairs result in return of sperm in the ejaculate in toli cells. High levels of exogenous gonadotropins will
following factors (1): spermia, urinary tract infection, epidydimitis, and a 85–90% of men and pregnancy in 60% of couples. Return decrease the endogenous gonadotropins, which helps
Age of the female partner. watery ejaculate due to the reflux of urine. of sperm in the semen following repeat reconstruction in improving the FSH and LH receptors in the Leydig
Shorter duration between vasectomy and reversal. has been noted in 75% of men and 43% pregnancy rates.8 and the Sertoli cells.9 A 3–6 months of treatment with
Shorter the resected segment—better success. Sperm retrieval is the treatment of choice for patients with gonadotropins is required to induce spermatogen-
Outcomes Vas Deferens and obstruction due to tuberculosis. esis.10,11
Presence of opalescent fluid in the intraoperative
fluid indicating the presence of sperm. • Following microsurgical vaso-vasostomy, 86% patency • Human chorionic gonadotropin—2,000 IU subcutane-
Shorter duration of obstruction. and 52% pregnancy outcomes are reported. ICSI ously thrice weekly or 2,500 IU twice weekly along with
Length of the vas segment between epididymis and • About 20–40% of couples achieve pregnancy following • As the number of good motile sperms retrieved by
supplementation with FSH-37.5–150 IU thrice weekly
vasectomised site. vaso-epididymostomy microsurgery through inter- after 3–6 months. Human chorionic gonadotropin
sperm retrieval techniques, ICSI is preferred in men
Presence of granuloma at the vasectomised site. course. helps in the initiation of spermatogenesis while FSH
with OA resulting in improved fertilization, clinical
• Postoperative patency and pregnancy rates are excel- helps in the completion of spermiogenesis.12
pregnancy, and delivery rates.6
lent after vaso-vasostomy. • Pulsatile administration of gonadotropin-releasing
Vaso-epididymostomy • Sperm retrieval /ICSI is preferred to surgical treatment
• After TURED, the sperms return to the ejaculate in hormone (GnRH) subcutaneously/intravenously/
in case of:
• Vaso-epididymostomy is performed when there is approximately 50–75% of men and approximately 20% intranasally can be given only in men with intact pitui-
1. advanced maternal age;
absence or presence of poor quality fluid with no men achieve pregnancy.6 tary glands and when treatment with gonadotropins
2. female factor infertility;
sperms indicates obstruction of the proximal vas. Good microsurgical techniques, selection of healthy vas 3. where the success with sperm retrieval/ICSI fails. Dose: 5–20 µg every 2 hours subcutaneously.
Modern techniques for vaso-epididymostomy aim at segments, precise mucosal-to-mucosal, and tension free exceeds the success with surgical treatment; Spermatogenesis results in 77% of azoospermic men
identifying and directly anastomosing a single pat- anastomosis result in excellent outcomes following vasec- 4. sperm retrieval/ICSI is preferred by the couple. after treatment for 12–24 months.13 Disadvantage: Very
ent epididymal tubule to the much larger and stur- tomy reversal,6 while outcomes following vasal recon- expensive and frequent usage.
dier lumen of the vas deferens. Vaso-epididymostomy struction for other etiologies are limited (Table 17.2). As Microsurgical Reconstruction Versus • Androgen excess: Excess testosterone in itself can
Sperm Retrieval with IVF/ICSI cause suppression of endogenous hormones. In these
cases, cessation of the hormones, endogenous pro-
Table 17.2. Advantages and disadvantages of different sperm retrieval techniques.* • Microsurgical vasovasostomy and vasoepididymos- duction of these hormones, and spermatogenesis may
Advantages Disadvantages tomy are more cost-effective than sperm retrieval with start after 1 year.
IVF/ICSI, as the latter requires intervention in both the • Aromatase inhibitors: Imbalance in the circulating
Microsurgical epididymal Large quantity of sperms obtained suitable for several
sperm aspiration (MESA) in vitro fertilization (IVF)/ intracytoplasmic sperm male and female. testosterone and estradiol has also been found in men
injection (ICSI) cycles in one procedure • Microsurgical reconstruction, if successful, allows cou-
with nonobstructive azoospermia, which suppresses
Perccutaneous epididymal No microsurgical skills required ples to have subsequent children without additional
the LH and FSH and testosterone levels. Aromatase
sperm aspiration (PESA) Fast medical treatment. Therefore, in most cases, microsur-
inhibitors such as testolactone, anastrazole, letro-
Minimum post-op discomfort gical reconstruction is more appropriate as an initial
zole block the conversion of androgens to estradiol
Testicular sperm extraction No microsurgical skills required except when micro Risk of testicular damage with treatment for OA.
which helps in correcting the imbalance. Many studies
(TESE) and micro TESE TESE performed multiple biopsies • If the duration of obstruction is long, sperm retrieval
Incision with post-op discomfort
done have shown an increase in the testosterone and
with ICSI is preferable.
Higher cost decrease in the estrogen but has not shown the return
of sperm in the ejaculate.14
Percutaneous testicular No microsurgical skills required Fewer sperms retrieved NONOBSTRUCTIVE AZOOSPERMIA • Antioxidants: Use is still debated though studies have
sperm a
spiration (TESA) Fast and easy
Minimum post-op discomfort It represents failure of spermatogenesis, which could be shown to that increased oxidative stress and lower anti
Minimally invasive due to lack of stimulation by gonadotropins (hypogonado- oxidant capacity in men with abnormal semen param-
*ASRM Practice Committee. Sperm retrieval for obstructive azoospermia. Fertil Steril. 2008. tropic hypogonadism) or intrinsic testicular impairment. eters.15
144 The Infertility Manual Current Trends in the Management of Azoospermia and Oligoasthenospermia 145
Surgical Management of Nonobstructive of lubricants, shorter or longer duration of abstinence, the risks of prednisolone therapy are weighed against the PROBABLE QUESTIONS
high-grade fever, history of sexually transmitted diseases pregnancy.
Azoospermia
or genitor-urinary infection, childhood diseases such as 1. What is the definition of azoospermia? How do you
Testicular Sperm Extraction (TESE) mumps, orchitis, cryptorchidism, testicular trauma or Androgens classify? What are the underlying pathophysiological
torsion, exposure to gonadotoxins, and occupational are mechanisms?
and Micro TESE Administration of androgens (Inj. Testosterone 200–500 mg
some of the causes. 2. Discuss the etiology of obstructive azoospermia.
• Removal of tiny tissues from the testes either by an Episode of fever can reduce the concentration of IM every 2 weeks) given to suppress spermatogenesis ini- 3. Evaluate the genetic causes involved in azoospermia.
open technique or under micro-dissection is done. The sperms by 0.4–7% and a decrease in the motility by 0–23% tially and with discontinuation the sperm production 4. How do you evaluate a case of OA?
microscopic approach of the testes helps in improving 2–6 weeks after fever episode. Post this, the values can is believed to rebound to the levels higher than the pre- 5. Management of OA.
the yield of spermatozoa and removal of lesser tissue come to normal by 2–3 months. So, any abnormal sperm suppression levels. Recent studies have shown that there is 6. Explain about various surgical sperm retrieval tech-
and decreased damage to the blood supply of the tes- test during this period should be repeated after 3 months. no benefit of androgens in improving the fertility.21 niques with appropriate diagrams.
ticles. Fine needle aspiration (FNA) mapping helps 7. Discuss the medical management in a case of non-OA.
Similar phenomenon can be seen in patients who are
to target biopsies for sperm retrieval and reduces the Gonadotropins and Gonadotropin-releasing 8. How do you evaluate and manage a case of non
exposed to high temperatures.
chances of deleterious effects. obstructive azoospermia?
• In men with atleast one area of hypo-spermatogene-
Finding the cause and offering the right treatment Hormones 9. How do you evaluate a case of oligoasthenospermia?
when available is the main step in the treatment of oligoas-
sis, sperm retrieval was 79%, while in men with matu- No role of gonadotropins (hCG, hMG) and GnRHs has been 10. Discuss the management of oligoasthenospermia.
thenospermia and when possible medical management
ration arrest only 47% sperm retrieval was seen with demonstrated in the treatment of oligoasthenospermia.22 11. Role of antioxidants in improving male fertility: an
should be offered over assisted reproductive procedures.
TESE (Table 17.2). In men with Sertolicell-only pat- evidence.
tern, sperm retrieval by TESE is 24%.16 Multiple sites The aim of pharmacological treatment is to improve the
sperm concentration and motility to improve the fertility Antiestrogens 12. Discuss various male surgical reconstruction proce-
with sperm on FNA mapping increases the success of dures and evaluate their practical usage.
TESE by 99% and if few areas are present the sperm potential of the sperms. Clomiphene citrate and tamoxifen exert their action by 13. ICSI after surgical retrieval of sperm—discuss on the
retrieval rates are up to 90%. The success of TESE is competing to block the estrogen receptors which in turn safety concerns with evidence.
debatable when no positive sampling is seen on FNA Evaluation of Oligoasthenospermia enhances the gonadal function by increasing GnRH activity,
mapping.17,18 gonadotropin secretion, and production of testosterone and
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Int Braz J Urol. 2011. • Ejaculatory disturbances noradrenaline combined with reduced tone of the
Zuckerman H. The prostaglandin inhibitor effect of anti-
16. Outcomes of micro dissection TESE in men with non- inflammatory drugs in the therapy of male infertility. Fertil smooth muscles of the blood vessels, which occurs
obstructive azoospermia. Weill Cornell Medical College, Steril. 1984;42(3):406-11. Biological Process of Response to due to the release of endothelin and neuropeptide Y.
Department of Urology. 2015. 25. Tesarik J, Thebault A, Testart J. Effect of pentoxifylline on
17. Diegido P, Jhaveri JK, Ghannam S, Pinkhasov R, Shabsigh R, sperm movement characteristics in normozoospermic and a Sexual Activity • Puborectalis, levator ani, external anal sphincter, and
Fisch H. Review of current varicocelectomy techniques asthenozoospermic specimens. Hum Reprod. 1992;7(9): urethral sphincter are the pelvic floor muscles involved
and their outcomes. BJU Int. 2011;108(7):1157-72.
• The dopamine (DA) release in a sexually active male is in establishing an erection.3
1257-63.
due to three integrative processes.1
• The nigrostriatal system enhances the somatomotor Nonconsummation of Marriage4
activity.
Common causes for inability to consummate are as
• The mesolimbic system is involved in various types of follows:
motivation.
• The medial preoptic area (MPOA) coordinates the • Premature ejaculation—23%
motivation to act on sexual targets, increases sexual • Erectile dysfunction—61%
activity and response, and also coordinates the genital • Combined factors—16%
reflexes. Some of the diagnostic tests used to detect erectile
• 5-Hydroxy tryptamine (5-HT) is considered to have an dysfunction are as follows (Table 18.1):
inhibitory response to sexual activity. Combined injection and stimulation test (CIS): After
• 5-HT is released after ejaculation at lateral hypotha- injecting vasodilators intracavernously, a subsequent
lamic area. erection is visual rated.
• Other factors that can modify the impact of hormones Nocturnal penile tumescence (NPT): NPT is based
in achieving libido are certain medications, alcohol, on physiological erection, which occurs in RWM sleep.
and health conditions. Various methods used are stamp test, strain gauges, snap
• Local response is seen in cavernosa due to the action gauges, NPTR (Rigiscan), and sleep lab NPTR.5
of acetylcholine and nitric oxide (NO), which are Blood flow detection studies: Commonly used are color
released from the endothelium, noradrenaline, vaso- duplex Doppler ultrasound, dynamic infusion cavernos-
active intestinal polypeptide (VIP), calcitonin gene- ometry/cavernosography (DICC), and selective internal
related peptide (CGRP), and prostaglandins.2 pudendal pharmacoangiography. Color duplex ultrasound
• NO and VIP are responsible in achieving erection. provides measurement of penile blood flow. It aids in eval-
• Erection occurs due to erotogenic stimuli that causes uating both arterial and venous pattern in the cavernosa
parasympathetic nerve stimulation, which leads to and forms an objective testing which is minimally invasive.6
148 The Infertility Manual Sexual Dysfunctions in Male Infertility 149
Table 18.1. Erectile dysfunction—classification, d
isorders, and problems. Ejaculatory Disturbances • Abdominal and trans-rectal ultrasonic examination of
genital organs
Category Disorders Problem Ejaculation is a dual phase mechanism7: Presence of more than 15 sperms/high power field
Psychogenic Performance anxiety, depression Loss of libido, overinhibition impaired nitric oxide release 1. Deposition of semen in prostatic urethra. (HPF) confirms retrograde ejaculation
Neurogenic Stroke, spinal cord injury, diabetes Lack of nerve impulse, or Interrupted transmission 2. Expulsion of semen during which smooth sphincter
mellitus, post-surgical of bladder neck closes and external striated sphincter Management of Retrograde Ejaculation10
opens.
Hormonal Hypogonadism, hyperprolactinoma Inadequate nitric oxide release • Drug therapy to convert retrograde into antegrade ejac-
Sympathetic nervous system via T10-L3 level along
Vasculogenic (arterial Atherosclerosis, trauma, hypertension, Impaired arterial or venous flow ulation
with contraction of pelvic floor and periurethral smooth
or venous) diabetes • Collection of postejaculatory urine and harvest sperms
muscles results in ejaculation.
Medication induced Antihypertensives, antidepressants, Central suppression, vascular Insufficiency • Surgical treatment
alcohol, cigarette use Ejaculatory disorders include Medical therapy: α-adrenergic agonists such as
• Premature ejaculation pseudoephedrine, ephedrine, phenylpropanola-
https://www.auanet.org/education/auauniversity/medical-student-education/medical-student-curriculum/ed
• Retrograde ejaculation mine, and imipramine.
• Anejaculation Careful regulation of pH and osmolarity of the
Flowchart 18.1: Erectile dysfunction: management flowchart. urine is achieved before collecting post-ejaculation
• Deficient or retarded ejaculation
urine. There are several protocols for urinary
alkalization.
Premature Ejaculation
Results due to an altered perception of genital sensations Protocol for Urinary Alkalization
in the afferent pudendal pathway.8
Two kinds of premature ejaculation are
and Collection of Sperms
• Biogenic and • Oral tablet of sodium bicarbonate 2 g TID and
• Psychogenic. α-adrenergic agonists for 3 days prior to collection and
Medications that are used commonly are fluox- monitoring his urinary pH with pH strips.
etine, paroxetine, and imipramine either singly or • pH should reach 7.3 and the osmolarity should be
in combination. Pelvic floor rehabilitation helps in 280 mOsmol.
certain situations. • Later, catheterize the bladder and drain it to completion.
Circumcision also improves the condition. • Then administer a small volume of mediainto bladder.
• Patient then ejaculates and collects his antegrade s ample.
• Re-catheterize, collect the bladder sample and if
Retrograde Ejaculation
needed irrigate the bladder with media.
• Defined as passage of seminal fluid from the posterior
urethra into the bladder instead of distal urethra.9 Anejaculation
• Defect can be due to anatomic, neurogenic, pharma-
• Inability of a man to have an ejaculation.
cologic, or idiopathic.
• Occurs due to spinal cord injury, retroperitoneal
• Anatomic causes may be due to prostatectomy (open
lymph node dissection, or other retroperitoneal sur-
or transurethral) or bladder neck surgeries (Y-V plasty,
gery, diabetes mellitus, transverse myelitis, multiple
transurethral incision of the bladder neck). sclerosis, psychogenic, or idiopathic.11
• Neurogenic causes may be due to spinal cord injury,
retroperitoneal surgery, and diabetes mellitus.
http://www.aafp.org/afp/2000/0101/p95.html TREATMENT
• Pharmacologic agents such as neuroleptics, tricyclic
antidepressants, α-blockers, and certain antihyper- Vibratory stimulation helps in cases where the afferent or
tensives can cause retrograde ejaculation.10 efferent neural pathways (reflex arc) is intact or else elec-
Neurological evaluation: It consists of performing the interpretations of the resulted electrical potentials helps in
troejaculation is used.
bulbocavernosus reflex (BCR) and somatosensory evoked differentiating various afferent nerve pathologies such as in
potentials (SSEPs). The reflex arc consists of somatosen- diabetes, spinal cord injury, and post radical pelvic surgery. Diagnosis is Established by
sory afferents via the pudendal and dorsal nerves and Treatment includes combined therapy such as sexual • Semen analysis demonstration
Vibratory Stimulation
autonomic efferents via the cavernous and pelvic nerves. education, psychotherapy, oral medications (sildenafil • Presence of sperm in the post-ejaculate urine • Vibratory stimulation is applied to the underside of the
Corpus Cavernosal EMGs (CC-EMGs) measure the citrate), intracavernosal injections, specific treatment for • Presence of markers of the adnexa secretion in urine glans penis and vibrates at a designated frequency and
corpora cavernosal smooth muscle electrical activity,5 and organic cause, and surgery as last resort (Flowchart 18.1). • Determination of the secretion wave amplitude.
150 The Infertility Manual Sexual Dysfunctions in Male Infertility 151
• This stimulation travels via the sensory nerves to the Treatment: Behavioral therapy—helps in psycho- • Ejaculatory disorders include premature ejaculation, cavernosometry and cavernosography in the evaluation
spinal cord and induces a reflex ejaculation.12 genic cases retrograde ejaculation, anejaculation, and retarded of erectile dysfunction. Int J Impot Res. 1999;11(1):47-51.
Electroejaculation—most often is successful ejaculation. 7. Hermabessiere J, Guy L, Boiteux JP. Human ejaculation:
• Frequency of 100 Hz and peak-to-peak amplitude of
physiology, surgical conservation of ejaculation. Prog Urol.
2.5 mm aids in ejaculation over a period which may Epididymal aspiration and ICSI is used in • Medications for premature ejaculation are fluoxetine,
1999;9(2):305-9.
vary from 10 to 180 minutes.13 resistant cases paroxetine, and clomipramine either singly or in com- 8. Metz ME, Pryor JL, Nesvacil LJ, Abuzzahab F Sr, Koznar J.
• Secondary anorgasmia is due to the usage of psy- bination. Premature ejaculation: a psychophysiological approach
chotropic drugs such as sertraline and gebapentin. • Principles of management of retrograde ejaculation for assessment and management. J Sex Marital Ther.
Electroejaculation Other medications such as antidepressants, especially include medications, sperm harvesting from postejac- 2000;26(4):293-320.
clomipramine or SSRIs. can also be causative. ulateurine, and surgical treatment. 9. Malossinni G, Ficarra V, Caleffi G. Retrograde ejaculation.
• Electroejaculation includes electrical stimulation of
• Medications for retrograde ejaculation include Arch Ital Urol Androl. 1999;71(3):185-96.
the sympathetic efferent fibers.14 It is performed under • Nefazadone, amantadine, cyproheptadine, silde-
10. Glander HJ. Disorders of ejaculation. Fortschr Med.
general anesthesia in all patients who have abdominal nafil, serotonin antagonist mianserin may help in α-adrenergic agonists such as pseudoephedrine, phe-
1998;116(26):26-8, 30-1.
and perirectal sensation. secondary anorgasmia. Electroejaculation or vibratory nylpropanolamine, and imipramine. 11. Witt MA, Grantmyre JE. Ejaculatory failure. World J Urol.
stimulation is also tried. • Treatment for anejaculation includes vibratory stimu- 1993;11(2):89-95.
• Stimulation is given by probe introduced per rectally
lation, electroejaculation, and surgical sperm retrieval. 12. Sonksen J, Biering-Sorensen F, Kristensen JK. Ejaculation
directing toward the obturator nerves, where both pre-
• Treatment for anorgasmia includes behavioral ther- induced by penile vibratory stimulation in men with spinal
ganglionic and post-ganglionic sympathetic fibers are SUMMARY cord injuries. The importance of the vibratory amplitude.
apy, electroejaculation, and surgical sperm retrieval.
found.15 Paraplegia. 1994;32(10):651-660.
Sexual dysfunction management forms a major part in
• Delivery of current is performed manually to increase treating male infertility as many of them will be suffering PROBABLE QUESTIONS 13. Ohl DA, Sonksen J, Menge AC, McCabe M, Keller LM.
the voltage delivery progressively for a few seconds in Electroejaculation versus vibratory stimulation in spinal
either primarily or secondarily. Proper evaluation with 1. What are the components of the male reproductive cord injured men: sperm quality and patient preference.
a sinus rhythm. the availability of new advanced therapies and medica- system? J Urol. 1997;157(6):2147-9.
• Voltage is gradually raised till ejaculation occurs. The tions has made the solution to these conditions quite pos- 14. Ohl DA, Bennett CJ, McCabe M, Menge AC, McGuire EJ.
2. What compounds mediate communication within
ejaculate is then collected. sible. Newer techniques such as electroejaculation and Predictors of success in electroejaculation of spinal cord
the male reproductive system?
• Next a catheter is placed into bladder and sperms from vibratory stimulation have helped for successful sperm injured men. J Urol. 1989;142:1483-6.
3. What is semen composition?
the bladder which have passed retrogradely is also retrieval. However, there are innumerable parameters 15. Eid JF. Electroejaculation. AUA Update Series 1992,
4. What factors determine the sex of an individual? Volume XI, Lesson 10: 73-80.
collected.16-19 both microscopic and macroscopic, which can result in 5. What are the types of anejaculation? 16. Chung PH, Verkauf BS, Mola R, Skinner L, Eichberg RD,
wide variation in the outcome of the assisted reproductive Maroulis GB. Correlation between semen parameters of
Retarded Ejaculation techniques of intrauterine insemination and in vitro ferti- REFERENCES electroejaculates and achieving pregnancy by intrauterine
lization. The need for improvement in semen recovery and 1. Hull EM, Lorrain DS, Du J, Matuszewich L, Lumley LA,
insemination. Fertil Steril. 1997;67(1):129-32.
• Difficulty or inability to ejaculate despite the presence processing is enormous, and constant research should aid 17. Hirsch IH, Sedor J, Jeyendran RS, Staas WE. The relative
Putnam SK, et al. Hormone-neurotransmitter interactions
of adequate sexual desire, erection, and stimulation in better results in future. Fertility issues in menis complex in the control of sexual behavior. Behav Brain Res. distribution of viable sperm in the antegrade and retrograde
which is persistent and nonsituational.10 1999;105(1):105-16. portions of ejaculates obtained after electrostimulation.
need a multidisciplinary management approach.
• Etiology may be organic-medical illness or drug 2. Hora M, Vozeh F. The physiology of erection. Urologicka Fertil Steril. 1992;57(2):399-401.
klinika LF UK a FN, Plzen Cas Lek Cesk. 1997;136(12):363-6. 18. Nehra A, Werner MA, Bastuba M, Title C, Oates RD.
induced (antiadrenergic agents), few of the surgical
interventions, or psychological factors. KEY NOTES 3. Shafik A. Pelvic floor muscles and sphincters during Vibratory stimulation and rectal probe electroejaculation
erection and ejaculation. Arch Androl. 1997;39(1):71-8. as therapy for patients with spinal cord injury: semen
• Medical therapy along with behavioral therapy or • In approximately 1% of infertile couples sexual dys- 4. Zaragooshi J. Unconsummated marriage: clarification of parameters and pregnancy rates. J Urol. 1996;155(2):
vibratory therapy is helpful. function is seen. aetiology: treatment with intracorporeal injection. BJU Int. 554-9.
• Causes of unconsummation: Premature ejaculation 2000;86(1):75-9. 19. Rajasekaran M, Hellstrom WJ, Sparks RL, Sikka SC. Sperm-
5. Broderick GA. Evidence based assessment of erectile damaging effects of electric current: possible role of free
Orgasmic Disorders (23%), erectile dysfunction (61%), and combination of
dysfunction. Int J Impot Res. 1998;(Suppl 2):S64-73; radicals. Reprod Toxicol. 1994;8(5):427-32.
factors (16%). discussion S77-9. 20. Michetti PM, Rossi R, Travaglia S, Barrese F, Franco G,
• Primary • Diagnostic tests for erectile dysfunction are combined 6. McMahon CG, Touma K. Predictive value of patient Leone P, et al. Primary absolute anorgasmy in the male.
• Secondary injection and stimulation test or pharmaco testing history and correlation of nocturnal penile tumescence, Report of three clinical cases. Minerva Urol Nefrol.
Primary absolute anorgasmia is the inability to (CIS), nocturnal penile tumescence (NPT), blood flow colour duplex Doppler ultrasonography and dynamic 1999;51(1):23-6.
have an orgasm and ejaculation while awake but studies, and neurological assessment.
has normal nocturnal erection and emission.20 • Management of erectile dysfunction: Combination
Etiology: Commonly it is psychogenic, occasion- of various treatments such as psychotherapy, sexual
ally a neurological basis such as occult spinal education, oral medications (sildenafil citrate), intra-
dysraphism with tethered cord, multiple sclerosis cavernosal injection agents, and specific treatment
is seen. directed for organic cause.
C HA PT E R
19
Surgical Sperm Retrieval 153
Table 19.2. MESA: Advantages and disadvantages. Table 19.4. PESA: Advantages and disadvantages.
Surgical Sperm Retrieval Advantages
•• Precise
Disadvantages
•• Time-
Complications
•• Bleeding
Advantages
•• Simple
Disadvantages
•• Cannot be pre-
Complications
•• Hematoma
•• Blood-free aspiration consuming •• Quick cisely controlled. •• Vascular
Madhu Lakshmikantha, Kamini A Rao •• Sperms can be •• Needs an •• Avoids open •• The chances injury
obtained from operating surgery of missing a
multiple sites microscope •• It can be sperm-containing
Chapter Outline
•• Adequate number •• A trained repeated ductule.
• Indications • Operative Techniques of motile sperm can microsurgeon many time •• Possibility of
• Contraindications • Testicular Retrieval Techniques
be recovered and is required when required puncturing a
• Sperm Retrieval Techniques
cryopreserved for nearby blood
future cycles of ICSI vessel
INTRODUCTION SPERM RETRIEVAL TECHNIQUES
The perspective of managing azoospermia has changed (TABLE 19.1)
Table 19.3. OFNA: Advantages and disadvantages. Table 19.5. TESA: Advantages and disadvantages.
from the time of reconstructive surgery and donor insemi- In obstructive azoospermia, sperms can be obtained from Advantages Disadvantages Complications Advantages Disadvantages Complications
nation to the recent advent of intracytoplasmic sperm the vas, epididymis, and/or the testis.
injection (ICSI). •• Fluid can be •• Open •• Bleeding •• Simple •• Blind •• Hematocele forma-
In nonobstructive azoospermia only testicular sperms aspirated under surgical pro- •• Infections •• No special procedure tion
Four distinct factors have made the surgical retrieval can be obtained. vision cedure training is •• More •• Intra-testicular
successful to a large extent. Nowsperms from the •• No microsurgi- required testicular hemorrhage
epididymis or testes are used to help the azoospermic men OPERATIVE TECHNIQUES cal dissection or •• No special tissue
become biological fathers. suturing is involved equipment damage
First: Epididymal transit is not required for ssuccessful Microsurgical Epididymal Sperm •• Very quick required •• The amount
fertilization. •• Does not need of cellular
Aspiration (MESA) (Table 19.2)2 special equipment material is
Second: Recognition and histologic demonstration or training scanty
that heterogenicity of spermatic tubules exists in biopsy Technique
specimens.
• Under an operating microscope.
Third: Development of ICSI.
• Scrotal incision is made.
• 26-G needle is used. Testicular Sperm Aspiration (TESA)5
Fourth: Availability of special extraction techniques. • The epididymal fluid can be aspirated from the
• An epididymal ductule is gently mobilized and opened.
epididymal surface. Technique(Table 19.5)
• The spermatic fluid is then aspirated.
• The ductular opening is not sutured.
INDICATIONS1 • Microsutures are used to close the ductule. Anesthesia: Local
• A 21/22-G butterfly needle is pricked into the testicular
1. Obstructive azoospermia—When reconstruction has
Open Fine Needle Aspiration (OFNA)3 Percutaneous Epididymal Sperm substance while applying suction with a 20 mL syringe.
failed
• Vas aplasia Aspiration (PESA) (Table 19.4)4 • The aspirated fluid is checked for sperm.
• Multiple blocks following tuberculosis
Technique (Table 19.3)
• If the couple chooses ICSI over surgery. Anesthesia: Local
Technique Needle Aspiration Biopsy (NAB)6
2. Nonobstructive azoospermia—in men with focal • The first step involves the exposure of epididymis. Anesthesia: Local Anaesthesia: Local (Table 19.6)
spermatogenesis. • A ductule is directly punctured through the tunica. • The head of the epididymis should be located and pal- • 18-G scalp vein needle.
3. Failure to ejaculate prior to an ICSI procedure. • No dissection is required. pated. • Gentle suction is applied using a 10 or 20 mL syringe.
4. Failure of vibrator stimulation.
• Then it is stabilized between thumb and forefinger. • The needle should be staying with in tunica all the time
5. Nonavailability of electro-ejaculation.
6. Total astheno/necrozoospermia. Table 19.1. Classification of sperm retrieval techniques. • Then it is punctured, directly through the scrotal skin, and is pushed in up to its hub, then pulled partly out
Procedure Open Percutaneous with a 26-G needle attached to a tuberculin syringe several times.
Epididymis MESA PESA containing 0.1 mL of sperm-washing medium. • The needle is rotated 180 degrees out-and-in motion
CONTRAINDICATIONS1 OFNA • The syringe should be staying within the epididymis of the needle is repeated a few times till the aspirate is
1. Active genital tract infection Testis Conventional TESE TESA and is rotated alround 180 degrees. seen in the tubing of the needle.
2. Bleeding diathesis MD-TESE NAB • The contents of the syringe are emptied onto a dish and • The needle is withdrawn slowly out of the testis after
SAT Tru-cut
3. Previous scrotal surgery within last 3 months examined for the presence of sperms under microscope. clamping the tube.
154 The Infertility Manual Surgical Sperm Retrieval 155
Table 19.6. Needle aspiration biopsy: Advantages and Table 19.7. CNB: Advantages and disadvantages. Table 19.10. MICRO-TESE: Advantages and disadvantages.
disadvantages Advantages Disadvantages Complications Advantages Disadvantages Complications
Advantages Disadvantages Complications •• Simple •• Causes more trauma •• Hematocele •• Less tissue removed •• Tunical incision is large •• More devascularization injury
•• Simple •• Blind proce- •• Hematocele method •• The amount of tissue •• Intra-testicular •• Less testicular damage •• More d issection of the testicu- •• Fibrosis of the testis
•• Quick dure •• Intra-testicular recovered may be hemorrhage •• Improved chances of finding sperm in cases lar tissue
•• Incisionless hemorrhage less •• Damage to with focal spermatogenesis
•• Damage to epididymis. epididymis •• Deeper sampling possible
• A long incision is made in the tunica to expose the tes- 2. What are epididymal sperm retrieval techniques?
Table 19.8. COB: Advantages and disadvantages. Table 19.9. SST:B: Advantages and disadvantages.
ticular parenchyma. Discuss the techniques with their advantages and
Advantages Disadvantages Complications Advantages Disadvan- Complica-
• The seminiferous tubules are separated and examined disadvantages.
•• Easy method •• Open •• Testicular ves- tages tions
under an operating microscope. 3. What are testicular sperm retrieval techniques?
•• Yields a good procedure sels may be •• Extensive sampling of the •• Open •• Blee
• Only the healthy tubules are biopsied and examined Discuss the techniques with their advantages and
amount of tes- damaged testis surgical ding
ticular tissue. •• Impairment of •• Small incision on the tunica. procedure •• Infec- for sperm. disadvantages?
testicular func- •• Only “healthy” tubules are tions • The tunica is closed with a prolene suture. 4. Discuss about the testicular sparing sperm retrieval
tion. biopsied. techniques with suitable examples and evidences.
•• No blood vessel is damaged SUMMARY
•• Multiple biopsies can be
• As the needle emerges from the scrotal skin, a loop of taken without affecting the There is “insufficient evidence to recommend any specific REFERENCES
the seminiferous tubule is pulled out and grasped with vascularity or parenchyma form of sperm retrieval technique,” according to a recent
nonserrated microsurgical forceps and more tissue is of testis. Cochrane meta-analysis.10 1. Current Management of Male Infertility, Niederberger
Craig S. (Ed). Urologic Clinics of North America. 2014;41(1):
pulled out of the testis. The clinical discretion with available resources and 169-80.
• The fluid and the tissue obtained are checked for Single Seminiferous Tubule— expertise should be used to decide on the simplest and 2. Patrizio P, Silber S, Ord T, Balmaceda JP, Asch RH. Two
sperm under microscope. least invasive technique whenever possible. births after microsurgical sperm aspiration in congenital
Biopsy (SST-B)9 absence of vas deferens. Lancet. 1988;2:1364.
Technique (Table 19.9) In Obstructive Azoospermia 3. Shah RS. Advanced Infertility Management. In:
Cutting Needle Biopsy7 PESA should be the first choice because of simplicity.
Hansotia M, Desai S, Parihar M (Eds). Surgical and Non-
Surgical Methods of Sperm Retrieval. New Delhi: Jaypee
Under local procedure
Brothers; 2002. pp. 253-8.
Technique (Table 19.7) • The scrotum is opened by a small incision and the tes-
If PESA fails then OFNA or NAB should be considered.
4. Shrivastav P, Nadkarni P, Wensvoort S, Craft I. Percutaneous
tis is exposed. epididymal sperm aspiration for obstructive azoospermia.
Under local anaesthesia In Nonobstructive Azoospermia Hum Reprod. 1994;9:2058-61.
A spring-activated tissue-cutting biopsy needle is • An avascular area on the tunica is punctured with a
Initially NAB is tried. 5. Schlegel PN, Li PS. Microdissection TESE: Sperm retrieval
used. 26-G needle.
in non-obstructive azoospermia. Hum Reprod Update.
• A micro-forceps is introduced into the puncture hole and Then microsurgical biopsies by the SST method are
The needle is placed against the testis and released, it 1998;4:439.
dilated until a loop of seminiferous tubule pops out. taken bilaterally and multiple sites. 6. Craft I, Tsirigotis M. Simplified recovery, preparation
enters the stroma of testicular. parenchyma, cuts a small If no sperms are found then micro-TESE is tried.
• The seminiferous tubule is held with the micro- and cryopreservation of testicular sperm. Hum Reprod.
slice of tissue and withdraws it into a sheath. When Operative Sperm Retrieval is Required For 1995;10: 1623-7.
forceps, pulled out and examined under the operating
The sample is examined for the presence of sperms. Men with failure to ejaculate, a NAB biopsy will provide 7. Morey AF, Deshon GEJ, Rosanski TA, Dresner ML.
microscope.
adequate number of sperm. Technique of biopsy gun testis needle biopsy. Urology.
• If no sperm are found, or if the tubule appears fibrous,
1993;42:325-6.
then another area is punctured and the tubule is pulled
TESTICULAR RETRIEVAL TECHNIQUES In Men with Total Asthenozoospermia
8. Silber SJ, Nagy ZP, Liu J, Godoy H, Devroey P, Van
out and examined under microscope. Steirteghem AC. Conventional in-vitro fertilization versus
Conventional Open Biopsy8 • The procedure is repeated at multiple sites in the testis NAB is preferred to PESA since testicular sperm are more intracytoplasmic sperm injection for patients requiring
till sperms are found or the entire testicular surface has microsurgical sperm aspiration. Hum Reprod. 1994;9:1705-9.
likely to be viable in these cases.
Technique (Table 19.8) been explored. 9. Girardi SK, Schlegel P. MESA: Review of techniques,
preoperative considerations and results. J Andrology.
Anesthesia: Local
• Tunica is not sutured. PROBABLE QUESTIONS 1996;17:5-9.
10. Proctor M, Johnson N, van Peperstraten AM, Phillipson G.
• A small scrotal incision is made. 1. What are the indications and contraindications of the
Microdissection TESE—Microsurgical surgical sperm retrieval techniques? Enumerate the
Techniques for surgical retrieval of sperm prior to intra-
• The tunica is incised, and a piece of testicular tissue is cytoplasmic sperm injection (ICSI) for azoospermia.
excised. Testicular Sperm Extraction (Table 19.10)3 various procedures. Cochrane Database Syst Rev. 2008;2:CD002807.
• The tunica is sutured, and the incision is closed. • The testis is exposed by a midline incision.
SE C T I O N
4
Female Factor Infertility
C HA PTE R
Female Age and Fertility Preservation
20
Nupur Garg
Chapter Outline
• Potential Outcomes of Cancer Therapy • Investigational Procedures
• Indications of Fertility Preservation Females • ASCO Guidelines
• Established Methods of Fertility Preservation
INTRODUCTION the remaining follicular pool in the ovary can be pre-
dicted. Knowing the extent of ovarian reserve can help
Fertility preservation has become an established branch women to make informed decisions for voluntary fertility
of reproductive medicine. Developed primarily with the preservation.
aim of helping cancer patient undergoing treatment to
preserve their fertility, this discipline is now no longer
restricted to cancer patients but to all those who face risk Fertility Preservation in Cancer Patients
of fertility failure or want to postpone child bearing for
some reason. Fertility preservation helps all the patients
Cancer Therapy and Fertility
facing fertility threatening treatments retain their fertility, Since rapidly dividing cells are the target of chemo- and
or ability to reproduce. With evolving times and changing radiotherapy, these treatments act not only on cancer cells
lifestyle many women are choosing to postpone childbear- but also on germ cells.2
ing for later in life for social, career, or financial reasons.
Postponement of fertility is emerging as one of the novel
POTENTIAL OUTCOMES OF
indications of fertility preservation. It is likely that in the
future women wishing to postpone childbearing may CANCER THERAPY (FLOWCHART 20.1)
constitute the main group of patients requesting fertility
Mechanism
preservation.
a. Apoptosis of growing follicles2
b. Vascular injury2
Age-related Fertility Decline c. Cortical fibrosis.2
Every women is born with a finite number of ovarian fol- The extent of damage to gonadal function and future
licles that continue to undergo atresia throughout her life fertility depend on following factors3 (Fig. 20.2):
time by apoptosis and ovulation. The fecundity of women
begins to decline gradually from the age of 32 years 1. Patient’s age
becoming much rapid after the age of 37 years1 as shown in 2. Stage and type of cancer being treated
3. Drug used
Figure 20.1.
4. Mode of administration
5. Extent and site of the radiation field
Biological Clock and Fertility Preservation 6. Radiation regimen intensity and dose.
With change in the demands of society, there has been
an increase in the mean age of mothers, as education INDICATIONS OF FERTILITY
and career have become important factors in women’s
decisions to delay marriage and motherhood. Age affects
PRESERVATION FEMALES
the natural ability of women to get pregnant. With the Fertility preservation will be beneficial to women at threat
development of sensitive tools to assess ovarian reserve, of future ovarian failure4 (Table 20.1).
160 The Infertility Manual Female Age and Fertility Preservation 161
Flowchart 20.1: Outcome of cancer therapy. Flowchart 20.2: Fertility-sparing interventions in female patients.6
Table 20.1. Indications for fertility preservation in
female patients.
Fig. 20.1: Decline of ovarian reserve with age.1 Indication Condition
Any cancer Ovarian surgery,
chemotherapy, radiotherapy 3. Alternatives available such as donor embryos, donor b. Mature oocyte cryopreservation: With improved
Exposure to gonado- Haematological disorder: sickle cell gametes, and adoption. cryopreservation techniques oocyte cryopreservation
loxic agent anaemia, thalassaemia major and 4. Potential safety of future pregnancy after the gives implantation and clinical pregnancy rates com-
aplastic anaemia treatment. parable to cryopreserved embryos.8 It is no longer a
Surgical menopause Prophylactic oophorectomy; 5. Possibility of gestational surrogacy, if receiving pelvic research technique but a valid option for postpuber-
BRCA1 and BRCA2 mutation car- radiation therapy. tal females without male partner.
riers 6. Disposition of cryopreserved gametes and embryos
Benign ovarian disease requiring in the event of death. Advantages
multiple operation: severe endome- 1. Spermatozoa not required
7. Infectious disease testing.
triosis 2. Avoids quandaries related to the storage and disposal
Mental health professionals, genetic and financial
Premature ovarian Chromosomal and genetic abnor- of embryos.
failure malities counselors should be available to counsel the patient and
Turner’s syndrome help them in decision making.6
Trisomy X syndrome (47, XXX) Ovarian Stimulation for Cryopreservation
Fig. 20.2: Risk of ovarian failure post chemotherapy depends on sev-
Fragile X syndrome (Martin-Bell
eral factors such as type of drug, cumulative dose of drug, and age
syndrome)
Fertility Preservation Strategies Ovarian Stimulation in Different
of patient. Risk of ovarian failure is greatest with pelvic radiation and
lowest with antimetabolites. Risk is also higher in women with lower
Autoimmune diseases Choice of fertility preservation strategy dependss on sev- Phases of Menstrual Cycle
ovarian reserve, higher age and high cumulative drug dose. Environmental factors eral factors7:
a. Patient’s age a. Ovarian stimulation in the menstrual phase: Stand-
Women wishing to Anticipated gamete exhaustion
postpone motherhood b. Type of disease ard Antag protocol. Addition of letrozole/tamoxifene
Criteria for Selection of Patient for c. Spread of the disease and mild stimulation protocol if estrogen positive
Fertility Preservation5 d. Planned treatment tumor.9
threatening therapies. Interdisciplinary collaboration is e. Time available b. Ovarian stimulation in the preovulatory phase. If
1. Age less than 40 years dominant follicle is present, it should be aspirated
important between oncologists, reproductive endocrinol- f. Whether she has a partner.
2. Tumor type, disease stage, overall health status of the There are several methods of fertility preservation
ogists, urologists, counselor, and reproductive surgeons and vitrified followed by GnRh antagonist for 5 days.
patient (Flowchart 20.2). Some are established methods others
trained in fertility preservation techniques to help the c. Ovarian stimulation in the luteal phase: GnRH
3. Time available investigational.
patient. antagonists for 5 days to induce immediate luteolysis
4. Cost
followed by gonadotropins.9
5. Greater than 50% chances of 5-year survival.
Reproductive Counseling ESTABLISHED METHODS OF
Fertility Management of Counseling reduces psychological distress in the patients. FERTILITY PRESERVATION GnRH Agonist Trigger to Reduce
Cancer Patients Health care providers should discuss: (FLOWCHARTS 20.3 AND 20.4) the Incidence of OHSS
1. Fertility regardless of age and marital status before According to ASRM established methods of fertility pres- Ovarian hyperstimulation syndrome should be avoided at all
Multidisciplinary Approach initiation of therapy. ervations are as follows:6 cost in Cancer patients by using GnRH agonist trigger instead
The ASRM practice committee recommends a multidis- 2. Possible reproductive consequences of cancer a. Embryo cryopreservation: With proven success of the traditional hCG trigger.10 It also prevents the risk of arte-
ciplinary approach in the care of patients facing fertility treatment. rate, this is an ideal option for post pubertal females. rial thrombosis11 associated with OHSS in cancer patients.
162 The Infertility Manual Female Age and Fertility Preservation 163
Flowchart 20.3: Options for ovarian tissue cryopreservation.15,18–20 Flowchart 20.5: Fertility preservation options post treatment.
sensitive tumors. While several live births reported, still protection during chemotherapy, with a discussion of the
considered investigational as safety and efficacy lacking.14 known benefits. GnRH analogs should commence at least
10 days before chemotherapy and should continue till
Ovarian Tissue Cryopreservation15-18 2 weeks after the end of chemotherapy.22
Three options (Flowchart 20.3)
1. Ovarian cortical tissues Post-treatment Fertility Preservation
2. Ovarian follicles The ideal time to preserve fertility is before chemotherapy
3. Whole ovary. as in some patients post chemotherapy ovarian failure
is permanent. In such cases adoption and egg donation
Indications
should be suggested (Flowchart 20.5).
1. Prepubertal females
2. Those who cannot delay cancer treatment in order to
Flowchart 20.4: Decision algorithm for fertility preservation. INVESTIGATIONAL PROCEDURES undergo ovarian stimulation and oocyte retrieval Pregnancy after Cancer Treatment
3. Estrogen sensitive tumors. Children born from cryopreserved embryos appear
Fertility-sparing Surgeries in Females healthy. Studies have shown no increased risk of con-
Limitations genital malformations in children of cancer survivors.23,24
The global rates of fertility-sparing surgery in females are
currently unknown. Young women presenting with bor- 1. Technically demanding, expertise required Risk of long-term damage to DNA after chemo—or
derline tumors and early-stage cancers may be offered 2. Two surgeries are required radiotherapy—unknown.
conservative surgeries like unilateral oopherectomy and 3. Loss of follicles due to ischemia19
4. Graft has limited life span • Present recommendations suggest a wait period of at
radical trachelectomy.12 Patient should understand the least 6 months post chemotherapy before undertaking
risk of recurrence and the need for strict follow-up. 5. Potential for reseeding tumor cells.20
oocyte and embryo cryopreservation and before con-
Oophoropexy (Ovarian Transposition) Ferto-protective Adjuvant Therapy ception.3
A surgical procedure, moving the ovaries as far as possible, “Ferto-protective adjuvant” is an attenuating agent that if
out of field of radiation, thus protecting the ovaries.13 administered during or prior to chemotherapy can pre- ASCO GUIDELINES
vent loss of ovarian follicle reserve.21
American Society of Clinical Oncology recently made rec-
Limitations of Oophoropexy Investigational drugs are as follows:
ommendations on fertility preservation strategies.25 Some
1. Sphingosine-1-phosphate (S1P)
1. In 60–90% of cases it leads to ovarian failure. of the principle recommendations are as follows:
2. Imatinib
2. Transvaginal oocyte retrieval becomes difficult if
3. Thalidomide 1. Fertility preservation should be discussed with all
in vitro fertilization is required in the future.
4. Granulocyte colony-stimulating factor patients of reproductive age group about to embark
Possible Drawbacks to Superovulation in 3. Cannot be done in the patients who require addi-
5. Tamoxifen.
tional chemotherapy. on cancer treatment.
Young Women with Cancer 4. Two surgeries are required with added complications
So far, the only drug used in clinical practice is the gon- 2. Interested patients should be referred to reproductive
adotropin-releasing hormone (GnRH) agonist.
1. Delay in initiating cancer treatment associated with surgery. specialists.
2. Risk of elevation of oestradiol concentration 3. Fertility preservation should be addressed before the
3. Most breast cancers in young women are ER positive In Vitro Maturation of Oocytes (IVM) GnRH Analogs treatment starts.
4. Increased risk of arterial thrombosis11 Circumvents the need for ovarian stimulation, indicated Data regarding benefits are lacking. Given the lack of 4. All the discussions pertaining to fertility preservation
5. Not useful in prepubescent female in women requiring urgent cancer treatment or estrogen apparent risk, they could be considered for ovarian should be documented in the medical record.
164 The Infertility Manual Female Age and Fertility Preservation 165
5. Concerns regarding impact of fertility preservation on CONCLUSION 6. American Society for Reproductive Medicine. Fertility 18. Bedaiwy MA, Jeremias E, Gurunluoglu R, Hussein MR,
cancer treatment should be addressed. preservation in patients undergoing gonadotoxic therapy Siemianow M, Biscotti C, et al. Restoration of ovarian
6. Appropriate referral to psychosocial support providers The scope of fertility preservation has widened from can- or gonadectomy: a committee opinion. Fertil Steril. function after autotransplantation of intact frozen–thawed
cer to all patients who anticipate gamete exhaustion natu- 2013;100:1214-23. sheep ovaries with microvascular anastomosis. Fertil Steril.
if they experience distress about potential infertility.
rally or due to any iatrogenic causes. Fertility preservation 7. Rodriguez-Wallberg KA, Oktay K. Fertility preservation 2003;79:594-602.
7. Patients should be encouraged to participate in regis- during cancer treatment: clinical guidelines. Cancer
gives hope to these patients of future reproduction. Multi- 19. Baird DT, Webb R, Campbell BK, Harkness LM, Gosden
tries and clinical studies. Manage Res. 2014;6:105-17. RG. Long-term ovarian function in sheep after ovariectomy
8. Patient should be informed about both established disciplinary team approach is the key to its success. Fertil- 8. Cobo A, Kuvayama M, Perez S, Ruiz A, PellIcer A, Remohi J. and transplantation of autografts stored at –196°C.
(embryo and oocyte cryopreservation) and experi- ity preservation should be discussed with all the patients Comparison of concomitant outcome achieved with fresh Endocrinology. 1990;140:462-71.
mental (ovarian tissue cryopreservation) methods of of reproductive age group about to embark on fertility and cryopreserved donor oocytes vitrified by cryotop 20. Sonmezer M, Shamonki MI, Oktay K. Ovarian tissue
fertility preservation. threatening therapy. Multiple techniques are available for method. Fertil Steril. 2008;89:1657-64. cryopreservation: benefits and risks. Cell Tissue Res.
9. Reddy J, Oktay K. Ovarian stimulation and fertility
9. Patient should be informed about conservative fertility fertility preservation among which embryo and oocyte 2005;322:25-32.
preservation with the use of aromatase inhibitors in
sparing surgeries and the option of ovarian transposi- cryopreservation are the only established techniques of 21. Woodruff TK. Preserving fertility during cancer treatment.
women with breast cancer. Fertil Steril. 2012;98:1363
tion (oophoropexy) should be discussed when pelvic fertility preservation. Many new promising methods will Nat Med. 2009;15:1124-5.
10. Oktay K, Türkçüoǧlu I, Rodriguez-Wallberg KA. GnRH
radiation therapy is performed as cancer treatment. 22. Blumenfeld Z. How to preserve fertility in young women
be available in the near future. agonist trigger for women with breast cancer undergoing
10. Patients should be informed that there is insufficient fertility preservation by aromatase inhibitor/FSH exposed to chemotherapy? The role of GnRH agonist
stimulation. Reprod Biomed Online. 2010;20(6):783-8. cotreatment in addition to cryopreservation of embryo,
evidence regarding the effectiveness of ovarian sup-
pression (gonadotropin-releasing hormone analogs) PROBABLE QUESTIONS 11. Somigliana E, Peccatori FA, Filippi F, Martinelli F, oocytes, or ovaries. Oncologist. 2007;12:1044-54.
Raspagliesi F, Martinelli I. Risk of thrombosis in women with 23. Winther JF, Boice JD, Jr, Mulvihill JJ, Stovall M, Frederiksen K,
as a fertility preservation method, and these agents 1. Define fertility preservation and indications for fertil- malignancies undergoing ovarian stimulation for fertility Tawn EJ, et al. Chromosomal abnormalities among offspring
should not be relied on to preserve fertility. ity preservation. preservation. Human Reprod Update. 2014;20(6):944-51. of childhood-cancer survivors in Denmark: a population-
2. Short note on strategies of fertility preservation in 12. Dargent D, Mathevet P. Radical laparoscopic vaginal based study. Am J Hum Genet. 2004;74(6):1282-5.
The Future post-pubertal female in preovulatory phase for urgent hysterectomy. J Gynecol Obstet Biol Reprod. (Paris). 24. Fosså SD, Magelssen H, Melve K, Jacobsen AB, Langmark F,
chemoradiotherapy after 5 days. 1992;21(6):709-10. Skjaerven R. Parenthood in survivors after adulthood
Creation of Gametes de Novo 3. Stimulation protocol in a 35-year-old woman with
13. Bisharah M, Tulandi T. Laparoscopic preservation of cancer and perinatal health in their offspring: a preliminary
ovarian function: an underused procedure. Am J Obstet report. J Natl Cancer Inst Monogr. 2005;(34):77-82.
In future, it would be possible to create gametes from stem estrogen receptor-positive stage I breast cancer with Gynecol. 2003;188:367; Ronn R, Holzer HE. Oncofertility in 25. Loren AW, Mangu PB, Beck LH, Brennan L, Magdalinski AJ,
cells in lab by means of “In vitro gametogenesis,” a prom- PCOS. Canada: gonadal protection and fertility sparing strategies.
Partridge AH, et al. Fertility preservation for patients with
ising fertility preservation option that is actively being 4. Short note on ASCO guidelines on fertility Curr Oncol. 2013;20:e602.
cancer: American Society of Clinical Oncology Clinical
developed for both males and females.26 preservation. 14. Practice Committee of American Society for Reproductive
Medicine. In vitro maturation: a committee opinion. Fertil Practice Guideline Update. J Clin Oncol. 2012;31(19):2500-
5. Short note on reproductive counseling in fertility 10. © 2013 by American Society of Clinical Oncology.
Steril. 2013;99:663-6.
In Vitro Follicle Maturation and Culture preservation. 15. Donnez J, Jaboul P, Squifflet J, Van Langendonckt A, 26. R. Vassena, C. Eguizabal, B. Heindryckx, K. Sermon, C.
6. Ethical and legal issues in fertility preservation. Donnez O, Van Eyck AS, et al. Ovarian tissue cryopreser- Simon, A.M.M. van Pelt, et al. on behalf of the ESHRE
Currently a research procedure, it holds promise for young
vation and transplantation in cancer patients. Best Prac Special Interest Group Stem Cells in Reproductive
cancer patients who wish to preserve their fertility. Medicine: Ready for the Patient? Human Reprod. 2015;
Res Clin Obstet Gynecol. 2010;24:87-100.
REFERENCES 16. American Society for Reproductive Medicine. Ovarian 1-8.
Artificial Ovaries27 1. Baker TG. A quantitative and cytological study of germ tissue and oocyte cryopreservation. Fertil Steril. 2006;86: 27. Amorim A, Shikanov A. The artificial ovary: current status
cells in human ovaries. Proc R Soc Lond B Biol Sci. S142-7. and future perspectives. Future Oncol. 2016;12(20):2323-32.
Ethical Issues 1963;158:417-33. 17. Bedaiwy MA, Hussein MR, Biscotti C, Falcone T. 28. Ethics Committee of the American Society for Reproductive
2. Meirow D, Dor J, Kaufman B, Shrim A, Rabinovici J, Schiff E, Cryopreservation of intact human ovary with its vascular Medicine. Fertility preservation and reproduction in cancer
Fertility preservation raises many complex ethical issues:28 pedicle. Human Reprod. 2006;21:3258-69. patients. Fertil Steril. 2005;83:1622-8.
et al. Cortical fibrosis and blood vessels damage in human
a. Disposition of stored tissue, gametes or embryos in ovaries exposed o chemotherapy. Potential mechanisms of
event of demise or divorce ovarian injury. Hum Reprod. 2007;22:1626-33.
b. Posthumous use of stored samples for reproduction 3. Roness H, Kalich-Philosoph L, Meirow D. Prevention of
or research chemotherapy-induced ovarian damage: possible roles
for hormonal and non-hormonal attenuating agents. Hum
c. Posthumous sperm retrieval in absence of consent
Reprod Update. 2014;20:759.
d. Informed consent for the harvesting, storage, and use
4. Gidoni Y, Holzer H, Tulandi T, Tan SL. Fertility preservation
of germ cells for fertility preservation in children and in patients with non-oncological conditions. Reprod
adolescents Biomed Online. 2008;16:792-800.
e. Future of offspring in case of demise 5. Noyes N, Knopman JM, Melzer K, Fino ME, Friedman B,
f. Risk of disability and increased risk of cancers in Westphal LM. Oocyte cryopreservation as a fertility
offsprings preservation measure for cancer patients. Reprod BioMed
g. Ethical issues associated with third party reproduction. Online. 2011;23:323-33.
C HA PT E R
21
Congenital Uterine Malformations and Reproduction 167
Congenital Uterine Malformations
and Reproduction
1. Thickening of lateral wall causing narrowing of cavity
Santosh Kumar Jena, Kamini A Rao 2. Inverse corpus to cervix ratio and cavity narrowing
but without associated lateral thickening
Chapter Outline
• Embryological Development • ESHRE Classification
• Prevalence • Impact on Reproductive Outcome
• Etiology • Diagnosis
• Classification • Management 1. Straight or curved
interostial line.
2. Internal indentation
at the fundal mid-
INTRODUCTION • The gold standard diagnostic modalities such as lap- line not exceeding
aroscopy and hysteroscopy are invasive procedures 50%
Congenital uterine malformations are a heterogeneous and thus cannot be applied to low risk population. Fig. 21.1: Class U0—normal uterus. Fig. 21.2: Class U1—dysmorphic uterus.
group of disorder that may adversely affect reproduc- The most recent prevalence study by YY Chan2 is men-
tive potential. Although in recent times there has been a tioned in (Table 21.2).
paradigm shift in diagnostic approach of congenital uter-
ine anomalies, from more invasive and expensive proce- ETIOLOGY
dures like endoscopy and MRI to 3-D USG but till today
evidences regarding its effect on reproductive outcome & • Although the exact etiology is still unclear, increased
management falls into the gray zone. In this chapter we will chances of anomalies arising from mesodermal origin in
be focusing on diagnostic approach & recent evidences in patients having uterine anomaly suggest a shared cause.3
management of the same.
CLASSIFICATION
EMBRYOLOGICAL DEVELOPMENT The basis of classifying uterine anomalies depending on
Mullerian developmental process is proposed by Acien P.4
Sequence of genital development is mentioned in accord-
1. Partial or total agenesis of one (unicornuate) or both
ance with Haley A1 in (Table 21.1).
Müllerian ducts (MRKH syndrome)
Internal indentation
2. Partial or total absence of fusion of both Müllerian at the fundal midline
PREVALENCE ducts (bicornuate uterus and didelphys uterus) exceeding 50%
It is indeed difficult to assess true prevalence of congenital 3. Partial or total absence of resorption of septum (par- of the uterine wall
thickness
uterine anomaly because: tial or complete uterine septum)
4. Lack of later development (T-shaped uterus and
• None of the available classifications is universally Fig. 21.3: Class U2—septate uterus.
hypoplastic uterus)
accepted.
5. Segmental defects and combination of different
anomalies. Among various available classifications, the most logical developmental process are categorized to
Table 21.1. Timetable of genital development.
recent one given by the European Society of Human Repro- different classes.
Time (weeks c. Classification into subclasses is based on clinically
Table 21.2. Incidence of congenital uterine anomaly.2 duction and Embryology/European Society for Gynaeco-
Phase of genital development1 of gestation)
Unselected population—5.5% logical Endoscopy (ESHRE/ESGE) is mentioned here.4 The significant variations among different anomalies
Indifferent gonadal phase 4–6 within a class.
Infertile women—8% same classification is illustrated in (Figs. 21.1 to 21.6) for
Gonadal differentiation 7 d. Vaginal and cervical abnormalities are mentioned
easy understanding.
Lateral fusion of both paramesonephric ducts 7–9 Miscarriage group—13.3% separately in the present classification.
Infertile women who also had a history of miscarriage—24.5%
Vertical fusion occurs in the eighth week 8 ESHRE CLASSIFICATION5
when the lower most fused parameso- Most common anomaly in an unselected group of women— IMPACT ON REPRODUCTIVE OUTCOME
nephric ducts fuse with the ascending arcuate uterus Main concepts of classification are as follows:
Altered volume and altered vasculature theory are the
endoderm of the sinovaginal bulb Most common anomaly in all of the high-risk groups— a. Anatomy is the basis of classification. two most accepted ones to understand the mechanism by
Resorption of midline septum 20 canalization defect b. Anatomical variations resembling similar embryo- which congenital anomaly affect reproductive outcome.6-10
168 The Infertility Manual Congenital Uterine Malformations and Reproduction 169
• Syndactyly
Table 21.3. Conception rate in different uterine anomalies.
• Scoliosis
Infertility perspective in terms of
Urologic abnormality conception rate in comparison with
• Unilateral renal agenesis normal uterus as control12
• Ectopic or horseshoe kidney Anomaly Risk ratio (95% CI)
External • Duplication of the collecting systems Unicornuate uterus 0.74 (0.39–1.41)
indentation • Hydronephrosis. Didelphys uterus 0.9 (0.79–1.04)
is more than
50% of wall
Conception rate in different uterine anomalies is men- Bicornuate uterus 0.86 (0.61–1.21)
thickness tioned below in accordance with study by YY Chan12 in Septate uterus 0.86 p value <0.05 (0.77–0.96)
at midline (Table 21.3).
fundus
Arcuate uterus 1.03 (0.94–1.12)
Fig. 21.4: Class U3—bicorporeal uterus.
DIAGNOSIS 13 2D sonography—86.6
Hysterosalpingography—86.9%
Unilateral uterine development Absent development • CT scanning is no longer considered to be a diag- • MRI is rarely used as a primary screening tool. MRI is
Contralateral part could be of either of the horns nostic modality due to poor depiction of soft tissue mainly used to subclassify the uterine anomaly.
either incompletely formed or structures
absent
• Different diagnostic modalities are discussed along
• Overall, diagnostic accuracy of different procedures with their advantage and disadvantage in Table 21.4.
3D sonography—97.6% • Diagnostic modalities in different category of patients
Sono hysterography—96.5% is mentioned in Table 21.5.
Table 21.4. Diagnostic modalities for uterine anomaly.
Diagnostic modality Advantage Disadvantage
Gynecological •• Still considered as first step in •• Blind procedure
examination approaching any gynecologic case •• It is always very subjective with a wide inter-observer
•• In experienced hands, it is often very variation
informative •• Cannot be applied to patients who have never been
Fig. 21.5: Class U4—hemiuterus. Fig. 21.6: Class U5—aplastic uterus. •• Inspection of cervix and vagina can add sexually active
information to reach a diagnosis
•• Local tenderness can be elicited much
Theories explaining obstetric • Second-trimester pregnancy loss precisely
complication in uterine anomaly
• Cervical incompetence X-ray hysterosal- •• Basic diagnostic modality and readily •• The procedure is painful as carried out mostly
• Intrauterine growth retardation pingography available in most centers without any anesthesia, preferably should
• Abnormal presentation •• Printable X-ray film can be re-evaluated prescribe some anti-spasmodic agents
• High presenting part later if required 30–60 minutes prior
•• Gives information regarding tubal •• Radiation exposure is a major risk so ideally should
• Preterm labor morphology and intra-cavitary lesion be done postmenstrual phase
• Dystocia (Asherman syndrome, fibroid, polyp) •• Cannot differentiate between bicornuate uterus and
Altered volume theory6-9 Altered vasculature theory10
• Stillbirth septate uterus
•• Diminished function of the •• Vascular supply is altered
myometrium and/or endo- and erratic in uterine ano • Retained placenta •• No information regarding noncommunicating rudi-
metrial cavity malies in comparison to mentary horn if present
Gynecologic problems •• The procedure has a very high false negative value
•• Reduced volume & normal uterus
unyielding cavity for foetal
• Dysmenorrhea so all tubal blocks should be reconfirmed with a
development • Dyspareunia laparoscopic chromopertubation
• Cyclic abdominal pain Two-dimensional •• Simple, easily available •• Getting a required plane and optimum image quality
• Endometriosis ultrasound •• OPD procedure is much dependent on experience of sonologist
Complications Associated with • Primary amenorrhea •• Still pictures and procedure video can be •• Transvaginal scan which gives better information
Congenital Uterine Anomaly11 Skeletal abnormality
saved and reviewed later regarding pelvic pathologies cannot be applied to
•• Additional information regarding uterine, patients who have never been sexually active
Obstetric complications • Hemivertebrae ovarian, and tubal pathology
• Recurrent first-trimester loss • Supernumerary vertebrae •• Associated renal pathology can be screened
• Ectopic pregnancy • Vertebral fusion Contd...
170 The Infertility Manual Congenital Uterine Malformations and Reproduction 171
Contd... Müllerian Agenesis • The option of surgical metroplasty should be indi-
Table 21.4. Diagnostic modalities for uterine anomaly. vidualized. Co-existing vaginal septum should be cor-
• Reassurance and support are integral part of the rected.19
Diagnostic modality Advantage Disadvantage management apart from surgical procedures such as
Three-dimensional •• It is becoming very popular imaging •• Experience of sonographer is the major determinant vaginal dilators, vaginal anastomosis, Mc Indole vagi-
ultrasound modality as it offer evaluation in coronal in getting desired image
noplasty, or tubal unification procedure depending of Bicornuate Uterus
plane and provision of rendering in differ-
ent angles to get desired image extent and site of Müllerian segment affected. • The detection of a bicornuate uterus without a signifi-
•• Stored data can be reevaluated later and • Affected women should be counseled that although cant adverse history does not mandate intervention.
rendering can be done later to reduce they are infertile, normal sexual function can be • Unification procedure can be considered in patients
procedure duration
expected and that genetic offspring can be achieved by with history of recurrent pregnancy loss and preterm
•• Information equivalent to laparoscopy can
be obtained using different software in vitro fertilization (IVF) with surrogate transfer. labor.
MRI •• No radiation exposure •• Expensive and not readily available
• Uterine transplantation although looks promising • Complexity of repair and need for abdominal approach
•• It provides objective information in •• Contraindicated in claustrophobia after recently reported live birth following the proce- should always be assessed during case selection.
different planes •• Need more experience for interpretation of result to dure in Sweden,14 still the debate continues whether
•• Complex anomalies and associated reach at a diagnosis to offer transplantation for such non-life threatening Evaluation of bicornuate uterus prior to IVF:20
abnormality can be better diagnosed •• Examination planes are fixed, predefined, and inde- • In a bicornuate uterus, two factors need to be consid-
conditions?
pendent of the examiner ered: ease of catheter placement into one cavity or the
• Surely uterine transplant is an option in countries
Hysteroscopy •• Detailed and reliable information is •• No information about outer contour of uterus and other and differential endometrial response in each
obtained about vagina, cervix, and uterine wall thickness where surrogacy is not legally allowed but more has to
be explored in regards to reduce the cost, complication side.
cavity •• Cavity evaluation is not possible in case of blind
•• The whole procedure can be recorded vagina or conditions where negotiation of hystero- rate, and training in this field for betterment of proce- • Single embryo transfer is highly recommended.
and stored for future evaluation scope is not possible dure.15
•• Minimally invasive procedure
•• Additional opportunity of treating at the Septate Uterus
same setting Unicornuate Uterus • The decision regarding intervention is based on extent
Combined hyster- •• Till now, it is considered as gold standard •• Anesthetic risk of the septum. Simple and subtle divisions that are
olaparoscopy procedure •• Objective estimation of the uterine wall thickness is • Pregnancy outcome is not affected by mere presence
•• Detailed evaluation of all pelvic struc- not possible of a rudimentary horn, when the pregnancy sac is restricted to the very top of the cavity are probably nor-
tures, upper abdominal structures, and •• It is often seen that diagnosis of complex anomalies located in unaffected horn.16 mal variants and require no intervention. Divisions that
peritoneum to reach at a diagnosis is often subjective and dependent on experience of • Presence or absence of functional endometrium is extend deeply into the cavity and result in two constricted
•• Minimally invasive procedure surgeon cavities require intervention and surgical correction.
deciding factor whether to treat or not.17
• Excision should be considered in presence of func- • Benefits of metroplasty in terms of clinical pregnancy
tional endometrium. rate are controversial.21
Table 21.5. Diagnostic workup. • A detailed description of the ovaries particularly on the • Previous pregnancy complications are major determi-
Asymptomatic Symptomatic high risk affected side is important. Location of the ovary on the nant for surgical correction.22,23
women population Complex anomaly Symptomatic adolescent affected side is frequently along the line of migration. • Hysteroscopic route should always be preferred given
•• Gynecological •• Gynecological examination •• Abdominal and/or tran- •• Gynecological examination Inclusion of this detail will be important should the the relative simplicity of the procedure.
examination •• 2D USG srectal 3D USG •• Abdominal and/or transrectal 2D • Interostial line should be taken as a guide to decide
patient elect to move to IVF. An abdominally located
•• 2D sonography •• 3D USG •• MRI and 3D US extent of resection.
•• HSG •• Hysterolaparoscopy with •• MRI as a first-line diagnostic ovary may be accessed for retrieval through transab-
•• HyCoSy, 2D or 3D surgical correction in same procedure dominal ultrasound guidance or laparoscopically. • Intrauterine adhesion formation is of major concern
sonohysterography setting •• Hysterolaparoscopy with surgical • Single embryo transfer is highly recommended. postseptal resection. Its occurrence can be reduced
•• Hysteroscopy and, in correction in same setting with concurrent use of any of available adhesion pre-
cases of suspected adnexal vention agents such as hormone therapy, mechanical,
pathology, laparoscopy Didelphys Uterus and chemical agents.
• Compared with other uterine anomalies, it has a good • A second-look hysteroscopy or a 3D ultrasonography
MANAGEMENT • Pain management and addressing issues such as prognosis in terms of pregnancy rate.18 is advisable to rule out adhesion formation and recur-
endometriosis which is more common in congenital • In a recent meta-analysis, pregnancy rate and miscar- rence before proceeding with embryo transfer.
Management of congenital uterine abnormalities focuses uterine. • Embryo transfer should be deferred for 4–6 weeks after
riage rate were unaffected in presence of didelphys
on the following:
Previous obstetric performance is a major determi- uterus but mal-presentation and preterm labor were resection.
• Improvement in reproductive outcome. nant to decide for a particular management strategy. increased.12 • Single embryo transfer is highly recommended.24
C HA PTE R
Tubal Factor Infertility and Ectopic
22
172 The Infertility Manual
Arcuate Uterus of congenital uterine anomalies. Ultrasound Obstet
Gynecol. 2003;21:578-82.
Pregnancy—Diagnosis and
• The arcuate uterus is considered as a normal variant
with no clinical significance.25
9. Saravelos SH, Cocksedge KA, Li TC. The pattern of
pregnancy loss in women with congenital uterine Management
• Its surgical correction is not advised.26 anomalies and recurrent miscarriage. Reprod Biomed
Online. 2010;20(3):416-22.
Richa Sharma
10. Buchell RC, Creed F, Rasoulpour M, Whitcomb M. Vascular
PROBABLE QUESTIONS anatomy of the human uterus in pregnancy wastage. Br J
Chapter Outline
Obstet Gynaecol. 1978;85:698-706.
1. Briefly discuss with diagram about different classi 11. Elyan A, Saeed M. Mullerian duct anomalies: clinical • Evaluation of Tubal Factor—Diagnosis • Management of Tubal Pathology
concepts. ASJOG. 2004;1:11-20. • RCOG Recommendations for Tubal Assessment • Diagnosis and Management of Ectopic Pregnancy
fi
cation systems used for congenital uterine • European Society for Human Reproduction and • Human Chorionic Gonadotropin Measurements in Women with
malformations. 12. Chan YY, Jayaprakasan K, Tan A, Thornton JG,
Embryology Guidelines Pregnancy of Unknown Location
Coomarasamy A, Raine-Fenning NJ. Ultrasound Obstet
2. Discuss with evidence regarding impact of Müllerian Gynecol. 2011;38:371-82.
anomalies on reproductive outcome. 13. Grimbizis GF, Di Spiezio Sardo A, Saravelos SH, Gordts S,
3. Discuss about evidence-based management of con- Exacoustos C, Van Schoubroeck D, et al. The Thessaloniki INTRODUCTION pregnancy to happen naturally, we need to have normal
genital uterine malformations. ESHRE/ESGE consensus on diagnosis of female genital parameters of both male and female along with the func-
4. Discuss about Müllerian development and its defects. anomalies. Hum Reprod. 2016;31:2-7. Tubal factor infertility (TFI)—tubal factor is one of the first tional and dynamic fallopian tubes:
14. Brannstrom M, et al. Livebirth after uterus transplantation. indication for in vitro fertilization (IVF), and it’s a type • Dynamic conduit and not a passive channel
5. Discuss about different diagnostic modalities used for
Published online in www.thelancet.com, October 5, 2014. • Role in sperm transport and capacitation
diagnosis of Müllerian anomaly. of female infertility factor in which fallopian tubes (uni-
15. Brannstrom M, Johannesson L, Dahm-Kähler P, Enskog A, • Ova retrieval and transport
Mölne J, Kvarnström N, et al. First clinical uterus transplantation lateral/bilateral) are damaged by infections, congenital
malformations, and scarring or may be blocked leading to • Fertilization
trial: a six-month report. Fertil Steril. 2014;101:1228-36.
REFERENCES 16. Spitzer RF, Kives S, Allen LM. Case series of laparoscopically impediment of descent of a fertilized ovum into the uterus • Embryo storage, nourishment, and transport.
1. Healey A. Embryology of the female reproductive tract. resected non-communicating functional uterine horns. J and the resulting pregnancy/live birth. Tubal factors con-
In: Mann GS, Blair JC, Garden AS, (eds). Imaging of Pediatr Adolesc Gynecol. 2009;22(1):e23-e28.
tribute to 25–30%.1 Chlamydia and Mycoplasma are pre- Causes and Risk Factors in Tubal Damage
Gynaecological Disorders in Infants and Children, Medical 17. Sadik S, Taskin O, Sehirali S, Mendilcioglu I, Onoğlu AS,
Radiology. Berlin Heidelberg: Springer-Verlag; 2012;21-30. Kursun S, et al. Complex mullerian malformation: report ventable causes with unfavorable pregnancy outcome. (Figs. 22.2A to C; Flowchart 22.1)
2. Chan YY, Jayaprakasan K, Zamora J, Thornton JG, of a case with a hypoplastic non-cavitated uterus and two Infections may cause the microcilia damage affecting
Raine-Fenning N, Coomarasamy A. The prevalence of rudimentary hors. Hum Reprod. 2002;17:1343-4. transportation of the oocyte through the tube.2,3 • Inflammatory conditions—such as endometriosis
congenital uterine anomalies in unselected and high-risk 18. Heinonen PK, Kuismanen K, Ashorn R. Assisted • Infections presenting as PID
populations: a systematic review. Hum Reprod Update. reproduction in women with uterine anomalies. Eur J
2011;17:761-71. Obstet Gynecol Reprod Biol. 2000;89:181-4.
Parts of a Fallopian Tube (Fig. 22.1) • Space occupying lesions in some cases, especially
toward the corneal end (submucosal fibroids)
3. Taylor E, Gomel V. The uterus and fertility. Fertil Steril. 19. Reichman DE, Laufer MR. Congenital uterine anomalies Parts of a fallopian tube are isthmus, ampulla, infundibu-
2008;89:1-16. affecting reproduction. Best Pract Res Clin Obstet Gynaecol. • Tubal ligation removal
lum, and fimbriae.
4. Acien P, Acien MI. The history of female genital tract 2010;24:193-208. • Complications from lower abdominal surgery such as
malformation classifications and proposal of an updated 20. Letterie GS. Management of congenital uterine cesarean section
system. Hum Reprod Update. 2011;17:693-705. abnormalities. RBM Online. 2011;23:40-52. Functions of a Fallopian Tube • Genital tuberculosis
5. Grimbizis GF, Gordts S, Di Spiezio Sardo A, Brucker S, De 21. Brucker SY, Rall K, Campo R, Oppelt P, Isaacson K.
Angelis C, Gergolet M, et al. The ESHRE/ESGE consensus Treatment of congenital malformations. Semin Reprod Fallopian tubes work as the connecting link between the • Pregnancy complications such as ovarian torsion and
on the classification of female genital tract congenital Med. 2011;29:101-12. male and female gametes (sperm and the ova) inside. For ectopic pregnancy with recurrence risk.
anomalies. Hum Reprod. 2013;28:2032-44. 22. Letterie GS. Management of congenital uterine
6. Propst AM, Hill JA. Anatomic factors associated abnormalities. Reprod Biomed Online. 2011;23:40-52. Incidence of Tubal Damage
with recurrent pregnancy loss. Semin Reprod Med. 23. Revel A. Defective endometrial receptivity. Fertil Steril.
2000;18:341-50. 2012;97:1028-32. Tubal factor is seen in 14% cases4 with proximal obstruc-
7. Salim R, Regan L, Woelfer B, Backos M, Jurkovic D. 24. Galliano D, Bellver J, Díaz-García C, Simón C, Pellicer A. tion contributing to 10–25%. Out of all factors, pelvic
A comparative study of the morphology of congenital ART and uterine pathology: how relevant is the maternal inflammatory disease (PID) is responsible for more than
uterine anomalies in women with and without a history side for implantation? Hum Reprod Update. 2015;21:13-38.
of recurrent first trimester miscarriage. Hum Reprod. 25. Olpin JD, Heilbrun M. Imaging of Müllerian duct anomalies.
half causes of tubal factor.
2003;18:162-6. Clin Obstet Gynecol. 2009;52:40-56.
8. Salim R, Woelfer B, Backos M, Regan L, Jurkovic D. 26. Devi Wold AS, Pham N, Arici A. Anatomic factors in recurrent Pathophysiology
Reproducibility of three-dimensional ultrasound diagnosis pregnancy loss. Semin Reprod Med. 2006;24:25-32.
Fallopian tubes work on the concept of a complex and
coordinated neuromuscular activity, microcilia action
along with the local endocrine factors working in
Fig. 22.1: Parts of the fallopian tube. cohort.
174 The Infertility Manual Tubal Factor Infertility and Ectopic Pregnancy—Diagnosis and Management 175
EVALUATION OF TUBAL Generally, it is done without anesthesia but in difficult
cases or on patient request can be done under anesthesia
FACTOR—DIAGNOSIS
with written informed consent.
Assessment of Fallopian Tubes (Table 22.1)
Tubal Cannulation with Advanced
Assessment of fallopian tubes is one of the most important Hysterosalpingography and Tubal
diagnostic test and very common also as part of the proto-
col, and there are various methods for this.5 Gold standard
Assessment Categories
test would be correct to predict and improve pregnancy It involves use of real time guidance with uterotubal
rates in a cost-effective manner.6 manipulation, tubal cannulation, fluoroscopy, and tubal
pressure assessments with better results such as:
A B Methods of Assessment with Reference Tubal cannulation and selective salpingography Its
Tubal blockage, hydrosalpinx (HSG) Ectopic pregnancy
fluoroscopic/hysteroscopic guided which not only assess
Hysterosalpingography
live visualization of patency but also tubal wall compli-
Hysterosalpingography (HSG) is the time tested and most ance8 by measurement of intratubal pressure.
cost-effective method for evaluating tubal patency.
Dye used—water /lipid soluble media is used. Endoscopic Methods9-11
Sensitivity is 78% with specificity of 96%. Laparoscopy and chromopertubation Its golden diag-
Useful in detecting— nostic test for checking tubal patency along with the over-
Proximal and distal tubal occlusion all condition of the pelvic organs and the peritoneal cavity.
Peritubular adhesions Dilute solution of methylene blue dye is introduced via
Fimbrial phimosis the cervix. It can identify blockage/occlusion along with
Rare conditions such as salpingitis isthmica nodosa (SIN).7 minor tubal factors such as hydrosalpinx, fimbrial phimo-
sis, endometriosis, or peritubal adhesions.
Table 22.1. Fallopian tube assessment tests.
C
Transcervical whalebone tubal catheterization,
Hysterosalpingo-contrast Sonography (HyCoSy)
Tuberculosis fallopian tube Smith (1849) It is an ultrasound-guided technique to check patency of
Figs. 22.2A to C: Causes of tubal damage.
Laparoscopy Jacobaeus (1910), Palmer (1947) tubes by using Echovist (solution of galactose and 1% pal-
Hysterosalpingogram, Carey (1914) mitic acid) or a mixture of air and saline. It has sensitivity
Flowchart 22.1: Causes and risk factors of tubal damage. of 93.3% and specificity of 89.7%.
Rubin’s test: tubal perfusion pressures
Oxygen Rubin, (1920) Salpingoscopy
Carbon dioxide, Rubin (1952) Salpingoscopy was originally performed during laparot-
Dye injections with culdocentesis, Decker (1952) omy for reconstructive tubal surgery to assess the mucosa
of the infundibulum and ampulla.
Injection of phenolsulfonphthalein, which has been
absorbed by the peritoneum if the Fallopian tubes were
patent, could then be detected in the urine, Speck (1970) Falloposcopy15
Injection of radiolabelled xenon solution with Pertynski Its transcervical microendoscopic procedure assessing
et al. (1977) from tubal ostia till the fimbrial end.
Gamma-camera screening
Transvaginal Hydrolaparoscopy (Fertiloscopy)
Selective salpingography and tubal catheterization,
Corfman and Taylor (1966) Fertiloscopy is a day case procedure done under sedation/
local anesthesia with sensitivity of 86%, reliability of 95%,
Salpingoscopy, Brosens et al. (1987)
and complications occur in 2% cases.
Falloposcopy, Kerin et al. (1990a) It is based on concept of staining the tubal cell nuclei
Tubal diseases can affect any segment of it depending obstruction (blockage), narrowing (stenosis), dilatation to assess functional capacity of the fallopian tubes such
Hystercontrast sonography Deichert (1993)
on the type of pathology and damage even if minimal and (hydrosalpinx), or may form a tuboovarian mass in some that more colored nuclei less functioning is the mucosa
superficial can damage it permanently. It can vary from situations. Fertiloscopy Watrelot et al. (1999)
and vice versa.
176 The Infertility Manual Tubal Factor Infertility and Ectopic Pregnancy—Diagnosis and Management 177
RCOG RECOMMENDATIONS FOR Cesarean Section and Tubal Infertility: Flowchart 22.2: Management of ectopic pregnancy.
TUBAL ASSESSMENT Is there an Association?
Women who are not known to have comorbidities (such Postsurgical adhesions in a cesarean section can affect the
as pelvic inflammatory disease, previous ectopic preg- tubal function leading to secondary subfertility later on.
nancy, or endometriosis) should be offered HSG to screen
for tubal occlusion because this is a reliable test for ruling MANAGEMENT OF TUBAL PATHOLOGY
out tubal occlusion, and it is less invasive and makes more
Deciding factors are as follows:
efficient use of resources than laparoscopy.
Where appropriate expertise is available, screening • Type of pathology
for tubal occlusion using HyCoSy should be considered • Grade/severity of pathology
because it is an effective alternative to hysterosalpingogra- • And segment of fallopian tube affected.
phy for women who are not known to have comorbidities.
Women who are thought to have comorbidities should
Tubal Surgery in the Era of Assisted
be offered laparoscopy and dye, so that tubal and other Reproductive Technology: Clinical Options
pelvic pathology can be assessed at the same time. Based on the evidence based medicine, here are the few
conclusions of the same:
EUROPEAN SOCIETY FOR HUMAN Tubal ligation reversal is the most common indica-
REPRODUCTION AND EMBRYOLOGY tion for tubal microsurgery.
Adhesiolysis has the best results. Laparoscopy is
GUIDELINES preferred over laparotomy. • Intramural pregnancy (in myometrium)
European Society for Human Reproduction and Embry- Proximal surgeries (tubocornual anastomosis) have • Vaginal pregnancy
ology (ESHRE) guidelines concluded that if there are no better results than the distal tubal disease due to • Multiple tubal pregnancy
concerns about pelvic or tubal health, it may be appropri- various confounding factors.12,13
ate to perform three cycles of ovulation induction prior to Early Pregnancy Assessment Services
checking tubal patency. DIAGNOSIS AND MANAGEMENT
Early pregnancy unit (EPU) is defined as a 5–7 days
OF ECTOPIC PREGNANCY
Recommendations by the American (per week) multidisciplinary service involving d octors,
(FLOWCHART 22.2) nurses, ultrasonographers, midwives, and support staff
Society for Reproductive Medicine for that provide early testing and care (up to 13 weeks) for
Tubal Factor Evaluation Definition pregnancy. Most of the EPUs are associated with gyne-
cology units, but are staffed separately.
Evaluation of tubal patency is a key component of the diag- An ectopic pregnancy is one in which the fertilized egg
Fig. 22.3: Female reproductive system showing ectopic pregnancy. Cases seen in EPUs—bleeding of any type (from light
nostic workup in infertile couples. All available methods implants in tissue outside of the uterus and the placenta
spotting to bloody clotting), brownish discharge, abdomi-
for evaluation of tubal factors have technical limitations and fetus begin to develop there (Fig. 22.3).
nal, pelvic or back pain, constant severe nausea, suspected
that must be considered when any one technique yields The most common site of occurrence is within a fallo- Classification
miscarriage, GP, or practice nurse referral (Figs. 22.4A to C).
abnormal results. Further evaluation with a second, com- pian tube; however, ectopic pregnancies can occur in the • Tubal—most common (96–98%) with ampullary being
plementary method is prudent whenever specific diagno- ovary, the abdomen, and in the lower portion of the uterus in 80–90% Symptoms and Signs of
sis or best treatment strategy is uncertain. (the cervix).14 • Abdominal in 1–2% (tubo-abdominal/abdomino-ovarian)
Incidence—ectopics happen in about 0.25–1% of all • Ovarian (0.5–1%) Ectopic Pregnancy and Initial Assessment
Induced Abortion and Tubal Subfertility pregnancies. • Cervical (<0.5%)
Symptoms are nonspecific, so it may create difficulty in ini-
The mortality rate is about 1 per 1,000 ectopics (10% • Heterotopic (combination of ectopic + intrauterine
Previous history of abortion can increase the risk especially pregnancy) tial diagnosis of an ectopic pregnancy and it may mimic gas-
of all maternal deaths). trointestinal conditions, urinary tract infections (UTI), etc.
when it is infected.
Ectopic pregnancy rate increased almost 4 fold (from
4.5 per 1,000 pregnancies to 16.8 per 1,000 pregnancies Uncommon Ectopics
Hydrosalpinx Impact Using Ultrasound for Diagnosis
since 1970). • Intraligamentous pregnancy (in broad ligament)
Hydrosalpinx (unilateral/bilateral) can decrease the implan- Age group—24 to 35 years of age (younger reproduc- • Pregnancy in a uterine diverticulum or sacculation Transvaginal scan (TVS) will help to locate pregnancy and
tation and pregnancy rates to half in IVF, so it is better to do sal- tive age group) with 75% detected and managed with in • Angular pregnancy (inside the uterotubal attachment) check for viability to decide the gestation and the resulting
pingectomy or tubal clipping before taking the case further.12 first trimester only. • Pregnancy in a rudimentary horn of uterus management.
178 The Infertility Manual Tubal Factor Infertility and Ectopic Pregnancy—Diagnosis and Management 179
Flowchart 22.3: Human chorionic gonadotropin (hCG) measurements in women with pregnancy of unknown location (PUL).
A B
C
Figs. 22.4A to C: (A and B) Uncommon ectopic pregnancies. (C) Right tubal ectopic pregnancy in 11th week of gestation.
A B
HUMAN CHORIONIC GONADOTROPIN • No signs of tubal rupture
• Adnexal mass <3 cm
MEASUREMENTS IN WOMEN WITH • No embryonic heartbeat.
PREGNANCY OF UNKNOWN L OCATION
Think first ectopic always whenever in pregnancy of Medical Management: Methotrexate
unknown location. (Antimetabolite)
Unruptured ectopic pregnancy
Management Options (FLOWCHART 22.3) Stable hemodynamics
Ectopic mass <4 cm
Expectant Management Indications β-hCG value <5,000 IU/L.
Expectant management for 7–14 days will be considered in
the following situations: Surgical Management Indications
• Minimal pain or bleeding in reliable patient Surgically, it made can be managed by laparoscopy or
• Beta-human chorionic gonadotropin (β-hCG) less laparotomy. Depending upon situation, we can resort to C D
than 1,000 IU/L and falling either salpingectomy or salpingotomy (Figs. 22.5A to D). Figs. 22.5A to D: Steps of Salpingotomy in Right sided Ectopic Pregnancy.
C HA PTE R
180 The Infertility Manual
•
•
Persistent ectopic pregnancy/worsening condition in
spite of medical management
Failure to locate with β-hCG >1,500
Ruptured ectopic gestation with unstable hemody-
namics.
6. Gleicher N, Karande V. The diagnosis and treatment of
proximal tubal disease. Hum Reprod. 1996;11:1823-34.
7. Papaioannou S, Afnan M, Girling AJ, Coomarasamy A,
Ola B, Olufowobi O, et al. The effect on pregnancy rates
of tubal perfusion pressure reductions achieved by guide-
wire tubal catheterization. Hum Reprod. 2002;17;2174-9.
The Prediction and Management of
Poor Responder
23
R Karthigayeni, V Swetha, Divyashree PS, Kamini A Rao
8. Confino A, Tur-Kaspa I, De Cherney A, Corfman R,
Coulam C, Robinson E, et al. Transcervical balloon tuboplasty.
PROBABLE QUESTIONS A multicentre study. J Am Med Assoc. 1990;264:2079-82. Chapter Outline
9. Lang EK, Dunaway HH. Recanalisation of obstructed • Incidence • Group 2
1. Discuss the various tubal assessment tests.
fallopian tube by selective salpingography and transvaginal • Variable Definitions Used in the Literature • How to Synchronize Follicle Development?
2. What is role of tubal surgery in the era of ART? • Bologna Consensus • Group 3
bougie dilatation: outcome and cost analysis. Fertil Steril.
3. Will you manage all ectopic pregnancies under knife— 1996;66:210-5.
• POSEIDON Strategy • Role of Androgens
explain with evidence? • Poor Ovarian Response • Drugs Used for Androgen Supplementation
10. Woolcott R, Petchpud A, O’Donnell P. Differential impact • Gonadotropin Receptor Polymorphism • Why to Increase Number of Oocyte?
4. What is pregnancy of unknown location (PUL) and on pregnancy rate of selective salpingography, tubal • Management • What is Considered as Good Number to Obtain?
how to manage? catheterization and wire-guide recanalisation in treatment • Group 1 • Ways to Increase Number of Oocytes
of proximal Fallopian tube obstruction. Hum Reprod. • Concept of Ovarian Sensitivity • Group 4
1995;10:1423-6. • Increasing Dosage of Gonadotropins • Growth Hormone
REFERENCES 11. Honore GM, Holden AE, Schenken RS. Pathophysiology • Importance of Addition of LH • Other Intervention in Poor Responders
and management of proximal tubal blockage. Fertil Steril. • Preferred COS Protocol in Poor Responders
1. Serafini P, Batzofin J. Diagnosis of female infertility.
1999;71:785-95.
J Reprod Med. 1989;34:29-40.
12. Papaioannou S, Afnan M, Girling AJ, Ola B, Hammadieh N,
2. Swart P, Mol BW, van der Veen F, van Beurden M, Redekop WK,
Bossuyt PM. The accuracy of hysterosalpingography in the
Coomarasamy A, et al. The learning curve of selective INTRODUCTION INCIDENCE
salpingography and tubal catheterization. Fertil Steril.
diagnosis of tubal pathology: a meta-analysis. Fertil Steril. 2002;77:1049-52. The trends of child bearing have changed in the past few The incidence of POR varies between 9 and 24% in the lit-
1995;64:486-91. 13. Holz K, Becker R, Schurmann R. Ultrasound in the decades with late pregnancies becoming an increasing eratures depending upon various definitions.4
3. Perquin DA, Beersma MF, de Craen AJ, Helmerhorst FM. investigation of tubal patency. A meta-analysis of three
The value of Chlamydia trachomatis-specific IgG antibody phenomenon in present days. Whether the reasons are
comparative studies of Echovist-200 including 1007
testing and hysterosalpingography for predicting tubal late marriage, sterility, lack of awareness of treatment, VARIABLE DEFINITIONS USED
women. Zentralbl Gynäkol. 1997;119:366-73.
pathology and occurrence of pregnancy. Fertil Steril. 14. Marchino GL, Gigante V, Gennarelli G, Mazza O, Mencaglia L. career development, or other social reasons, more and
2007;88:224-6. Salpingoscopic and laparoscopic investigations in relation more women are crossing the age of 35 or even 40 before IN THE LITERATURE
4. Perquin DA, Dorr PJ, de Craen AJ, Helmerhorst FM. Routine to fertility outcome. J Am Assoc Gynecol Laparosc. thinking of pregnancy. Various definitions were used in the past like with one pre-
use of hysterosalpingography prior to laparoscopy in the 2001;8:218-21. As the age increases, the need for in vitro fertilization vious cancelled IVF cycle, age greater than 40 years, day
fertility workup: a multicentre randomized controlled trial. 15. Kerin J, Daykhovsky L, Segalowitz J, Surrey E, Anderson R, (IVF) increases and the ovarian reserve is compromised. 3 follicle-stimulating hormone (FSH) greater than 7–15
Hum Reprod. 2006;21:1227-31. Stein A, et al. Falloposcopy: a microendoscopic technique
5. Novy MJ. Transhysteroscopic techniques for tubal
In such patients poor response (POR) is a pesky problem. IU/L, etc.
for visual exploration of the human fallopian tube from
catheterization. References en Gynecologie Obstetrique. the uterotubal ostium to the fimbria using a transvaginal It represents one of the few unresolved problems of mod- A study analyzed 47 randomized trials and found that
1995:67-71. approach. Fertil Steril. 1990;54:390-400. ern infertility care. 41 different definitions were used in the past. In that not
First described in 1983 as reduced follicular response more than three trials used the same definition. Even dif-
and E2 levels following controlled ovarian stimulation ferent definitions were used by same research group across
Dedicated to My Parents Sh D. N. Basnotra and Smt Shakuntla Sharma and Brother Dr Amit Basnotra (COS), which lead to production of less oocytes and trans- different trials. And none of the criteria used was adopted
ferable embryos.1 in more than 50% of the trials.5
But all were inconclusive, insufficient, inadequate,
What is Meant by Poor Ovarian Response? inconsistent, and uncertain. So there came an urgent need
Poor response in IVF can be defined as development of for uniform definition.
mature follicles in insufficient number following stimula-
tion with gonadotropin leading to retrieval of few oocytes BOLOGNA CONSENSUS
or cycle cancellation.2
In order to provide a uniform definition worldwide ESHRE
Goal of ovarian stimulation in IVF is multiple follicular
consensus in 2011 proposed a definition for POR and
recruitment so that inefficiencies in embryology culture,
named it to bologna criteria.6
embryo selection, and implantation are compensated. But
It should meet at least two of three following criteria:
poor responders fail to serve this goal.3
But one of the main problems for POR in literature is 1. Advanced maternal age or any risk factors for POR
the absence of a standard definition. (≥40 years).
182 The Infertility Manual The Prediction and Management of Poor Responder 183
2. A previous POR (<3 oocytes with conventional stimu- Advanced Maternal Age Table 23.2. Gonadotropin receptor polymorphism.
lation protocol).
As age advances there occurs a decrease in number and LH Receptor FSH Receptor
3. An abnormal ovarian reserve tests (i.e. AFC—5–7 fol-
quality of oocyte.10 v-βLH FSH-R
licles or AMH 0.5–1.1 ng/mL). Trp8-Arg/Ile15-Thr Asn680/Ser—most common (75%)
The quality of the oocyte is affected by the following
Of all the above parameters, minimum of one previous Short T1/2–5 to 9 min High AFC
ways:11
More potent action Slightly elevated basal FSH
stimulated cycle is essential to make a diagnosis. Resistance to FSH Resistance to FSH stimulation
• Impairment of mitochondrial function (less energy)
And also if previous episode of poor response at least stimulation
• Increased granulosa cell apoptosis
twice after maximal IVF stimulation is present it is suffi- Fig. 23.1: Poseidon strategy for poor responders. • Increased oxidative stress.
cient to a make diagnosis.
Advanced maternal age with reduced ovarian reserve Because of theca cell ageing there is reduced produc- GONADOTROPIN RECEPTOR
Table 23.1. POSEIDON strategy for poor responders. tion of androgen.
is defined as expected poor responder in the absence of POLYMORPHISM
IVF stimulation. Good ovarian reserve Poor ovarian reserve There is decrease in percentage of euploid blastocyst as
Studies in poor responder patients categorized accord- Group 1 Group 2 Group 3 Group 4 age increases irrespective of increase in number of blastocyst.12 Table showing gonadotropin receptor polymorphism
ing to bologna criteria were found to have low prognosis Age <35 years Age >35 years Age <35 Age >35 (Table 23.2).13
consistently irrespective of age or number of criteria used, AMH >1.2 AMH >1.2 years years Lifestyle-related Factors Carriers of v-beta LH variants and FSH-R Ser680 were
AFC >5 AFC >5 AMH <1.2 AMH <1.2
live birth rate per cycle of about 6%.7 • Smoking found to have higher FSH consumption.14,15
(Sub Groups (Sub groups AFC <5 AFC <5
Although Bologna criteria gave uniform definition Ia <4 Oocytes Ia <4 Oocytes • Obesity Ser/Ser carriers of FSH receptor polymorphism
worldwide, there exist some drawbacks for the same. Ib 4–9 Oocytes) Ib 4–9 Oocytes require 75IU more than Asn/Asn carriers to achieve the
Acquired Factors same steroidogenesis.16
Limitations of Bologna Criteria • Endometriosis
• Consideration of ovarian sensitivity to gonadotropins Predicting a Poor Response
• Heterogeneous populations with different prognosis into account. • Ovarian surgery
are clubbed together. • Previous pelvic inflammatory diseases (PID) to IVF Stimulation
• More clarity in implementing COS in impaired response.
• Specific characteristic profiles of unexpected poor— • Chemotherapy/radiotherapy The ability to accurately assess and predict ovarian
suboptimal responders are not included. Include four groups with difference in degree of low response would reduce the burden imposed by failure
• No definition for hypo/suboptimal responders. prognosis, so each requires a more personalized treatment Genetic Factors because of inadequate response to stimulation.
• It failed to distinguish between alteration of oocyte approach (Fig. 23.1, Table 23.1). • Structural and numerical abnormalities of X chromo-
quantity and those of oocyte quality as reflected by some Interpretation of Various Ovarian
ART outcome, the real oocyte quality is not included.
POOR OVARIAN RESPONSE Turners syndrome/Turner mosaicism Reserve Tests17
• ORT predicts magnitude of COS response but do not Fragile X syndrome (FMR1 premutation)
foretell pregnancy chances. • Balanced translocation Both AMH and AFC correlate well with response of the
Pathophysiology • Autosomal functionally relevant genetic variants ovary to COS. The ideal test is the ovarian response to COS
FSH receptor mutation itself. AFC and AMH alone or in combination were not
POSEIDON STRATEGY 1. Depletion of ovarian follicle pool9
found to improve the prediction of response of the ovary
• Insufficient initial follicle number FSH receptor single nucleotide polymorphisms (SNPs)
(POSEIDON—Patient-Oriented Strategies Encompassing • Accelerated follicle loss Luteinizing hormone (LH) receptor mutation to COS. Most important factor related to live birth rates is
Individualized Oocyte Number) • Iatrogenic LH beta polypeptide (v-LHβ). the age of the women (Fig. 23.2, Table 23.3).18
In order of improve treatment outcome and guide the
2. Ovarian follicle dysfunction9
physicians in more detailed way, a new more clinically rel-
• Signaling defect
evant criteria were introduced.8
• Enzymatic deficiency
It combines quality and quantity for stratification of
• Autoimmunity
patients as low prognosis in ART cycle.
• Idiopathic
The following factors are considered:
• New categorization of patients based on their impaired
Risk Factors
response to COS.
• Includes both qualitative and quantitative ovarian 1. Advanced maternal age
response into consideration. 2. Lifestyle-related factors
• Ability to generate number oocytes to produce at least 3. Acquired factors
one euploid embryo. 4. Genetic factors Fig. 23.2: Various parameters for predicting POR.
184 The Infertility Manual The Prediction and Management of Poor Responder 185
Table 23.3. Parameters for predicting POR and their interpretation. r-hFSH an initial slow response (stagnation between Table 23.5. Role of LH in folliculogenesis.
Poor pregnancy Non response
day 7 and 10 or no follicle >10 mm on day 8 of stimula-
Early Follicular Phase Intermediate Follicular Phase
tion with normal follicular cohort).19,20 (Induction of Androgen (Expression of LH Receptors
Sensitivity Specificity Sensitivity Specificity
• Reflects hyposensitivity of the granulosa cell to FSH. Production in Theca Cells) in Granulosa Cells)
TEST Cut point % % % % Reliability Advantages Limitations
• These are the women with good ovarian reserve and •• FSH receptor induction •• Sustain of FSH-depend-
FSH 10–20 IU/L 10–80 83–100 7–58 43–100 Limited Widely used Reliable, low sensitivity can achieve adequate number of retrieved oocytes. in Granulosa Cells ent granulosa activities,
AMH 0.2–0.7 ng/mL 40–97 78–92 * * Good Reliable Do not predict non • But they need higher dosage of FSH (i.e. 3,000 IU) and •• Increases responsive- including aromatase
pregnancy prolonged stimulation cycle with low FORT, unex- ness induction and growth fac-
AFC 3–10 9–73 73–100 8–33 64–100 Good Reliable, Low sensitivity •• Acts synergistically with tors release
pected poor response (i.e. <3 eggs retrieved) and lower IGF-1 •• Regulation of final follicle/
widely used
PRs. •• Increases recruit ability oocyte maturation
Inhibin 40–45 pg/mL 40–80 64–90 * Limited Reliable, do not predict • If hyporesponse is identified early (i.e. day 5–8 of COS), of pre antral and antral •• Optimization of steroido-
B non pregnancy follicles genesis
r-h LH is effective in rescuing follicle/oocyte number
CCCT 10–22 IU/L 35–98 68–98 23–61 67–100 Limited High sensitiv- Reliable limited addi- (FORT) and embryo competence.20
ity than FSH tional value
*Limited evidence A meta-analysis26 done with 43 trials and 6,443
Source: Practice Committee. Ovarian reserve testing. Fertil Steril. 2015. Sub-optimal Responders patients evaluated the benefit of addition of adding r-LH
• Women with good ovarian reserve who obtain less with r-FSH. Bologna criteria were not used. They found
number of oocyte (4–9) compared to normal respond- that addition of r-LH increased the pregnancy rate by 30%
Table 23.4. POSEIDON strategy—Group 1 (Young
women with good ovarian reserve). ers (10–15). in patients with poor response but not so in patients with
• They have a lower prognosis compared to normal normal response.
Reason Interventions
responders in terms of cumulative live birth.21 ESPART trial27 randomized 939 poor responder
•• FSH dose does not •• Change the protocol
patients selected according to bologna criteria to receive
reach the threshold •• Increase FSH dose
•• Genetic polymorphism •• Add LH activity either r-FSH+ r-LH (2:1) (Pergovaris) or r-FSH alone. No
of FSH-R, LH-R, V-LH-β •• hCG or agonist trigger
INCREASING DOSAGE difference in outcome was observed in terms of pregnancy
•• Trigger problem OF GONADOTROPINS rate and live birth rate.
A meta-analysis28 showed that addition of r-LH in COS
Useful in patients with good ovarian reserve and
MANAGEMENT FSH threshold not reached or due to FSH receptor
protocol improves implantation rate and clinical pregnancy
rate in patients with advanced maternal age (>35 years).
Treatment will be based on these low prognoses category polymorphism.
(Fig. 23.3). There is a little benefit in increasing the daily dose of
gonadotropins to 450 IU in patients who poorly respond to PREFERRED COS PROTOCOL
Fig. 23.3: POSEIDON strategy for low prognosis patients. GROUP 1 standard protocol.22 IN POOR RESPONDERS
No benefit in increasing the starting dose of FSH above
Young women with good ovarian reserve. Oocyte quality A study29 analyzed regarding the preferred protocol in poor
300 IU in terms of live birth rate.23
will be good (Table 23.4). responder patients over 45 countries worldwide in total of
Study published regarding personalized protocol in
196 centers. Majority were in favor of GnRH antagonist
FSH starting dose in patients, categorized according to
CONCEPT OF OVARIAN SENSITIVITY their expected response to COS. They in patients expected
protocol (56%) followed by short agonist protocol (20%).
In a Cochrane review,30 it was concluded that there is
to have poor response there is no benefit in increasing FSH
FORT Index (Follicular Output Ratio) not much evidence to promote any particular intervention
starting dose to more than 300 IU.24
• Ratio of preovulatory follicle to small antral follicle either in adjuvant therapy, pituitary down-regulation or
(Fig. 23.4). ovarian stimulation in poor responder patients.
IMPORTANCE OF ADDITION OF LH Two meta-analyses31,32 compared GnRH antagonist and
long agonist protocol in poor responder patients. They found
Hyporesponders Role of LH in Folliculogenesis25 no difference in number of oocyte retrieved, pregnancy rate,
• About 15% of normogonadotropic good prognosis Basically LH through androgen production improves the and cancellation rate. However, with antagonist protocol,
patient are hyporesponders. sensitivity of the granulosa cell to FSH by increasing the there is reduction in dose and duration of gonadotropins.
• Incidence with long agonist protocol is 19.8 and 15.2% number of FSH receptor (Table 23.5). A randomized controlled trial (PRINT) performed in
in antagonist protocol. If hyporesponse is identified early (i.e. day 5–8 of poor responders33 categorized the patients according to
• FSH hyposensitivity is defined as normogonadotropic COS), r-h LH is effective in rescuing FORT and embryo bologna criteria into three groups each receiving either
Fig. 23.4: FORT index (Follicular output ratio). normo-ovulatory young patients who will have to competence.20 antagonist, long and short protocol. Higher numbers of
186 The Infertility Manual The Prediction and Management of Poor Responder 187
oocytes were retrieved in long and antagonist protocol Table 23.6. POSEIDON strategy—Group 2 (Older Contd...
compared to short protocol. So they concluded that long women with good ovarian reserve). Table 23.7. Treatments used to synchronize the follicle development.
agonist and antagonist protocol are the suitable ones in Reason Interventions Drugs Rationale Regimen Advantages Disadvantage Evidences
poor responder. •• Ageing—reduced androgen •• Protocol GnRH Causes immediate, 3 mg on D25 Aides in Large scale reduced size disparity
Using Corifollitropin-alfa in older women was found to production •• Add LH activity antagonist rapid gonadotropin follicular syn- studies required of early antral follicle41
be equally effective compared to daily recombinant FSH in •• Asynchronous development •• Synchronize follicle suppression by com- chronization
terms of, number of oocytes retrieved, pregnancy rate, and of follicle wave petitively blocking
•• Genetic polymorphism of •• Increase FSH dose GnRH receptors in
live birth rates.34
FSH-R, LH-R, LH β (rare) •• Aneuploidy anterior pituitary,
If conventional protocol fails we can try with mild screening—PGS thereby preventing
stimulation protocol. A meta-analysis found compared to FSH rise and follic-
micro flare protocol, in antagonist/letrozole protocol CPR ular discrepancy in
was lower.35 HOW TO SYNCHRONIZE early follicular phase
AACEP Uses agonist flare Pretreatment with OCPs Focuses on Large well de- Helps in Estrogenic
By natural cycle IVF overall treatment burden to the FOLLICLE DEVELOPMENT? (GnRH ago- effect for recruitment with GnRH-agonist promoting signed studies dominance in the stim-
couple is reduced. However, there is an increased risk nist antago- of follicles and estro- overlapping last 5–7 Estrogenic required ulated cycles, avoiding
of cycle cancellation. So it should be considered before The following treatment will synchronize the follicle devel-
nist conver- gen supplementation days of OCPs till onset dominance in the ill effects of LH flare
deciding for donar oocyte, especially in less than 38 years.36 opment (Table 23.7). sion with E2 increases estrogen of menses, on D2—Low the stimulated and androgens42
• OCP pretreatment priming) dominance in folli- dose GnRH-antagonist ovaries thus
• Luteal phase manipulation: cles leading to better (0.125 mg/day) and Es- avoiding the
GROUP 2 Estradiol priming quality oocytes in tradiol 2 mg IM 2 doses ill effects of
Older women with good ovarian reserve. The oocyte qual- GnRH antag POR every 3 days along with LH flare and
FSH/hMG stimulation, androgens—
ity may be affected due to age factor (Table 23.6). AACEP F/B Estrogen supple- may improve
mentation till dominant prognosis in
follicle POR
Table 23.7. Treatments used to synchronize the follicle development.
Drugs Rationale Regimen Advantages Disadvantage Evidences
OCPs Pituitary suppression Day 5–25 of menstrual Decrease in Due to profound A meta-analysis found Table 23.8. POSEIDON strategy—Group 3 (Young WHY TO INCREASE NUMBER
women with poor ovarian reserve).
cycle of previous month ovarian cyst pituitary sup- that there is increase OF OOCYTE?
No increase in FSH formation pression there in dose and duration Reason Interventions
level in luteal phase is an increase of gonadotropins and A study analyzed the association between number of
•• Poor ovarian reserve •• Protocol
Increase in in need of dose no difference in other oocytes and cumulative live birth. There exists a significant
•• Asynchronous develop- •• Maximum FSH dose
So no early recruit- homogeneity and duration outcome of IVF37
ment of follicle •• Synchronize follicle wave increase in cumulative live rate if more number of oocytes
ment of follicles of cohort of the gonado-
•• Androgens
tropin A Cochrane review are retrieved.48
•• Dual stimulation
Role in enhance- showed improved There exist a nonlinear relationship between live
ment of ER sensiti- pregnancy outcomes
birth rate and number of oocytes obtained following COS
zation with progesterone pre- GROUP 3
treatment and poorer regardless of age. The number of oocytes to maximize the
outcomes with OCP Young women with poor ovarian reserve. In spite of poor live birth rate is 15.49
pretreatment38 ovarian reserve, the quality of oocytes is good (Table 23.8). A study was performed analyzing the relationship
Luteal Phase Manipulations between number of mature oocytes retrieved and chance
Estradiol With its negative 4 mg daily from D20 to Aides in Further large A meta-analysis ROLE OF ANDROGENS of obtaining a euploid blastocyst. Found that each supple-
feedback effect on D2 of next cycle follicular syn- scale studies showed increased • Follicular recruitment, growth, and survival mentary mature oocyte increased the chance of obtaining
pituitary, E2 levels chronization required number of retrieved
prevents early rise oocytes but no
• Increases intraovarian concentration of androgens euploid blastocyst by 11%.50
in FSH levels in difference in clinical • Act via androgen receptor predominant on the granu-
late luteal phase pregnancy rate (CPR)39 losa cells
thereby preventing • Up regulation of IGF1, IGF1-R, FSH-R. WHAT IS CONSIDERED AS GOOD
early recruitment of Another meta-analysis
follicles in follicular found decrease in cycle
NUMBER TO OBTAIN?
DRUGS USED FOR ANDROGEN
phase cancellation and an The below chart explains how many oocytes are required
increase in CPR40 SUPPLEMENTATION to get an euploid blastocyst across women of different age
Contd... See Table 23.9. group (Fig. 23.5).51
188 The Infertility Manual The Prediction and Management of Poor Responder 189
Table 23.9. Drugs used for androgen supplementation. WAYS TO INCREASE NUMBER They observed that a good number of oocytes are
retrieved in poor responders by using this protocol.52
Drug MOA Regimens Advantages Disadvantages Evidence OF OOCYTES
A study was performed using Duplex protocol and they
DHEA Act during early 75 mg/d 6-8 Provides Still large- Cochrane review found that T and
follicular growth weeks before substrate for scale studies DHEA might be useful in terms of
1. Increasing FSH Dosage analyzed the outcome of COS in both phase of menstrual
before the gon- stimulation folliculogenesis are required increasing live birth rate, but if we • Useful if ovarian reserve is good and FSH thresh- cycle. COS was done in both follicular and luteal phase by
adotropin sensitive and helps in to prove the removed high risk bias trials then it old not reached or FSH receptor polymorphism. classical antagonist protocol with GnRH trigger for follicu-
phase improvement effectiveness showed no significance43 • Not of much use if antral follicle counts is low. lar maturation.54
Increases the of antral follicle There is increase in CPR but no Higher dose of gonadotropins will not create fol- COS in both phase of menstrual cycle provided a simi-
recruitment of fol- count improvement in other outcome as licles in de novo. lar number of oocytes, zygotes, and blastocyst.54
licles shown in a meta-analysis44
• If ovarian reserve is low and FORT is high no gon- Hence, duplex protocol compared to single COS cycle
Increases IGF & Another recent meta-analysis found adotropin can compensate. doubled the final blastocyst yield.54
serves as precur- improvement in CPR significantly but
sor for steroids not so if we consider only RCT45 • In such cases dual stimulation is an option.
TESTOS- Enhancing FSH 20 µg/kg/day Local Still controver- A meta-analyses showed an increase
2. Double Stimulation or Shanghai Protocol:52 GROUP 4
TERONE sensitivity during from D15 in application sial in CPR and LBR as well as reduction in • Based on concept that there occur 2 or 3 waves of
GEL early Gn sensitive the preceding No systemic dose and duration of gonadotropins46 follicle recruitment (Fig. 23.6).53 Older women with poor ovarian reserve. Again the quality
phase cycle side effects • It is a combination of two stimulation protocol in of oocytes is affected due to age factor (Table 23.10).
A recent randomized clinical trial in
Improves follicu- bologna poor responders concluded no one menstrual cycle.
lar function and improvement in ovarian parameters47 • It targets the antral follicles in the both follicular GROWTH HORMONE
steroidogenesis Ongoing RCT is TTRANSPORT phase and luteal phase.
Its use may be effective in group 4 women (Table 23.11).
(Testosterone TRANSdermal Gel for • Two OPU is done in a single menstrual cycle.
Poor Ovarian Responders Trial) • So, more number of oocytes and viable embryos
Letrozole Competitive inhibi- 2.5 mg for first Improves Mono follicular The CPR was significantly lower and are obtained.
tion of Aromatase 5 days of COS intraovarian growth duration of gonadotropin stimulation Table 23.10. POSEIDON strategy—Group 4 (Older
• Studied in 38 POR (Bologa criteria). The number women with poor ovarian reserve).
enzyme—↑ intrao- micro is lower with the antagonist/letrozole
of oocytes harvested was 167.
varian androgens environment group compared with microflare Reason Interventions
and decreases and endometrial protocol35 • 26/38 (68.4%) succeeded in producing 1–6 cryo-
•• Poor ovarian reserve •• Protocol
estradiol levels, receptivity preserved embryos.
•• Asynchronous develop- •• Maximum FSH dose
thereby inhibiting Other uses— • 21 underwent 23 frozen ET resulting in 11 ongoing ment of follicle •• Synchronize follicle wave
negative feedback oncofertility pregnancies (47.8%). •• Androgens
on FSH production
[2nd phase of stimulation if at least 2 AFC (2–8 mm) •• Dual stimulation
post OPU] •• Growth hormone
Fig. 23.5: Mean number of oocytes needed across different age groups to get one euploid blastocyst. Fig. 23.6: Double stimulation or Shanghai protocol.
190 The Infertility Manual The Prediction and Management of Poor Responder 191
Table 23.11. Growth hormone in poor responder. Future in Poor Responders follicles were found in 13/27 patients. About 8/13
patients had follicular growth secondary to this
Rationale Regimen Evidences • Ovarian fragmentation and in vitro activation method. About 5/8 patients had mature oocytes.
•• IGF is GH dependent and is involved •• Varies from 4 IU daily to A meta-analysis showed that the probability of • Ovarian PRP application (ovarian rejuvenation)
in potentiating the effect of FSH 24 IU on alternate days pregnancy is increased by addition of GH or by In that 2 had pregnancy following FET. One
• Application of mitochondrial activation. healthy baby was born after this treatment.70
•• In vitro GH increase estradiol •• Started on the day of doing day 2 embryo transfer55
production gonadotropin or from day GH was found to increases live birth rate in 1. Ovarian fragmentation and in vitro activation (IVA) 2. Ovarian PRP application (ovarian rejuvenation)
•• Oocytes from follicles having higher 21 in LBG protocol poor responders, but in which subgroup was not • Reproductive lifespan of the women is determined • PRP is rich in several growth factors that have a
antral fluid GH levels have better •• Continued till day of hCG shown in a cochrane review56
developmental potential
by her primordial follicular pool. It is maintained significant role in tissue regeneration.
•• Enhances nuclear and cytoplasmic Although GH does not increase COS response or through a balance between the tensin homolog • In the ovarian cortex, it can possibly stimulate the
maturation no of oocytes, three meta-analysis concluded the (PTEN) and phosphatidyl inositol 3-kinase (PI3K) germ cell line to develop into an oocytes.
•• Stimulates DNA repair positive effect of adding GH in terms of preg- (Fig. 23.7). • Studied in total of eight perimenopausal women.
•• Improves normal fertilization and nancy and live birth rate thus showing its effect on
oocyte quality57 • PTEN is break and P13K is a gas pedal. If PTEN is • PRP was injected into the ovary by transvaginal
embryo development
inhibited follicular growth is enhanced. Release of ultrasound guidance.
Meta-analysis done in poor responders showed
no benefit of GH in improving implantation or live PDK-1 (a protein regulated by the PI3K pathway) • Return of ovarian function was observed within
birth rate58 leads to premature death of primordial follicles. 3 months.
A recent RCT regarding the benefit of adding GH • Ovarian fragmentation and IVA promote follicle • But large scale data on ovarian rejuvenation and
in poor responders diagnosed according to bolo- growth via different mechanism.69 pregnancy outcomes will be required before a
gna criteria showed some improvement in ovarian • Ovarian fragmentation and IVA treatment was conclusion can be drawn.
parameters but found no difference in pregnancy given to 27 patients with ovarian failure. Residual 3. Application of mitochondrial activation (Fig. 23.8)
rate59
• Oocyte is the cell which have large amount of
mitochondria.
OTHER INTERVENTION IN 4. Assisted Hatching (AH) • Mitochondria the power house of the cell is
• AH compared to control group showed no differ- needed by oocyte to be competent for fertilization
POOR RESPONDERS and for embryo development.
ence in terms of live birth.63
1. Pyridostigmine • In patients with repeated IVF failure or in FET • Mitochondrial application at time of ICSI can
• Acetylcholinesterase inhibitor. cycles AH was found to increase clinical pregnancy be used in patients with repeated IVF failure or
• Increases GH secretion by enhancing the action of rate and multiple pregnancy. But it is of no benefit advanced age group.
acetylcholine. in women of advanced age or when performed in • It increases oocyte energy without altering fetal
• Was evaluated as a cheaper alternative to GH sup- unselected patients in fresh embryo transfer cycle.64 genome.
plementation (dose—120 mg/day). • Currently, there is an insufficient evidence to rec-
• Very limited no of studies and study population. ommend AH to patients with AMA or POR.
• Addition of pyridostigmine does not appear to 5. Day of Embryo Transfer
CONCLUSION
improve the ongoing pregnancy/delivery rate in • A randomized trial concluded that there is a sig- • Hyporesponse (impaired response) and poor response
poor responders undergoing IVF. nificant increase in pregnancy rate for day 2 trans- are not the same.
2. Aspirin fer (27.7%) compared to day 3 transfer (16.3%).65 • Hyporesponders and poor responders are associated
• Poor ovarian response might be due to impaired • Another randomized trial including total of 250 Fig. 23.7: Ovarian fragmentation and in vitro activation. with lower chance of live birth rate.
ovarian blood flow. patients showed that based on the day of embryo
• A meta-analysis concluded that there is no differ- transfer there is no difference in outcome.66
ence in clinical pregnancy rate by adding aspirin.60 6. Role of PGS
• Due to lack of evidence aspirin cannot be recom- • Its benefit in poor responder patients is controver-
mended in women undergoing IVF.61 sial.
3. L-Arginine • Some studies have shown in diminished ovar-
• It is involved in formation of nitric oxide (NO). ian reserve there is a chance of having genetically
• NO is an intra- and intercellular modulator that abnormal pregnancy or miscarriage.67 But some
plays a role in follicular maturation and ovulation. say that there is no increased risk of aneuploidy or
• In poor responders, the addition of L-arginine miscarriage in patients with poor ovarian reserve.68
increases the number of retrieved oocytes but has • So further RCTs are required before suggesting
no benefit on terms of pregnancy outcome.62 its role. Fig. 23.8: Application of mitochondrial activation.
192 The Infertility Manual The Prediction and Management of Poor Responder 193
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science behind 25 years of ovarian stimulation for in vitro 18. Broer SL, Dólleman M, van Disseldorp J, Broeze KA, in-vitro fertilisation (IVF). Cochrane Database Syst Rev. failure. Fertil Steril. 2008;89(2):342-7.
fertilization. Endocr Rev. 2006;27(2):170-20. Opmeer BC, Bossuyt PM, et al. Prediction of an excessive 2010;(1):CD004379. doi: 10.1002/14651858.CD004379.pub3 43. Nagels HE, Rishworth JR, Siristatidis CS, Kroon B. Androgens
4. Filippo U, Alberto V, Rosario D’A, Laura R. Management response in in vitro fertilization from patient characteristics 31. Pu D, Wu J, Liu J. Comparisons of GnRH antagonist (dehydroepiandrosterone or testosterone) for women
of poor responders in IVF: is there anything new? BioMed and ovarian reserve tests and comparison in subgroups: versus GnRH agonist protocol in poor ovarian responders undergoing assisted reproduction. Cochrane Database Syst
Res Int. 2014;10. an individual patient data meta-analysis. Fertil Steril. undergoing IVF. Hum Reprod. 2011;26(10):2742-9. Rev. 2015 Nov 26;(11):CD009749. doi:10.1002/14651858.
5. Polyzos NP, Paul D. A systematic review of randomized 2013;100(2):420-9. 32. Xiao J, Chang S, Chen S. The effectiveness of gonadotropin- CD 009749.pub2.
trials for the treatment of poor ovarian responders: is 19. De Placido G, Alviggi C, Perino A, Strina I, Lisi F, Fasolino A, releasing hormone antagonist in poor ovarian responders 44. Li J, Yuan H, Chen Y, Wu H, Wu H, Li L. A meta-analysis
there any light at the end of the tunnel? Fertil Steril. et al. Recombinant human LH supplementation versus undergoing in vitro fertilization: a systematic review and of dehydroepiandrosterone supplementation among
2011;96(5):1058-61. recombinant human FSH (rFSH) step-up protocol during meta-analysis. Fertil Steril. 2013;100(6) 1594601. women with diminished ovarian reserve undergoing in
C HA PTE R
24
194 The Infertility Manual
vitro fertilization or intracytoplasmic sperm injection. Int
J Gynaecol Obstet. 2015;131(3):240-5.
57. de Ziegler D, Streuli I, Meldrum DR, Chapron C. The value
of growth hormone supplements in ART for poor ovarian
Endometriosis, Adenomyosis,
45. Qin JC, Fan L, Qin AP. The effect of dehydroepiandrosterone
(DHEA) supplementation on women with diminished
responders. Fertil Steril. 2011;96(5):1069-76.
58. Yu X, Ruan J, HeL-P, Hu W, Xu Q, Tang J, et al. Efficacy of and Infertility
ovarian reserve (DOR) in IVF cycle: evidence from a meta- growth hormone supplementation with gonadotrophins in
analysis. J Gynecol Obstet Hum Repro. 2017;46(1):1-7. vitro fertilization for poor ovarian responders: an updated
46. Luo S, Li S, Li X, Qin L, Jin S. Effect of pretreatment with Anjali Gahlan
meta-analysis. Int J Clin Exp Med. 2015;8(4):4954-67.
transdermal testosterone on poor ovarian responders 59. Bassiouny YA, Dakhly DMR, Bayoumi YA, Hashish NM.
undergoing IVF/ICSI: a meta-analysis. Exp Ther Med. Does the addition of growth hormone to the in vitro Chapter Outline
2014;8(1):187-94. fertilization/intracytoplasmic sperm injection antagonist • Management • Adenomyosis
47. Bosdou JK, Venetis CA, Dafopoulos K, Zepiridis L, • Management of Endometriosis-Associated Infertility
protocol improve outcomes in poor responders? A
Chatzimeletiou K, Anifandis G, et al. Transdermal testosterone randomized, controlled trial. Fertil Steril. 2016;105(3):
pretreatment in poor responders undergoing ICSI: a 697-702.
randomized clinical trial. Hum Reprod. 2016;31(5):977-85.
48. Drakopoulos P, Blockeel C, Stoop D, Camus M, de Vos M,
60. Gelbaya TA, Kyrgiou M, Li TC, Stern C, Nardo LG. Low- INTRODUCTION anomalies. Some reports have also suggested that patients
dose aspirin for in vitro fertilization: a systematic review with leiomyomas are predisposed to endometriosis, per-
Tournaye H, et al. Conventional ovarian stimulation and and meta-analysis. Hum Reprod Update. 2007;13(4): Endometriosis is an inflammatory disorder in women
single embryo transfer for IVF/ICSI. How many oocytes haps due to excessive menstruation.16
357-64. of reproductive age group, which is characterized by the
do we need to maximize cumulative live birth rates after
61. Siristatidis CS, Basios G, Pergialiotis V, Vogiatzi P. Aspirin
The true pathophysiology of endometriosis is poorly
utilization of all fresh and frozen embryos? Hum Reprod. presence of endometrial glands and/or stroma in sites
for in vitro fertilisation. Cochrane Database of Systematic understood. There are several theories explaining the
2016;31(2):370-6. other than the uterine cavity. It is an estrogen-dependent
Rev. 2016(11). Art. No.: CD004832. doi: 10.1002/14651858. pathophysiology of endometriosis which includes trans-
49. Sunkara SK, Rittenberg V, Raine-Fenning N, Bhattacharya S, disease, which usually involves the peritoneum or
CD004832.pub4. plantation, induction, coelomic metaplasia, and vascular/
Zamora J, Coomarasamy A. Association between the number
62. Battaglia C, Salvatori M, Maxia N, Petraglia F, Facchinetti ovaries.
of eggs and live birth in IVF treatment: an analysis of 400 135 lymphatic dissemination.
F, Volpe A. Adjuvant L-arginine treatment for in-vitro The prevalence of endometriosis has been estimated
treatment cycles. Hum Reprod. 2011;26(7):1768-74.
fertilization in poor responder patients. Hum Reprod. • Transplantation theory—according to this, endome-
50. Capalbo A, Rienzi L, Cimadomo D, Maggiulli R, Elliott T, to be around 10–15% in the reproductive age women.1
1999;14(7):1690-7.
Wright G, et al. Correlation between standard blastocyst Women with endometriosis can be either asymptomatic or trial cells from the uterine cavity are refluxed into the
morphology, euploidy and implantation: an observational 63. Carney SK, Das S, Blake D, Farquhar C, Seif MM, Nelson
L. Assisted hatching on assisted conception (in vitro can present with dysmenorrhea, chronic pelvic pain, dys- peritoneal cavity via fallopian tubes. It develops in
study in two centers involving 956 screened blastocysts.
Hum Reprod. 2014;29(6):1173-81. fertilisation (IVF) and intracytoplasmic sperm injection pareunia, infertility, and adnexal mass.2,3 It has been seen women with a disordered immune system, i.e. one that
51. Sunkara SK, Rittenberg V, Raine-Fenning N, Bhattacharya S, (ICSI)). Cochrane Database Syst Rev. 2012. that around 25–50% of infertile women may be affected cannot identify and destroy menstrual endometrial
Zamora J, Coomarasamy A. Association between the 64. Martins WP, Rocha IA, Ferriani RA, Nastri CO. Assisted cells reaching the peritoneal cavity.
by endometriosis and 30–50% patients with endometrio-
number of eggs and live birth in IVF treatment: an analysis hatching of human embryos: a systematic review and
meta-analysis of randomized controlled trials. Hum sis may suffer from infertility.4 Despite all the research • Induction theory—it is similar except that the endo-
of 400 135 treatment cycles. Hum Reprod. 2011;26(7):
Reprod Update. 2011;17(4):438-53. accrued over the years, there is no consensus on most metriotic lesions occur due to metaplasia as a result of
1768-74.
65. Bahceci M et al. Efficiency of changing the embryo transfer aspects of the disease. irritation from the refluxed endometrium.
52. Kuang Y, Chen Q, Hong Q. Double stimulations during
time from day 3 to day 2 among women with poor ovarian • Coelomic metaplasia theory—it is based on the fact
the follicular and luteal phases of poor responders in IVF/ Factors leading to reduced fecundity in minimal/mild
response: a prospective randomized trial. Fertil Steril. 2006
ICSI programmes (Shanghai protocol). Reprod BioMed. endometriosis are as follows: that the coelomic cell is the common embryologi-
Jul;86(1):81-5.
2014;29(6):684-91.
66. Shahine LK, Milki AA, Westphal LM, Baker VL, Behr B, 1. Impaired uterotubal transport of sperm5 cal precursor which gives rise to the cells of the peri-
53. Oktem O, Urman B. Understanding follicle growth in vivo.
Hum Reprod. 2010;25(12):2944-54. Lathi RB. Day 2 versus day 3 embryo transfer in poor 2. Ovulatory disturbances6 toneum, ovarian surface, and endometrium. There is
54. Ubaldi FM, Capalbo A, Vaiarelli A, Cimadomo D, responders: a prospective randomized trial. Fertil Steril. 3. Subtle impairment of oocyte and embryo quality7–9 spontaneous metaplastic change in the mesothelial
Colamaria S, Alviggi C, et al. Follicular versus luteal 2011;95(1):330-2.
67. Srebnik N, Margalioth EJ, Rabinowitz R, Varshaver I,
4. Implantation defects10–12 cells resulting in the disease.
phase ovarian stimulation during the same menstrual
Altarescu G, Renbaum P, et al. Ovarian reserve and PGD 5. Antiendometrial antibodies13 • Invagination theory—it is the most widely accepted
cycle (DuoStim) in a reduced ovarian reserve population
results in a similar euploid blastocyst formation rate: treatment outcome in women with myotonic dystrophy. 6. Progesterone resistance14 theory which says that the blood and menstrual debris
new insight in ovarian reserve exploitation. Fertil Steril. RBM Online. 2014;29:94-101. 7. Increased risk of recurrent miscarriage.15 gets trapped by the adhesions and gets collected on
2016;105(6):1488-95. 68. Tremellen K, Savulescu J. Ovarian reserve screening: a
the surface of the ovary resulting in superficial endo-
55. Kyrou D, Kolibianakis EM, Venetis CA, Papanikolaou EG, scientific and ethical analysis. Hum Rep. 2014;29(12): In advanced stages of endometriosis, it is mainly the
Bontis J, Tarlatzis BC. How to improve the probability 2606-14. distorted tubo-ovarian axis along with above factors which metriosis on the ovarian surface. This leads to progres-
of pregnancy in poor responders undergoing in vitro 69. Hsueh AJW, Kawamura K, Cheng Y, Fauser CJM.
contributes to infertility. sive infolding of the ovarian cortex, and ultimately
fertilization: a systematic review and meta-analysis. Fertil Intraovarian control of early folliculogenesis. Endocr Rev. forms a pseudocyst.
Steril. 2009;91(3):749-66. 2015;36(1):1-24.
• Ovarian cyst theory—according to this, endometriotic
56. Duffy JMN, Ahmad G, Mohiyiddeen L, Nardo LG, Watson A. 70. Suzuki N, Yoshioka N, Takae S, Sugishita Y, Tamura M, Etiopathogenesis cyst starts as a functional ovarian cyst which becomes
Growth hormone for in vitro fertilization. Cochrane Hashimoto S, et al. Successful fertility preservation following
Database of Systematic Rev. 2010(1). Art. No.: CD000099. ovarian tissue vitrification in patients with primary ovarian Risk factors for the development of endometriosis includes gradually infiltrated by endometriotic implants.
doi: 10.1002/14651858.CD000099.pub3. insufficiency. Hum Reprod. 2015;30(3):608-15. early onset of menstruation, increased menstrual blood • Vascular/lymphatic spread is supported by the pres-
flow, and excess menstrual reflux as seen in Müllerian ence of endometriotic lesions at distant locations.
196 The Infertility Manual Endometriosis, Adenomyosis, and Infertility 197
Diagnosis Nezhat et al.19 have divided endometriomas into three adherent at various sites adjacent to the areas of
groups as follows: superficial endometriosis.
• Asymptomatic in about 1/5th of the patients
Type I—these are the small cyst present of less Endometriosis has been classified by AFS into
• Dysmenorrhea affects 85% of patients—congestive
than 2 cm on the ovarian surface with a densely four stages depending upon the laparoscopic findings
and spasmodic
adherent cyst wall which is difficult to remove. (Table 24.1):
• Pain severity does not correlates with the disease
Type II—they begin as functional cysts which gets Stage I (minimal) 1–5
severity
A B invaded by endometrial glands and stroma, hence Stage II (mild) 6–15
• Three main hypothesis have been suggested for pain
including inflammatory cells and cytokines, bleed- Figs. 24.1A and B: Ultrasound images of ovarian endometriomas. their cyst wall is easily removable. Stage III (moderate) 16–40
ing within the implants, and nerve irritation from Type III—these are large cysts and their cyst walls is Stage IV (severe) >40.
implants17 The morphology of peritoneal and ovarian implants
• It is possible that in any individual, more than one or all should be categorized as follows (Fig. 24.2):
of these mechanisms may be responsible for the pain Table 24.1: Classification of endometriosis.
• Patients with symptoms of severe dysmenorrhea and • Red (red, red–pink, and clear lesions)
Endometriosis <1 cm 1–3 cm >3 cm
deep dyspareunia have tender nodules in the areas of • White (white, yellow–brown, and peritoneal defects)
• Black (black and blue lesions). Peritoneum Superficial 1 2 4
the uterosacral ligaments and cul-de-sac region
Deep 2 4 6
• Patients developing dyschezia are those with infil-
tration of the uterosacral ligaments and/or diseases R superficial 1 2 4
directly adjacent to or invading the rectal wall Deep 4 16 20
Ovary
• The most severe pain is seen when the disease extends L superficial 1 2 4
6 mm below the peritoneal surface. However, the clini- Deep 4 16 20
cal examination may have false-negative results Posterior cul-de-cac Partial Complete
• CA-125 levels are elevated in moderate to severe endo- 4 40
metriosis due to the associated inflammation but it has
Adhesions <1/3 1/3–2/3 >2/3 enclosure
very low sensitivity (25–50%) for diagnosis of endome- enclosure enclosure
triosis.18
Ovary R filmy 1 2 4
Role of Imaging in the Diagnosis Dense 4 8 16
L filmy 1 2 4
Transvaginal and transrectal ultrasound are an important
Dense 4 8 16
tool for diagnosing ovarian endometrioma and rectovagi- Fig. 24.2: Endometriotic implants.
nal nodules. R filmy 1 2 4
Tube Dense 4* 8* 16
Ovarian endomerioma appears as a unilocular cyst
with acoustic enhancement and diffuse homogeneous L filmy 1 2 4
ground-glass echoes as a result of the hemorrhagic debris Dense 4* 8* 16
(Figs. 24.1A and B). If the fimbriated end of the fallopian tube is completely enclosed, change the point assignment to 16.
Gold standard for diagnosis is laparoscopy. Inspec-
tion of the pelvis begins in a clockwise or anticlockwise Additional Associated
Endometriosis Pathology
direction so that no lesions are missed. Number, size, and
_______________________ ______________________
location of endometrial plaques, lesions, implants, endo-
metriotic cyst, and/or adhesions are noted. To be used with normal tubes and ovarian To be used with normal tubes and ovarian
• Ovarian endometrioma should be confirmed by his-
tology or by the presence of the following features
(Fig. 24.3):
1. Adhesion to pelvic side wall and/or broad ligament
2. Endometriotic spots on the ovarian surface
3. Thick, tarry, and chocolate-colored fluid inside the
Fig. 24.3: Ovarian endometrioma.
cyst.
198 The Infertility Manual Endometriosis, Adenomyosis, and Infertility 199
MANAGEMENT significant loss of primordial follicles which are pre- metaplasia from ectopic intramyometrial endometrial tis- 1. Focal or diffuse myometrial bulkiness, typically
sent in the ovarian cortex. sue produced de novo.28 involving the posterior wall
Endometriosis can be managed either medically or 3. Ovarian cystectomy—if endometrioma is larger 2. Transition zone can thickened which appears as a
surgically. than 3 cm, cystectomy is preferred prior to IVF as it Diagnosis hypoechoic halo surrounding the endometrial layer
improves the endometriosis-associated pain and the of more than or equal to 12 mm thickness
Medical Management accessibility of follicles during oocyte pickup. Cystec- It has a reported incidence that ranges widely from 5 to 3. Subendometrial echogenic linear striations which
tomy is better than the drainage and electrocoagu- 70%, depending on the histological definition used or the appear as a rain in forest type of picture
It is advocated for the relief of pain and other symptoms,
lation of the endometrioma wall as it increases the imaging modality used.29 It may be silent causing no symp- 4. Subendometrial echogenic nodules
and not for infertility treatment due to its contraceptive
effect. Also, deep infiltrating endometriosis and endome- chances of spontaneous conception.21 Also, cystec- toms in some patients; in others, it may result in menor- 5. Small myometrial cysts/subendometrial cysts (most
triomas do not respond to hormonal therapy. Other dis- tomy is associated with less chances of recurrence as rhagia, dysmenorrhea, or dyspareunia. In 20% of cases, it specific sign31)
advantages are the side effects and it cannot be used for compared with other procedures.22 is associated with coexistent endometriosis.30 While doing 6. Myometrial echotexture appears heterogeneous
long term. a pelvic examination, uterine tenderness can be demon- 7. There is a hyperplastic reaction caused by endo-
Rationale of hormonal treatment in endometriosis has MANAGEMENT OF ENDOMETRIOSIS- strated during menstruation. metrial tissue, a “Venetian blind” appearance may
be seen due to subendometrial echogenic linear
been to induce a status that mimics either— ASSOCIATED INFERTILITY
• Pregnancy, i.e. prolonged exposure to high levels of Imaging Modalities Used to striations and acoustic shadowing where endometrial
progestogens which induces decidualization and ulti- Evidence suggests that ovarian cortex adjacent to endo- tissues cause a hyperplastic reaction.
metrioma’s shows reduced follicular number and activity Diagnose Adenomyosis
mately the degeneration of ectopic endometrium
Or with less rates of spontaneous ovulation.23
Ultrasound Role of MRI in Diagnosis of Adenomyosis
• Menopause, i.e. estrogen-deprived state to suppress Stage I/II endometriosis-associated infertility—
younger patients who are less than 35 years either expect- Pelvic ultrasound is usually the first imaging modality MR imaging is an accurate, noninvasive modality for diag-
the estrogen-dependent endometriosis.
ant management or superovulation with or without IUI employed to investigate menorrhagia and dysmenorrhea. nosing adenomyosis with a high sensitivity (78–88%) and
specificity (67–93%).32
Surgical Management can be offered after laparoscopy.24 Women 35 years of age The sonographic features of adenomyosis are variable and
or older, superovulation with IUI or IVF should be done. may be absent. Three forms can be seen as follows: Adenomyosis appears as either diffuse or focal thick-
Surgery should aim at the clearance of all the endometri- Stage III/IV endometriosis-associated infertility— ening of the junctional zone forming an ill-defined area of
a. Diffuse adenomyosis resulting in a globular enlarge-
otic lesions along with the restoration of pelvic anatomy. IVF should be offered to these patients. There is support- low signal intensity, occasionally with embedded bright
ment of the uterus: most common.
For peritoneal disease, either excision or ablation using ing evidence, which suggests that ultra-long protocol in foci on T2-weighted images.
b. Localized adenomyoma which typically involves the
electrocoagulation or laser vaporization is advocated. severe endometriosis increases the clinical pregnancy Histologically, areas of low signal intensity corre-
posterior wall displacing the endometrium anteriorly.
Excision of the peritoneal lesions is the only way to rates.25 However, a meta-analysis by Harb et al. showed sponds to smooth muscle hyperplasia, and bright foci
c. Cystic adenomyosis and myometrial cyst.
ensure complete treatment, as the exact depth of the peri- that in women with severe endometriosis (stage III/IV) on T2-weighted images corresponds to islands of ectopic
toneal implants cannot be assessed. undergoing IVF treatment, the implantation rates and The spectrum of findings includes the following endometrial tissue and cystic dilatation of glands. When
Ablation therapy claims that it is as effective as exci- clinical pregnancy rates are severely affected.26 (Figs. 24.4 and 24.5): menstrual hemorrhage occurs within these ectopic
sion and has the advantage of shorter operating time and
lesser bleeding. While treating the lateral pelvic wall, cau-
ADENOMYOSIS
tion is advised to avoid the thermal damage to the ureter.
Adenomyosis has been defined as the benign penetra-
Surgery for Ovarian Endometrioma tion of endometrium into the myometrium, resulting in
a globally enlarged uterus which microscopically exhib-
1. Ultrasound-guided aspiration—it is not the first line its ectopic, non-neoplastic, endometrial glands, and
of treatment. It may be used as an alternative approach stroma surrounded by the hypertrophic and hyperplastic
in recurrence and before IVF. Its major drawbacks are myometrium.27
inadequacy of cytology, adhesions, and infections. Pathogenesis of adenomyosis has been explained by
However, in situ injection of tetracycline, ethanol, or two theories—according to the first theory, it originates
methotrexate may prevent recurrence. from the invagination of the deepest portion (basalis) of the
2. Drainage and ablation of cyst wall—this can be endometrium between bundles of smooth muscle fiber of
achieved by using either CO2 laser or bipolar elec- the myometrium or along the intramyometrial lymphatic.
trocautery. It improves the endometriosis associated Second theory is based on the common origin of the
pain.20 The only disadvantage is that the cyst wall JZ myometrium and endometrium from müllerian ducts,
contains normal ovarian tissue which, if damaged, composed of pluripotent cells, and postulates that aden-
Fig. 24.4: Myometrial cyst. Fig. 24.5: Myometrial bulkiness with echogenic striations.
can potentially reduce the ovarian reserve due to the omyosis may originate and ultimately develop through
200 The Infertility Manual Endometriosis, Adenomyosis, and Infertility 201
Other modalities of treatment include uterine artery auto-antibodies in women with endometriosis. Hum
embolization and magnetic resonance image-guided Reprod. 1993;8:310-5.
14. Young SL, Lessey BA. Progesterone function in human
focused ultrasound surgery. Evidence is lacking regarding
endometrium: clinical perspectives. Semin Reprod Med.
there beneficial role in infertile patients. 2010;28:5-16.
15. Tomassetti C, Meuleman C, Pexsters A, Mihalyi A, Kyama C,
Simsa P, et al. Endometriosis, recurrent miscarriage and
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associated or unexplained infertility: a controlled
1. Impaired uterine contractility interfering with sperm myometrial tissue intact, along with the uterine recon- a randomized clinical trial with a five-year follow-up. Fertil
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C H AP T ER
25
202 The Infertility Manual
27. Bird CC, McElin TW, Manalo-Estrella P. The elusive 34. Kissler S, Zangos S, Wiegratz I, Kohl J, Rody A, Gaetje R,
adenomyosis of the uterus. Am J Obstet Gynecol. 1972;112: et al. Uterotubal sperm transport and its impairment in
583-93.
28. Benagiano G, Brosens I, Carrara S. Adenomyosis: new
endometriosis and adenomyosis. Ann N Y Acad Sci.
2007;1101:38-48. Role of Fibroids in Infertility
knowledge is generating new treatment options. Women’s 35. Silva PD, Perkins HE, Schauberger CW. Live birth after
Health. 2009;5:297-311. treatment of severe adenomyosis with a gonadotropin- Amit J Upadhyay, Kamini A Rao
29. Taran FA, Stewart EA, Brucker S. Adenomyosis: releasing hormone agonist. Fertil Steril. 1994;61:171-2.
epidemiology, risk factors, clinical phenotype and 36. Hirata JD, Moghissi KS, Ginsburg KA. Pregnancy after
surgical and interventional alternatives to hysterectomy. Chapter Outline
medical therapy of adenomyosis with a gonadotropin-
Geburtshilfe Frauenheilkd. 2013;73(09):924-31. • Pathophysiology • Effect of Fibroid on ART
releasing hormone agonist. Fertil Steril. 1993;59:444-5.
30. Chudleigh P, Thilaganathan B, Chudleigh T. Obstetric • Myomas and Infertility • Investigation
37. Osada H, Silber S, Kakinuma T, Nagaishi M, Kato K, • Myomas and Pregnancy • Management
ultrasound, how, why and when. Churchill Livingstone; Kato O. Surgical procedure to conserve the uterus for • Myoma and Obstetrical Outcomes • New Methods in Treatment of F
ibroid
2004.
future pregnancy in patients suffering from massive
31. Exacoustos C, Brienza L, Rienza A, Di Giovanni A,
adenomyosis. Reprod Biomed Online. 2011;22:94-9.
Szabolcs B, Romanini ME, et al. Adenomyosis: three-
dimensional sonographic findings of the junctional
38. Ozaki T, Takahashi K, Okada M, Kurioka H, Miyazaki K. Live
INTRODUCTION menopause), thus hypothecating that its intake might
birth after conservative surgery for severe adenomyosis decrease the risk of myomas.17 There is a probability that
zone and correlation with histology. Ultrasound Obstet
Gynecol. 2011;37:471-9. following magnetic resonance imaging and gonadotropin- • Myomas are benign gynecological tumor occurring stress may have an impact on the adrenal activity, which
32. Bazot M, Cortez A, Darai E, Rouger J, Chopier J, Antoine JM, releasing hormone agonist therapy. Int J Fertil Womens mostly in women during the reproductive age.1
Med. 1999;44:260-4.
might raise progesterone levels leading to increase in the
et al. Ultrasonography compared with magnetic resonance • Its occurrence is rare before menarche and commonly
39. Wang PH, Yang TS, Lee WL, Chao HT, Chang SP, size of fibroid (Table 25.1).18
imaging for the diagnosis of adenomyosis: correlation with regresses after menopause.2
histopathology. Hum Reprod. 2001;16:2427-33. Yuan CC. Treatment of infertile women with adenomyosis
• The lifetime incidence of fibroid is approximately 70%
33. Outwater EK, Siegelman ES, Van Deerlin V. Adenomyosis: with a conservative microsurgical technique and a gonad-
in white and 80% in black women.3 MYOMAS AND INFERTILITY
current concepts and imaging considerations. AJR Am J otropin-releasing hormone agonist. Fertil Steril. 2000;73:
Roentgenol. 1998;170:437-41. 1061-2. • The incidence of Uterine myomas are 20–50% in The impact of intramural fibroid on the fertility outcome is
women of reproductive age.4 still a dilemma; on the other hand, there is no doubt that
Various sign and symptom of fibroid such as heavy submucosal fibroids play a role in altering the reproductive
bleeding, prolonged bleeding, pelvis discomfort, and
altered reproductive function are seen in only 25% of
patients suffering from fibroid in their reproductive age.5,6 Table 25.1. The presence of fibroids may probably
affect fertility by the following mechanism.19
A review showing relationship of fibroids with infer-
•• Altering the position •• May decrease the chance of
tility reported that myomas might be responsible for only
of cervix sperm entering the cervical
2–3% of patients suffering from infertility.7 Submucous canal
myomas which deform uterine cavity have adverse effect
•• Increase in the size •• May hamper movement and
on reproductive outcomes,8 but this may not be the case or deformity of the transport of sperm
for those women affected with intramural fibroids, where uterine cavity
it is still an argument regarding the potential effects of •• Block the proximal •• Causes mechanical
intamural fibroids on the reproductive outcomes.9 end of tubes interference preventing the
sperm oocyte adhesion and
PATHOPHYSIOLOGY fertilization
•• Changed tubo-ovarian •• Interfere with ovum capture
• Genetic changes in the myometrial cell can give rise to
relation
fibroid and so it is also said to be clonal in origin.10,11
•• Altered uterine •• Might affect sperm or embryo
• Progesterone and various local growth factors also reg-
contractility movement or implantation
ulate growth of myomas.12–15 However, growth factors,
•• Degeneration or •• Interferes with implantation
cytokines, and chemokines are considered as potential
venous dilatation of embryo
effectors of estrogen and progesterone. above or adjacent a
• Genetic alterations, epigenetic mechanisms, and submucous fibroid
extracellular matrix (ECM) components are thought to •• Impaired endometrial •• Prevent endometrial
be important in the initiation and development of this blood flow development and implantation
tumor.16 •• Endometrial inflam- •• May alter endometrial
Soy protein have an anti-estrogenic effect when mation or secretion of receptivity
the level of estrogens in the body are high (i.e., prior to vasoactive substances
204 The Infertility Manual Role of Fibroids in Infertility 205
outcome. On the other hand, it is known that subserosal for implantation after ART when compared to infertile Advantages of MRI are as follows: Table 25.2. Recommended treatment options for
fibroids do not change fertility outcome. women without fibroids.8 women with uterine fibroid tumors.41
• Has the highest sensitivity for identifying uterine
• Intramural fibroids have less pronounced effect with Patient characteristics Treatment options
myomas, particularly in detecting small fibroids.39
an odds ratio of 0.62 for implantation rate and 0.7 for
MYOMAS AND PREGNANCY • Sensitivity and specificity of MRI in diagnosing a leio- Asymptomatic women Expectant manage-
delivery rate per transfer cycle.33 ment and follow-up
myoma is 88–93% and 66–91%, respectively.40
• Between 2.7–12.6% of pregnant women are seen to suf- • There is a negligible impact of subserosal fibroid on
• “Bridging vascular sign” which consists of vessels and/ Symptomatic women who desirous Non-surgical treat-
fer from leiomyoma.20 fertility with ART.32 of future fertility ment or myomectomy
or signal voids that extend from the uterus to supply a
• Growth of uterine fibroids usually occurs in the first Symptomatic women who do not Non-surgical treat-
pelvic mass is a useful sign to identify fibroids.41
trimester under the influence of raised sex steroids INVESTIGATION (FLOWCHART 25.1) desire future fertility but wish to ment or myomectomy
• MRI can predict and assess the response of fibroids to
hormone during pregnancy and many myomas, com- preserve the uterus
uterine artery embolization (UAE).
monly the bigger ones, often shrinks in the latter half Women who desire fertility preser- Myomectomy
• Fibroids showing a high signal on T1W images prior to vation and have had a pregnancy
of pregnancy.21,22
embolization are likely to have a poor response to UAE complicated by uterine fibroid tumors
• 71.4% of fibroids are seen to grow during the first and
as they may already have outgrown their blood supply Infertile women with distortion of Myomectomy
second trimesters, whereas 66% grow between the sec-
and undergone hemorrhagic necrosis.36 uterine cavity
ond and third trimesters.23
• Decrease in the size of fibroid after live birth has been In infertile women with uterine myomas, prior to any Women with severe symptoms who Hysterectomy
desire definitive treatment
observed in 72% of women.24 operative procedure, patient should be evaluated for other
concomitant causes of infertility and a plan should be
MYOMA AND OBSTETRICAL OUTCOMES
formed for the same.30 Medical Therapies
• Myomas associated with pregnancy may show compli- MANAGEMENT The ideal medical management in the treatment of fibroid
has remained elusive. There are various medical manage-
cation in approximately 10–40% of cases.25,26 Management of a patient with uterine fibroids is dependent
ment options available (Flowchart 25.2).
• Fibroids can cause increased risk of miscarriage and on clinical symptoms and is done in line of the wishes of the
preterm delivery. patient. There are number of treatment options available • GnRH agonist is known to reduce volume of fibroid by
• In cases where placenta is situated close to fibroid, it may which include expectant management, medical manage- 35–65% on treatment for 3 months. This is particularly
result in faulty trophoblastic reaction resulting in antepar- ment, noninvasive such as MR-guided ultrasound, mini- useful in women who are severely anemic, and hence,
tum bleeding and early rupture of membranes.27,28 mally invasive procedure such as uterine artery emboliza- surgery has to be deferred. Also, useful to make uterus
• They may result in abnormal presentations of the baby tion (UAE), and surgical management which could be open accessible by vaginal approach.42 However, there is a
resulting in increased chances of caesarean section. or laparoscopic or hysteroscopic myomectomy or hyster- study which found no effect of GnRH agonist on intra-
• Myomas with more than 3 cm in size show higher risk ectomy. Guidelines have been developed for clinicians to operative blood loss, postoperative fever, length of stay,
of developing complications during pregnancy. How- assist patient in making the optimum choice for manage- and pregnancy rates.43 Treating patient with GnRH
ment of fibroids (Table 25.2).41 agonist prior to surgery might increase the chances of
ever, there are cases reported in literature of normal
recurrent or persistent fibroids.44 There is no evidence
vaginal deliveries even with fibroids as large as 10 cm.29
Expectant Management of improving fertility with medical management.45
A meta-analysis done recently to review difference in
Asymptomatic women with serosal or subserosal or intra- Preoperative treatment with GnRH agonist has its own
outcomes between patients with fibroids at any site to no disadvantages such as:
mural fibroids can be managed expectantly.19 They are
fibroids suggests no difference in outcome.8 Contrarily, a
advised annual follow-up. In case of infertile women with • GnRH agonist causes softening of the fibroid and
study done on outcome of pregnancy in patients with or
infertility, expectant management is preferred unless the obscures its plane resulting in difficulty in removal.19
without myomectomy suggested that the risk of miscar-
fibroid is known to distort the uterine cavity or is very large • Thus, the operative time increases substantially.
riage was twice in patients without myomectomy.30
making conception difficult. • There are likelihood of recurrent/persistent myomas.44
EFFECT OF FIBROID ON ART Flowchart 25.2: Medical management in the treatment of fibroid.
Extensive studies have been done in patients to demonstrate
relationship and effects of fibroid on fertility outcome.8,31–33
• Location of fibroid is of critical importance in ART Results of sonohysterography correlate well hyst-
outcomes,34 as we are aware that submucosal fibroids eroscopic findings.37,38 Ultrasound with and without fluid
have poor fertility outcomes. Those distorting the cav- infusion is the best alternative for mapping of fibroid pref- ∑, non-steroidal anti-inflammatory drugs; £, gonadotropin-releasing hormone agonists; €, progester-
ity carry a relative risk of 0.3 for pregnancy and 0.28 erably when the uterus is not excessively enlarged. one receptor agonists; ≠, selective progesterone receptor modulators.
206 The Infertility Manual Role of Fibroids in Infertility 207
• The effect on reduction in volume is temporary and 3. Morcellation SOGC recommendation for abdominal myomectomy NEW METHODS IN TREATMENT
after discontinuing GnRH agonist the fibroid regrows.9 4. Laser technique. is as follows:46
OF FIBROID (FLOWCHART 25.3)
Medical therapy should not be recommend for the Various complications associated with the surgi- • It is preferable to take anterior uterine incision so as to
treatment of fibroids in cases of infertility as it may delay cal procedure should be explained to the patient such as minimize risk of postoperative adhesions. Flostat (Vascular Control Systems)
the use of more effective available treatment options.19 follows: A newer noninvasive technique (Flostat; Vascular Con-
Medical management is based on removing the stimula- Laparoscopic Myomectomy trol Systems) for management of fibroid utilizes applica-
1. Perforation of uterus
tion factors for fibroid such as estradiol and progesterone. It is a minimally invasive approach for removal of fibroids. tion of a device vaginally to the cervix under guidance
2. Bleeding
This can be achieved by avoiding ovulation or blocking Various studies have shown advantage of laparoscopic of Doppler to mechanically squeeze and obstruct the
3. Fluid overload
of estradiol and progesterone receptors. Hence, medical approach of over abdominal for removal of myoma. uterine arteries for few hours. Normal myometrium rep-
4. Electrolyte imbalance such as hyponatremia (when
treatment cannot be offered to patients with infertility.46 Two RCTs with a combined 267 patients compared erfuses after the removal of the device, but fibroid do
saline is not used as a distension media)
5. Large fibroid and severe endometrial disruption reproductive outcomes of laparoscopic myomectomy and not, and it later degenerate.19 There is decline in the vol-
Surgical Treatment myomectomy by laparotomy. ume of fibroid by approximately 40–50%, but data on the
increases the risk of postoperative intrauterine
• In patients with infertility with large intramural fibroids adhesion. In the first RCT, there was no significant difference in effect of this technique on future reproductive outcome
or submucosal fibroids indenting the uterus, myomec- the pregnancy rates between the endoscopy and laparot- are inadequate.51
tomy is the treatment of choice. Various techniques used to prevent development of omy groups (53.6 vs. 55.9%) of infertile patient who under-
• Also, in women with symptomatic fibroids who do not
adhesions after hysteroscopic surgeries are19 as follows: went myomectomy in view of infertility having minimum MRgFUS
wish to remove their uterus, myomectomy is routinely • Placement of intrauterine balloon catheter to preclude of one fibroid of greater than 5 cm in size. United States Federal Drug Administration approved
performed.19 juxtaposition of bald endometrial surfaces for a period Fewer cases of postoperative fever was seen in the application of MRgFUS for management of myomas in
• Since, myomectomy is a major surgery with inher- of 1 week postoperative endoscopic group.48 2004. It involves the destruction of uterine fibroid tissue
ent risks of blood loss and associated complications, • Increased dose of estrogen is given to facilitate growth Second RCT did not find any difference in the out- by coagulative necrosis by heating tissue to over 70°C by
counselling is a pre-requisite for these patients. of the endometrium, and come of cumulative pregnancy rates 1 year following focusing many high-frequency ultrasound beams on the
• Early “second-look” hysteroscopy for lysis of recurrent the procedure in laparoscopy and laparotomy groups target tissue. For maximum accuracy, the ultrasound
Society of Obstetrician and Gynaecology of Canada
adhesions. of patients (52.9 vs. 38.2%) and there was no significant beams are guided with MRI, which has the best resolution
(SOGC) recommendation for myomectomy are46 as follows:
difference in the cumulative pregnancy rates 1 year post and sensitivity to detect uterine fibroids.52
1. Removal of submucosal fibroid is associated with better A prospective cohort study showed that 72% of women
procedure.49
pregnancy rates in women with unexplained infertility.
were pregnant in less than 4 years following surgery and
SOGC recommendation for laparoscopic myomec-
Uterine Artery Embolization (UAE)
without further intervention.
2. There is no advantage of removing subserosal fibroids. tomy is as follows:46 Studies have been conducted on uterine embolization as
3. In women with unexplained infertility, intramural In women with history of previous miscarriage, the
an option to myomectomy and hysterectomy, mainly in
fibroids (not involving the uterine cavity, confirmed rate of miscarriage was reduced to 26% as compared to • Decision for laparoscopic myomectomy is d ependent women who no longer desire children, and it has progres-
62% before hysteroscopic myomectomy.47 on various criteria such as the total count, position,
on hysteroscopy) should not be removed regardless of sively become one of the most widely used noninvasive
SOGC recommendation for submucosal fibroids are as and dimension of uterine fibroids and also on the skills
the size of the fibroids. conservative methods of treatment of uterine fibroids after
follows:46 of operating surgeon.
4. The treatment of intramural fibroid should be indi- myomectomy.46
vidualized, and the benefits of surgery should be 1. It should be managed hysteroscopically. MRI study have shown transient ischemia changes
weighed against the risks in cases where conservative 2. The fibroid size should be less than 5 cm, although
Laparoscopic-assisted Myomectomy in the inner and middle layers of myometrium few hours
management is not an option. larger fibroids have been managed hysteroscopically, 1. Laparoscopic-assisted myomectomy has been rec- after UAE, but these changes decreases 48–72 hours
but repeat procedures are often necessary. ommended to decrease the operative time and asso- postprocedure, although the myoma shows irreversible
Hyteroscopic Myomectomy ciated complication such as uterine rupture. infarction.53
Indication: Abdominal Myomectomy 2. Procedure involves removal of myoma laparoscopi- Studies on outcome of fertility and pregnancy after the
cally followed by minilaparotomy to facilitate removal procedure are still limited.54,55
1. Myomas indenting the uterine cavity • The current indication for abdominal myomectomy of the fibroid and suturing of the myometrium.50 SOGC recommendation for UAE46 is as follows:
2. Minimum 50% of the volume of myomas is present is for infertile patients with large (>5 cm) type II sub-
within the uterine cavity. mucosal fibroids or type II fibroids with less than1 cm Flowchart 25.3: Newer methods for the treatment of fibroid.
between the external surface of the fibroid and the
Various methods for performing hysteroscopic
uterine serosa.
myomectomy are as follows:
• The goal, similar to that of hysteroscopic myomec-
1. Hysteroscopic scissors tomy for infertility, is to remove the fibroid in its
2. Electrosurgical techniques such as monopolar and entirety and to restore normal uterine cavity size and
bipolar cautery architecture.46 *MRgFUS, magnetic resonance-guided focused ultrasound surgery, #UAE, uterine artery embolization.
208 The Infertility Manual Role of Fibroids in Infertility 209
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• Recommendation for UAE by American College of
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9. Metwally M, Farquhar CM, Li TC. Is another meta-analysis myomas and complications in pregnancy. Obstet Gynecol.
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human myometrium and fibroids: changes during the and reproductive outcomes: a systematic literature ultrasonography in the diagnosis, mapping, and College of Obstetricians and Gynecologists (ACOG)
menstrual cycle and gonadotropin-releasing hormone review from conception to delivery. Am J Obstet Gynecol. measurement of uterine myomas. Am J Obstet Gynecol. Committee Opinion. Uterine artery embolization. Obstet
treatment. J Clin Endocrinal Metab. 1998;83:4092-6. 2008;198:357-66. 2002;186:409-15. Gynecol. 2004;103:403-4.
C HA PT E R
Reproductive Tract Infections
26A Contd...
RTI Causative agent
Incubation
period Clinical features and diagnosis Complications
Reproductive Tract Infections 211
Pelvic inflammatory
disease, salpingitis
Treatment
Tetracycline 500 mg oral
four times a day for 1
Vyshnavi A Rao
and endometritis week
leading to infertility Ofloxacin 400 mg two
Chapter Outline and ectopic times a day for 1 week
• Management of Sex Partners pregnancy
• Pelvic Inflammatory Disease Amoxicillin 500 mg oral
Cervicitis, urethritis/ three times a day for 1
bartholinitis week
INTRODUCTION cervical cancer, and increased risk of HIV trans- PROM leading to
mission. preterm delivery
• Reproductive tract infections (RTIs), sexually trans- • Adolescents are also a susceptible group of population In men, urethritis
mitted infections (STIs), and HIV/AIDS are becoming toRTIsduetotheirlackofknowledgeandunawarenessof epididymitis leading
a serious issue among public health problems. to infertility
risk factors, inability to access medical care when
• RTIs cause suffering to both sexes but appear to have Trichomo- Trichomonas Incubation Yellow or light colored abundant, Untreated: Symp- Metronidazole 2 g
required.
more devastating consequences in women. RTIs often niasis vaginalis (it is period— frothy vaginal discharge with foul toms1 remain orally after food single
• There are a lot of studies that covers epidemiological, venereal in origin) 5–28 days smell and sometimes itching, for years with dose/400–500 mg (AF)
go unrecognized and not treated leading to pelvic clinical, and diagnostic extent to the spectrum of RTIs painful urination, and dyspareunia exacerbation after 1-0-1 × 7 days along
Up to 80% of Most common
inflammatory disease, miscarriage, ectopic pregnancy, (Table 26A.1). menses with antiemetics
female partners form of vaginitis Diagnosis: Whipping motion on
have infected Incidence—Ap- direct microscopical examination Men: resolve with-
men as their prox 50% of of a swab of vaginal fluid of an out treatment
Table 26A.1: Causes, agents, clinical features, compilations and treatment of reproductive tract infections.
partners, women with infected woman (or) Can cause per-
Incubation suffer from vaginal dis- Urine of infected man examined sistent urethritis,
RTI Causative agent period Clinical features and diagnosis Complications Treatment trichomoniasis charge on a saline wet mount prostatitis, and
Gonor- Neisseria 2–5 days, Dysuria, frequency of micturition Salpingitis lead- Treatment is directed 5% of girls infertility
rhea gonorrhea associated with ing to infertility, toward both Gonorrhea 60%—proven
Purulent vaginal discharge being to infected
Chlamydia and increased occur- and Chlamydia with gonorrhea
Causing soreness but not pruritus rence of ectopic mothers become
Trichomoniasis1 Tab. Ciprofloxacin 500
Post coital bleeding Inflamed infected during
pregnancy, endo- mg orally (single dose)
vagina/urethral discharge. metritis, urethritis, delivery (IPPF,
(OR) 2004)
In men, it causes painful urination bartholinitis fever Tab. Cefixime 400 mg
and urethral discharge or pus rash associated orally Candidi- Yeast-like Incubation pe- Pruritus that is severe is associ- In men: It produces Clotrimazole 500 mg
with arthritis (blood (OR) asis organism riod of 12 hours ated with white discharge and itching and sore- high up in the vagina
Microscopy: reveals Gram stained
borne spread) Tab. Ofloxacin 400 mg Also
C andida albicans to 5 days sometimes with dyspareunia ness of the penis (single dose) or
urethral or endocervical discharge
specimen—Gram-negative PO known as (Gram-positive Thick, white, cheesy and forms after intercourse clotrimazole 200 mg tabs
intracellular diplococci culture: (OR) vaginal fungus) plaques and severe dys- vaginally once daily for
organism is isolated Spectinomycin 2 g IM thrush Sexually transmit- pareunia Small a week
Diagnosis: Clinical examination
(OR) ted papules with broken Fluconazole 150 mg
Specific: Direct microscopy of a skin may be seen
Tab. Azee 2 g Orally Frequently orally (stat dose) for both
saline/KOH wet mount prepara-
Ciprofloxacin seen in diabetic prepuce—painful partners
tion demonstrates pseudohyphae
and ofloxacin are women, during Vaginal paint—
contraindicated in pregnancy and Gentian violet 1%
pregnancy and breast women on oral in aqueous solution
feeding contraceptive pills repeated after 7 days
Chlamydia Chlamydia Associated Same as gonorrhea. Difficult to Cervicitis and Azithromycin 1 g oral
In Men: Usually do
trachomatis with Gonorrhea differentiate the two. absence of Gram- one dose
not require treatment,
(serovirus D toK) transmission of Diagnosis may be made by ob- negative diplococci (OR)
Antifungal cream on
infection can serving growth of organism in cul- on direct micro- Doxycycline 100 mg oral
occur even scopic examination. two times a day for 1 penis (Prepuce)
ture of cervical/vaginal discharge
if patient is or urethral discharge in men. Confirmed by week Treatment is indicated
asymptomatic serological tests/ (OR) only in cases of
Asymptomatic in 50–80% of
culture of causative reinfection of female
cases
organism partner
Contd... Contd...
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212 The Infertility Manual Reproductive Tract Infections 213
Contd... Contd...
Incubation Incubation
RTI Causative agent period Clinical features and diagnosis Complications Treatment RTI Causative agent period Clinical features and diagnosis Complications Treatment
Bacterial Caused by com- Incubation pe- Homogeneous thin milk white Preterm birth, low Metronidazole 500 mg Herpes Herpes simplex Incubation pe- Tertiary stage: Recurrence of No curative treatment,
Vaginosis mensal vaginal riod: 12 hours vaginal discharge birth weight babies BID for 7 days genitalis (HSV) types 1 riod is 3 weeks In not treated syphilis, secondary herpes occurs in hence recurrence is a
organisms to 5 days Vaginal walls are coated with the Sexually transmit- 0.75% gel per vaginally and 2 causes approx. 1 week stage occupies many years during half of the infected possibility
discharge ted infections (or) Herpes genitalis (IPPF, 2004) which spirochaetes attack bones, women, less severe Analgesics for relief of
Diagnosis: Fishy odor after Pelvic inflammatory Clindamycin 300 mg BID Spreads by joints eyes, blood vessels, heart, features, Infected pain.
addition of 10% KOH. pH 4.5 (or) diseases for 7 days Sexual contact and CNS causing neurosyphilis mothers—sponta-
Wash genital areas with
higher with infected and cardiovascular syphilis after neous abortions
(Or) soap and water regularly
partner 1–20 years of primary infection and premature
Clue cells: epithelial cells that 2% cream vaginally QID Acyclovir—200 mg oral
often fatal not amenable to labor
are studded with bacteria on light for 7 days five times a day for 7
microscopical examination of a treatment
For a patient with poor days in first episode
saline wet mount compliance, single dose Diagnosis: Dark ground
Recurrent episodes,
metronidazole 2 g orally illumination microscopic
same doses for even 10
may be used (may not be examination of serum from base
days partner should also
very effective) of primary chancre or from most
be treated
Chancroid Haemophilus Incubation Pain on urination/defecation, rectal Ulcer disappears if Azithromycin 1 g oral as lesions of secondary syphilis
period— bleeding dyspareunia Vaginal treatment not initi- a single dose (or ) Cip- shows T. pallidum
(soft core) ducreyi
Spreads through 2–7 days discharge painful shallow ulcers ated in a month but rofloxacin 500 mg twice Specific/nonspecific tests:
sexual/asexual with offensive pus on labia minora may be present till daily for 3 days Nonspecific tests:
contact and majora, on the introitus and 12 weeks (Or) Erythromycin
around the anus–bright red zone Wasserman test (WR)
500 mg four times for
of congestion, surrounded by complement fixation test
a period of 1 week or
edema, bleeds on touch ceftriaxone 250 mg IM venereal disease research
Diagnosis: by clinical features, single dose laboratory (VDRL) test
painful ulcers Inguinal buboes when flocculation type test for
Gram-negative bacilli in chains infected with pus needs nonspecific antibody
(Ducreyi bacilli on direct micros- to be aspirated Specific:
copy after Gram stain or by culture) (a) TPHA test (Treponemal
Syphilis Treponema Incubation pe- Primary stage: ulcer or chancre Tertiary stage of Early syphilis: hemagglutination test)
spread pallidum riod = 10 days can be seen at infected site syphilis—tiny, rub- Benzathine penicillin (b) FTA—ABS test (Fluorescent
by sexual 40% spontane- to 3 months Woman: vulva, cervix, mouth/anus bery lesions may G 2.4 million IU in two treponemal antibody test)
contact ous abortions, Approximately ulcer—painless sharply defined be seen on bones, intramuscular injections
skin, nervous sys- blister like painful ulcers in and
Post stillbirths, or 3 weeks serpiginous outline with brownish during one clinic setting
tem tissue around vagina, anus/thighs,
exposure, neonatal death it red base cervix erosion (Or) pain on urination, watery vaginal
60% of causes congenital Men: ulcer that is painless on penis, More prone to heart Aqueous procaine
syphilis attacks, paralysis, discharge, cervicitis or proctitis,
sexual anus, testicle, mouth, and nose benzathine penicillin G
blindness, stroke, headache, backache malaise,
partners Inguinal glands enlarge when 1.2 million IU daily by IM
and dementia fever
are af- ulcer develops in the external injections for 10 days
fected. first episode—clears up in
genitalia of either male or female For patients who are
Transmis- 2–4 weeks
Hard, but painless primary lesion allergic to penicillin
sion from of chancre—unnoticed nonpregnant patients: Diagnosis: is by clinical features,
mother to Doxycycline 100 mg oral tissue culture
Secondary stage:
fetus can two times a day for 2
occur at 9 Primary chancre takes about in
1–8 weeks to heal, blood spread, weeks
weeks of
gestation secondary stage develops (Or) PELVIC INFLAMMATORY DISEASE Causative Agent
Anorexia, headache, and pyrexia Tetracycline 500 mg oral
along with macular, pustular, or QID for 2 weeks • It is the infection of the upper portion of female repro- Neisseria gonorrheae (or) Chlamydia trachomatis.
papular skin rashes being nonir- For penicillin—allergic ductive tract that affects uterus, fallopian tubes, ova-
ritant seen bilaterally symmetrical pregnant patients: ries, and inside of the pelvis.
in distribution copper colored Erythromycin 500 mg
Clinical Features
• It comprises a spectrum of inflammatory disorders
occasional mucus patches—gen- oral QID daily for
eralized symmetrical glandular of upper part of female genital tract a combination of Fever, mucopurulent vaginal discharge, dyspareunia (or),
15 days
enlargement endometritis, salpingitis, tubo-ovarian (TO) abscess, irregular menses. Untreated PID can lead to ectopic preg-
and pelvic peritonitis.2,3 nancy, infertility, and chronic pelvic pain.
Contd...
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214 The Infertility Manual Reproductive Tract Infections 215
Diagnostic Considerations Recommended IM/Oral Treatment Flowchart 26A.1: Treatment of pelvic inflammatory disease (PID).
Diagnosis of acute PID is difficult. Late diagnosis and treat- Ceftriaxone 250 mg IM in a single dose
ment will lead to inflammatory sequelae. PID producing Plus
symptoms has a positive predictive value of 65–90%4-6 for Doxycycline 100 mg oral two times a day for 14 days
salpingitis. With or without
Metronidazole 500 mg twice a day for 14 days
Minimum Criteria (OR)
Cefoxitin 2 g IM single dose8 and Probenecid 1 g oral
One or more of the following
• Cervical motion tenderness administered concurrently in a single dose plus doxycy-
(OR) cline 100 mg orally twice a day for 14 days
• Uterine tenderness With or without
(OR) Metronidazole 500 mg orally twice a day for 14 days
• Adnexal tenderness (OR)
Other third-generation cephalosporin (e.g. Ceftizox-
Specific Criteria ime/Cefotaxime plus Doxy 100 mg BID for 14 days)
With or without
• Oral temperature more than 101°F or 38.3°C Metronidazole 500 mg BID for 14 days.7
• Abnormal cervical mucopurulent discharge or cervi-
cal friability
• Presence of abundant numbers of WBC on saline Parenteral Treatment
microscopy of vaginal fluid
Cefotetan 2 g IV every 12 hour
• Increased erythrocyte sedimentation rate (ESR),
Plus
c-reactive protein (CRP)
Doxycycline 100 mg orally (or) IV every 12 hours
• Laboratory documentation of cervical infection with
(Or)
N. gonorrheae (or) C. trachomatis.
Cefoxitin 2 g IV every 6 hours
Plus
Most Specific Criteria for Diagnosing PID
Doxycycline 100 mg orally (or) every 12 hours
• Endometrial biopsy with histopathology evidence of (OR)
endometritis. Clindamycin 900 mg IV every 8 hours MANAGEMENT OF SEX PARTNERS PROBABLE QUESTIONS
• Transvaginal sonography (or) MRI showing thickened, Plus • Men who have had sexual contact with a woman 1. Write short notes on Trichomonas vaginalis.
fluid filled tubes with or without free pelvic fluid (or) Gentamycin loading dose IV (or) IM (2 mg/kg) with PID during the 60 days preceding her onset of 2. Etiology of pelvic inflammatory disease and
TO complex (or) Doppler studies suggestive of pelvic followed by maintenance dose (1.5 mg/kg) every 8 hours. symptoms should be evaluated, tested, and presump- management.
infection. Single daily dosing (3–5 mg/kg) can be substituted. tively treated for chlamydia, gonorrhea, regardless of 3. Diagnosis of bacterial vaginosis and complications
• Laparoscopic findings consistent with PID.
the etiology of PID (or) pathogens isolated from the associated with it.
woman. 4. Short notes on Gonorrhea and Chlamydia.
Treatment of PID (Flowchart 26A.1) Alternative Parenteral Regimen • If woman has had last intercourse more than 60 days 5. Candida albicans—diagnosis and management.
PID regimens must provide empiric broad spectrum cov- Ampicillin/sulbactum 3 g IV every 6 hours before onset of symptoms, treatment of most recent
erage of likely pathogens. Plus partner is mandatory.
REFERENCES
Indications for hospitalization in a case of PID: Doxycycline 100 mg orally (or) IV every 12 hours • Male partners of women who have PID caused by
If the culture for gonorrhea is positive, treatment must C. trachomatis and/or N. gonorrheae frequently are 1. Chaudhuri SK. Practice of fertility control: a comprehensive
• Surgical emergencies (e.g. appendicitis cannot be
manual seventh edition 2008, Reprinted 2011;312-21.
excluded) be directed toward antimicrobial susceptibility. asymptomatic, arrangements should be made to link
2. www.cdc.gov/std/tg2015/pid.htm
• Tubo-ovarian abscess If the isolate is determined to be quinolone-resistant male partners to treatment. If linkage is delayed (or) 3. Wiesenfeld HC, Sweet RL, Ness RB, Krohn MA, Amortegui AJ,
• Pregnancy N. gonorrhea (QRNG) or if antimicrobial susceptibility unlikely, enhanced referral to treat these men is essen- Hillier SL. Comparison of acute and subclinical pelvic
• Severe illness, nausea, and vomiting/high-grade fever cannot be assessed [e.g. if only nucleic acid amplification tial to treat chlamydial (or) gonococcal infections.10,11 inflammatory disease. Sex Transm Dis. 2005;32:400-5.
• Unable to follow/tolerate an oral regimen test (NAAT) is available, an infectious-disease specialist
• No clinical response to oral antimicrobial treatment has to be consulted].
Kamini_Ch26.indd 214 21-12-2017 21:00:27 Kamini_Ch26.indd 215 21-12-2017 21:00:27
C H APT E R
216 The Infertility Manual
4. Jacobson L, Westrom L. Objectivized diagnosis of acute
pelvic inflammatory disease. Diagnostic and prognostic
value of routine laparoscopy. Am J Obstet Gynecol.
1969;105:1088-98.
5. Sellors J, Mahony J, Goldsmith C, Rath D, Mander R,
Hunter B, et al. The accuracy of clinical findings and
azithromycin or doxycycline for pelvic inflammatory disease:
a randomized controlled trial. Obstet Gynecol. 2007;110:
53-60.
9. Hosenfeld CB, Workowski KA, Berman S, Zaidi A,
Dyson J, Mosure D, et al. Repeat infection with chlamydia
and gonorrhea among females: a systematic review of the
Genital TB and Infertility
26B
Vyshnavi A Rao
laparoscopy in pelvic inflammatory disease. Am J Obstet literature. Sex Transm Dis. 2009;36:478-89.
Gynecol. 1991;164(1 Pt 1):113-20. 10. Golden MR, Whittington WL, Handsfield HH, Hughes Chapter Outline
6. Bevan CD, Johal BJ, Mumtaz G, Ridgway GL, Siddle NC. JP, Stamm WE, Hogben M, et al. Effect of expedited
• Incidence • Hydrosalpinx
Clinical, laparoscopic and microbiological findings in treatment of sex partners on recurrent or persistent • Pathophysiology of Genital TB • Male Genital Tuberculosis in Infertile Population
acute salpingitis: report on a United Kingdom cohort. Br J gonorrhea or chlamydial infection. N Engl J Med. 2005;352: • Clinical Presentation of Genital TB • Epididymal and Testicular TB Dr. Mandeep Kaur
Obstet Gynaecol. 1995;102:407-14. 676-85. • TB of Fallopian Tube • Prostatic TB
7. Source: Walker CK, Wiesenfeld HC. Antibiotic therapy for 11. Schillinger JA, Kissinger P, Calvet H, Whittington WL, • Tuberculosis of the Ovary • Penile TB
acute pelvic inflammatory disease: the 2006 CDC Sexually Ransom RL, Sternberg MR, et al. Patient-delivered • Tuberculosis of Cervix • Urethral TB
Transmitted Diseases Treatment Guidelines. Clin Infect partner treatment with azithromycin to prevent repeated
Dis. 2007;28(Supp 1):S29-36. Chlamydia trachomatis infection among women—a
8. Savaris RF, Teixeira LM, Torres TG, Edelweiss MI, randomized, controlled trial. Sex Transm Dis. 2003;30: INTRODUCTION The common site of female genital TB is fallopian tube.
Moncada J, Schachter J. Comparing ceftriaxone plus 49-56. The incidence of female genital TB reported is given in
• World Health Organisation (WHO) declared tubercu- Table 26B.1.
losis (TB) as a global emergency in 1993 as it affects
individual’s health and India at large. Table 26B.1: Frequency of tuberculosis in female genital organs.
• India accounts for about one-fifth of the global inci- Site Percentage
dence and about 1.9 million cases are reported every Fallopian tube 92–100
year. Every day, 5,000 people develop the disease and Endometrium 35–50
1,000 people die due to TB.1 Ovaries 10–30
Vulva and vagina <1
INCIDENCE Sharma et al. observed violin string type of adhesion
Incidence of infertility: 40–75.6%. between liver, diaghragm, or anterior abdominal wall in
TB affects postmenopausal women (62%) in devel- severe type of FGTB (Fig. 26B.1).3 Tubal surface and mes-
oped countries; 28% in developing countries belong to the osalpinx may show appearance of yellowish gray tubercu-
postmenopausal category.2 les. The most typical feature of TB salpingitis is with fim-
However, its incidence is underreported due to: brial ends of the tube remaining open.
• Latency of the organism
• Asymptomatic picture and varied presentation in
many cases and due to lack of accurate diagnostic
tool.
PATHOPHYSIOLOGY OF GENITAL TB
1. Hematogenous miliary spread from a primary
pulmonary lesion.
2. Hematogenous spread from a secondary miliary site.
3. Lymphatic spread from a primary pulmonary site to
intestinal lymph node and then spread into the pelvis.
4. Direct extension from adjacent abdominal organs.
A venereal transmission can also occur.
Female genital tuberculosis (TB) is almost always sec-
ondary to TB elsewhere in the patient’s body. Fig. 26B.1: Laparoscopic appearance of Fitz Hughs Curtiz syndrome:
Genital TB can coexist with pulmonary TB in about Violin string type of adhesion between liver, diaghragm.
8–15% of women. Source: Milann—The Fertility Center, Jayanagar, Bengaluru.
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218 The Infertility Manual Genital TB and Infertility 219
TUBERCULOSIS OF THE OVARY Table 26B.2. Different factors responsible in the
causation of TB.
Usually bilateral involvement
Tubal factor Defective ovarian function
Two forms of ovarian TB are as follows:
Blockage of fallopian tube Due to mass formation, adhe-
1. Perioophoritis: ovary is surrounded by or encased Loss of tubal function due sions, anovulation, and poor
in adhesions and studded with tubercles, caused by to ciliary damage causing ovarian reserve
direct extension from the tube. infertility and ectopic gesta- Endocrine disruption, chronic
2. Oophoritis: infection starts in the stroma of the ovary tion anovulation, Mycobacterium
by hematogenous spread. Perisalpingitis causing tuberculosis has antigonado-
adhesions and to mass tropic effect
formation Poor oocyte quality (intrinsic
TUBERCULOSIS OF CERVIX Tubercular hydrosalpinx oocyte defect)
with or without o
bstruction The basal production of
1. No specific macroscopic changes. progesterone is inhibited by
Fig. 26B.2: Symptomatology of tuberculosis.
2. May appear normal or inflamed, resemble invasive mycobacteria and the stimu-
carcinoma. latory effect of hCG being
CLINICAL PRESENTATION OF 3. Most common type: ulcerative form. opposed on corpus luteum
causing LPD, implantation
GENITAL TB failures, lower pregnancy
Causes of Infertility in FGTB rates and higher miscarriage
GTB presents as a chronic granulomatous inflammatory rates. Poor embryo quality
Infertility is very common in FGTB (40–80%) and can be
disease usually with low-grade symptoms (Fig. 26B.2). due to “intrinsic oocyte defect”
both primary and secondary (Table 26B.2, Fig. 26B.4).
Some patients present with specific complaints. It is
asymptomatic in 11% of patients.4
TB OF FALLOPIAN TUBE
• TB endosalpingitis
• TB exosalpinigitis
• Interstitial TB salpinigitis
• Salpingitis isthmica nodosa
Tubes affected with TB bacilli occurs usually in
patients with everted fimbriae (Fig. 26B.3), enlarged tube
causing distension, typically described as “tobacco pouch Fig. 26B.3: Microscopic appearance of the female genital tuber-
appearance.” culosis.
Flowchart 26B.1: Tuberculosis of the endometrium.
Fig. 26B.4: Failure of implantation in female genital TB.
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220 The Infertility Manual Genital TB and Infertility 221
Flowchart 26B.2: Diagnosis of tuberculosis.
Adverse Effect on Endometrial Markers: Genital TB is an asymptomatic disease and its diagno-
sis requires a high degree of suspicion.
Defective Vascularization of Endometrium Fig. 26B.7: Hysterosalpingogram showing adhesions in genital TB. Fig. 26B.8: Hysteroscopic appearance of TB endometritis.
Secretion of aPL Antibodies Ultrasonography Source: www.conceptionadvice.com/hysterosalpingogram-test-hsg-test/ Source: Milann—The Fertility Center.
Altered immune response → activate aPL antibodies. • Persistently thin endometrium 2–3 mm broken, bright,
Endometrial micro thrombus formation resulting in im- and echogenic.
appearance” when imaged in cross section, pathogno- • Poor distensibility
paired receptivity and implantation failure (See Flowchart • Periovarian inflammation and specs of calcification on
mic of a hydrosalpinx. • Narrowing of uterine cavity
26B.1). ovarian surface (Fig. 26B.5, Flowchart 26B.2).
• Waist sign (indentations on opposite sides).
Decreased subendometrial blood flow: repeated
• Incomplete septations give a “beads on string sign.”
implantation failure in patients undergoing antiretroviral HYDROSALPINX (Facilitates discrimination between hydrosalpinx
Laparoscopy Findings (Table 26B.3)
therapy (ART) treatment.
• Thickened longitudinal folds of fallopian tube and other adnexal masses.) Poor vascularization on • Peritoneal surface studded with tubercles
Antitubercular drugs can increase endometrial thick-
(Fig. 26B.6). Folds produce a characteristic “cog wheel Doppler. 3D imaging helpful in cases with uncertain • Bluish discoloration of uterus/inflammation
ness, increase in subendometrial peak systolic blood
features on 2D images. • Salpingitis, oophoritis, or a TO mass
flow with significant increase in ongoing pregnancy, and
• Occlusion of tubes causing hydrosalpinx
delivery rates.
Hysterosalpingogram (Fig. 26B.7) • Free peritoneal fluid looking like blood
• Caseation in POD
• HSG should not be performed in the presence of recent
• “Frozen pelvis”
acute pelvic infection.
• Omental adhesions
• Fistulous tracts between the genital tract and other
pelvic organs.
• Occlusion of the distal end of the fallopian tubes is
common, although marked hydrosalpinx is usually
Table 26B.3. Laparoscopic evidence of genital TB.
uncommon.
• Calcification of the organs. Suspicious: subtle signs of
Clear evidence of GTB chronic inflammation
Hysteroscopy (Fig. 26B.8) •• Multiple granulomas or •• Patent tubes with pelvic
exudates congestion
There is no exact appearance that can be suggestive of TB
•• Loculated fluid, adhe- •• Few scattered small
on hysteroscopy. sions granulomas
• Pale endometrium •• Hydrosalpinges •• Fimbrial agglutination
• Intrauterine synechiae of varying grade •• Beaded blocked tubes and phimosis
•• Tubal sacculations, peri-
• Completely obliterated cavity (80%) by adhesions hepatic adhesions
Fig. 26B.5: Thin endometrium on ultrasonography. Fig. 26B.6: Hydrosalpinx on ultrasonography. • Granulomas
Source: Milann—The Fertility Center. (Source: https://radiopaedia.org/articles/hydrosalpinx)
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222 The Infertility Manual Genital TB and Infertility 223
Laparoscopic Criteria (Table 26B.4) the form of growth index. A strong suspicion of TB is seen Table 26B.5. Medications used to treat tuberculosis.
when the growth index is greater than 10. It is advanta-
Maximum adult dose
Diagnosis of Latent TB Infection (LTBI) geous in terms of reducing culture time of 2 weeks; drug
Twice
sensitivity can be achieved quickly.9
• Mantoux-Tuberculin skin test (TST) Medication Daily weekly Adverse reactions Drug interactions
• Interferon Gamma Release Assay (IGRA) Isoniazid (H) 300 mg 900 mg Hepatatis, hepatic enzyme elevation, Phenytoin, carbamazepine, diazepam,
• Quatiferon-TB or QuatiFERON-Gold—detection of
Polymerase Chain Reaction peripheral neuropathy, rash disulfiram, triazolam, valproate, warfrarin
release of IFN-g on stimulation of sensitized T cells by It is a quick assessment of rapidly detecting and quan- Rifampin (R) 600 mg 600 mg Hepatatis, rash, fever, thrombocyto- Protease inhibitors, non-nucleoside
M. tuberculosis antigens. tifying only few DNA copies with high sensitivity and penia, orange discoloration of body reverse transcriptase inhibitors, metha-
• T-spot—detection of the T cells themselves. fluids done, warfarin, glucocorticoids, anticon-
specificity. Sensitivity is markedly high requiring only
vulsants, estrogens, azole antifungals
less than 10 bacteria per mL of specimens to get a posi-
tive result. Rifabutin 300 mg 300 mg Rash, hepatitis, fever, thrombocyto- Same medications as rifampin but milder
Menstrual Blood Analysis Bhanu et al. did a study on improved diagnostic penia, orange discoloration of body interactions
fluids
• Acid fast bacilli (AFB) smear (Flowchart 26B.3): value of PCR in the diagnosis of female genital TB lead-
ing to infertility. The study reported 56% of patients suf- Rifapentine NA 600 mg Hepatatis, rash, nausea, vomiting, Same medications as rifampin
Ziehl Neelsen
fering from infertility were PCR positive for TB while AFB weekly orange discoloration of body fluids
Carbol fuschin
Auromine Rhodomine (one smear) positivity was only 1.6% and positives were Pyrazinamide (Z) 2g 4g GI upset, hepatitis, rash, arthralgias, Cyclosporine
Stains bind to mycolic acid of the cell wall—it only 3.2%. Among samples tested, endometrial aspi- hyperuricemia
requires 10 organisms/mL for a positive test rate yielded 47% positivity, and POD fluid showed 16%
Ethambutol (E) 1.6 g 4g Optic neuritis, peripheral neuritis, Aluminum hydroxide
• QuatiFERON-Gold positivity.10 cutaneous reactions
M. tuberculosis is incubated with plasma separated
from whole-blood-specific antigens, ESAT-6, RD1, Comparison of Various Diagnostic
and CFP-10, and the levels of interferon gamma Modalities with Studies Table 26B.6. New categorization of patients.
(IFN-G) are measured. Treatment groups Type of patient Intensive phase Continuation phase
Positive test greater than 0.35 IU/mL Jindal et al. studied the efficacy of laparoscopic visual
inspection versus endometrial TB-polymerase chain reac- New Sputum smear positive sputum smear 2 H3R3Z3E3 4H3R3
Good corroboration with PCR negative extrapulmonary
Diagnosis tion (PCR) for an early diagnosis and management of
Others
Evaluating response to ATT in women with latent FGTB in India. Both laparoscopy and endometrial TB-PCR
were performed on 162 infertile women. Previously treated Smear positive relapse 2H3R3Z3E3S3/1H3R3Z3E3 5H3R3E3
GTB is a negative predictor to ATT therapy
A total of 52 endometrial TB-PCR-positive patients Smear positive failure
Sensitivity-89%, specificity-98% (Mori et al. Am J
were diagnosed as definite GTB, of whom 44 (84.6%) also Smear positive treatment after default
Respir Crit Care Med. 2004).
showed laparoscopic findings suspicious of TB. PCR posi- Others
Flowchart 26B.3: AFB culture methods. tivity is found in 54.5% of strongly suspicious and 54.1%
of mildly suspicious patients. Hence, they concluded that
endometrial TB-PCR had higher sensitivity to diagnose TB-PCR-positive patients for diagnosis but may be still and gynecological examination, and use of interventions
GTB as in laparoscopy. Laparoscopy may be avoided in required to rule out GTB in PCR-negative cases.11 such as endometrial biopsy along with imaging methods
Sharma et al. studied the laparoscopic evaluation of and endoscopic visualization, especially with laparos-
genital TB. In this study, a total of 85 women who had geni- copy (Tables 26B.5 and 26B.6).
Table 26B.4. Evaluation of diagnostic procedures. tal TB underwent diagnostic laparoscopy for infertility or
Sensitivity Specificity PPV NPV chronic pelvic pain was done retrospectively. Most women MALE GENITAL TUBERCULOSIS IN
Procedure (%) (%) (%) (%) were from low socioeconomic status (68.1%), past history
of TB (34.1%), women with pulmonary TB (22.3%). and
INFERTILE POPULATION
Smear 87.5 86.3 70 95
women with extra pulmonary TB(11.7%). Primary infertil- • Male genital TB is the most common extrapulmonary
BACTEC Histopathology 82.3 84.6 87.5 78.5
ity (72.9%) and secondary infertility (17.6%) and rest had site for infection with tubercle bacilli. TB can affect the
Culture 91.6 88.8 84.8 94.1
It is a radiometric culture method. Growth of AFB uses chronic pelvic pain (9.4%).3 entire male reproductive system: epididymis, testis,
PCR 96.4 100 100 66.66
palmitic acid as a substrate. The carbon atoms are radi- Interventions such as endometrial biopsy, PCR, HSG prostate, seminal vesicle, vas deferens, scrotal skin,
olabeled causing release of CO2, which is measured in Culture + PCR 100 100 100 100 with or without other tests, and laparoscopy are invalua- bulbourethral glands, and penis.
ble in arriving at a correct diagnosis of GTB. The final diag- • Socioeconomic factors, inflammation, and scar-
nosis is based on a good history taking, careful systemic ring that will follow infection results in distorted
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224 The Infertility Manual Genital TB and Infertility 225
normal anatomy causing fibrosis around the repro- Clinical Features URETHRAL TB • Patient compliance toward treatment is difficult due
ductive tract structures, which will ultimately lead to to increased duration and associated adverse effects of
obstruction. It may be asymptomatic at times, and some patients present It is secondary to genital TB. drugs.
with dysuria, frequency, hematuria, and hemospermia. In Acute urethritis presents as mycobacterial discharge,
Incidence
severe form of presentation, the formation of abscess and which leads to chronic stricture formation. Urethral stric- Standard Treatment of TB
perineal sinuses may occur, and decreased semen volume tures or fistulas may cause prevention of deposition of the
• Monitoring culture and sensitivity reports, altering
Male factor is solely responsible in 30% of cases of infer- may give an indication toward extensive spread of prostatic semen into vagina during intercourse.
antibiotic as necessary.
tility, out of which 15% is related to genital tract infection disease or ejaculatory duct obstruction. About 85% of cases Diagnosis is made on:
• Six months course of chemotherapy is recommended
including TB (IJSAR 2015) Agarwal et al. International with prostatic TB may have associated renal TB. a. Suggestive history
for genitourinary TB.
Journal of Sciences & Applied Research coexist with renal On examination: on per rectal examination, prostate b. Presence of nodules within epididymis or nodules in
• HIV-positive cases require treatment for a total of
TB—60–65% cases of pulmonary TB in 34% cases 2002 appears to be nodular with irregular enlargement being vas deferens
9 months.
(Indian J Allergy). nontender on palpation. c. by performing a fine needle aspiration cytology
• Malnourished patients require a highly nutritious diet
(FNAC)/epididymal excision
for maintaining their general health and body habitus.
Spread PENILE TB d. Presence of granulomatous infection on a tissue
specimen
• Hematogenous Mode of Spread • Positive culture/histological analysis of biopsy
Prevention of Genitourinary TB and its
Lymphatic specimens, possibly along with PCR—establishes a Transmission
From urinary tract Infected female is responsible for causation of TB of the
diagnosis of TB. • Screening of sex partners
To testis locally from epididymis penis by the sexual route who has genitourinary TB, and
• Leukospermia—early symptom of TB-related i nfertility. • Condom use has to be adopted to prevent possible
Dissemination from the prostate or the skin of penis, glans, and cavernous bodies are also
• Vasogram revealed multiple level blocks. transmission to sexual partners.
Seminal vesicles with retrograde involvement affected.
• Ultrasound shows diffuse hypoechogenicity in
throughout vas deferens. epididymal and testicular inflammation. CONCLUSION
Incidence • Transrectal ultrasonography: identifies site of
EPIDIDYMAL AND TESTICULAR TB TB is a rare cause of male infertility, and quick diagnosis
Less than 1% can result in disfigurement, ulcers, and bul- obstruction.
should be made.
(FLOWCHART 26B.4) bous enlargement with deformity leading to infertility. • Other imaging techniques: radiology: plain X-ray
There are no studies evaluating the validity of screen-
chest and abdomen with intravenous pyelography and
ing in men in the presence or absence of female genital TB.
retrograde pyelography.
PROSTATIC TB Clinical Features Most of the cases diagnosed to have TB will need ART,
• Contrast-enhanced CT scan.
It appears as a papulonecrotic tuberculid or as an ulcer at which provides results comparable to those in men with-
Mode of Spread out the disease.
the glans. Ulcer of the penis may cause cavernositis that Urine and Semen Analysis
It spreads hematogenously. extends to the urethra.
• Microscopic hematuria, albuminuria sterile pyuria.
• Early morning urine cultures (atleast three samples)
PROBABLE QUESTIONS
and semen AFB. 1. Discuss in detail regarding the etiology, pathophysiol-
• Semen analysis: low volume in 89% of patients. ogy, and management of female genital tuberculosis.
Flowchart 26B.4: Effects of epididymal and testicular tuberculosis. • Azoospermia/oligospermia in 53% of patients. 2. Medical management of genital tuberculosis.
• Significant leukocytospermia was identified in 77.6% 3. Male genital tuberculosis.
of patients with prostatic TB. 4. Adverse effects of antitubercular therapy.
Treatment
REFERENCES
Primary aim is totreat infection: 1. Revised National Tuberculosis Programme 2006. Directorate
• Requires conventional courses of TB chemotherapy. General of Health Services, Ministry of Health and Family
• Surgery is needed in case of abscess, obstructive symp- Welfare. [Cited 2011 July 1] Available from http://www.
toms, or failure of chemotherapy. tbcindia.org/pdfs/Procurement%20Manual.pdf
• Genitourinary TB has a better response to a short 2. Marcus SF, Rizk B, Fountain S, Brinsden P. Tuberculosis
infertility and invitro fertilization. Am J Obstet Gynecol.
course of treatment than pulmonary TB as it carries a
1994;171(6):1593-6.
lower mycobacterial load. 3. Sharma JB, Roy KK, Pushparaj M, Kumar S, Malhotra N,
• To avoid emergence of resistant organisms, a multid- Mittal S. Laparoscopic findings in female genital tuberculosis.
rug regimen has to be adopted. Arch Gynecol Obstet. 2008;278(4):359-64.
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226 The Infertility Manual
4. Sharma JB. Tuberculosis and Obstetric and Gynecological 8. Parikh FR, Nadkarni SG, Kamat SA, Naik N,
Practice. In Studd J Tan SL, Chervenak FA (Eds). Progress
in Obstetrics and Gynecology, Churchill Living stone,
Edinburgh 2008;18:395-427.
Soonawala SB, Parikh RM. Genital tuberculosis—a major
pelvic factor causing infertility in Indian women. Fertil
Steril. 1997;67:497-500.
SE C T I O N
5
5. Nogales-Ortiz F, Tarancon I, Nogales FF Jr. The pathology of 9. Tripathy SN, Tripathy SN. Infertility and pregnancy
female genital tuberculosis. A 31 year study of 1436 cases. outcome in female genital tuberculosis. Int J Gynecol
Obstet Gynecol. 1979;53(4):422-8. Obstet. 2002;76(2):159-63.
6. De Vynck WE, Kruger TF, Joubert JJ, Scott F, van der 10. Bhanu NV, Singh UB, Chakraborty M, Suresh N, Arora J,
Merwe JP, Hulme VA, et al. Genital tuberculosis associated Rana T, et al. Improved diagnostic value of PCR in the
with female infertility in the Western Cape. S Afr Med J. diagnosis of female genital TB leading to infertility J Med
1990;77:630-1. Microbiol. 2005;54:927-31.
7. Oosthuizen AP, Wesssels PH, Hefer JN. Tuberculosis of 11. Jindal UN, Bala Y, Sodhi, Verma S. Female genital tuberculosis:
the female genital tract in patients attending an infertility early diagnosis by laparoscopy and endometrial polymerase
clinic. S Afr Med J. 1990;77(11):562-4. chain reaction. Int J Tuberc Lung Dis. 2010;14(12):1629.
Ovarian Stimulation
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C HA PTE R
Drugs Used in Ovarian Stimulation
27
Arveen Vohra
Chapter Outline
• Clomiphene Citrate • Chorionic Gonadotropin Preparations
• Aromatase Inhibitors • Long-acting FSH
• Other SERMs • Future Developments in Gonadotropin Preparations
• Metformin • Newer FSH Preparation
• Gonadotropins • GnRH Analogues
• Development of Gonadotropin Preparations for Follicular Stimu- • GnRH Agonist Preparations
lation • Adjuncts for Ovarian Stimulation
• Recombinant LH Preparation • Key Notes
INTRODUCTION CLOMIPHENE CITRATE
This chapter overviews the development, structure, and CC is the most commonly prescribed ovulation-induction
mode of action of the medication used for controlled ovar- drug used to stimulate ovulation in women with infre-
ian stimulation (COS) in assisted reproductive techniques. quent ovulation or amenorrhea. It is also used to induce
The drugs commonly used in ovarian stimulation are more than one follicle to develop as a treatment for unex-
described below. plained infertility and for those who are unable or unwill-
• Oral ovulogens—Clomiphene citrate, Aromatase ing to pursue more aggressive therapies.
inhibitors, Tamoxifen and Metformin Clomiphene is the best initial treatment for the large
• Gonadotropins majority of anovulatory infertile women.1
• GnRH Agonists and antagonists Combination therapies involving CC and other agents
• Adjuncts for ovarian stimulation (metformin, glucocorticoids, exogenous gonadotropins)
The discovery by Aschheim and Zondek of a substance may be effective when treatment with CC alone fails to
in the urine of pregnant women with the same action as induce ovulation.
the gonadotropic factor in the anterior pituitary in 1927
started the advent of the new era of assisted reproduc- Pharmacology
tive technologies (ART). The treatment has today been • Chemically, CC is a nonsteroidal triphenylethylene
enhanced owing to the development in the characteriza- derivative that exhibits both estrogen agonist and
tion and purification of hormones. From the use of clo- antagonist properties.
miphene citrate (CC) and gonadotropins in 1961, to the • Estrogen agonist properties manifest only when endog-
present numerous combinations of the drugs in use, and enous estrogen levels are extremely low otherwise, CC
their various types, there has been a change aiming at bet- acts solely as a competitive estrogen antagonist.
ter results. • Metabolized through the liver and excreted in stool.
Ovarian stimulation protocols have been evolving • About 85% of an administered dose is eliminated after
since the first successful in vitro fertilization (IVF) proce- approximately 6 days, although traces may remain in
dure was performed in 1978, and with it have evolved the the circulation for much longer.
drugs used. • As currently manufactured, CC is a racemic mixture of
This chapter overviews the development, structure, two distinct stereoisomers, enclomiphene and zuclo-
and mode of action of the medication used for controlled miphene (Fig. 27.1).
ovarian stimulation (COS) in ART. • Available evidence indicates that enclomiphene is the
The drugs commonly used in ovarian stimulation are more potent isomer and the one primarily responsible
described below. for the ovulation inducing actions of CC.
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230 The Infertility Manual Drugs Used in Ovarian Stimulation 231
Table 27.1. Differences between the isomers
of clomiphene.
Enclomiphene Zuclomiphene
62% 38%
More anti-estrogenic Estrogenic
Short t1/2 (2.5–11.8 days) Cleared in weeks (t1/2
14.2–33.4 days)
More potent in OI Less potent
Fig. 27.3: Feedback mechanism of clomiphene. GnRH, gonadotropin-releasing hormone; FSH, follicle-stimulating hormone; LH, luteinizing
hormone.
• Ovulation induction rate: 70–80% evaluation, empiric treatment with CC may be justified,
• Cycle fecundability: 15–22% particularly in young couples with a short duration of
• Cumulative pregnancy rates: 70–75% (over 6–9 cycles). infertility and in those unwilling or unable to pursue
• CC will induce ovulation in about 80% of properly more aggressive therapies involving greater costs,
selected patients. risks, and logistical demands.
Fig. 27.1: Structure of clomiphene and the two isomers. Fig. 27.2: Mechanism of action of clomiphene.
• Once the CC dose that induces ovulation is established, • Hypothalamic/pituitary dysfunction—In PCOS, CC is
three ovulatory CC cycles are an adequate trial for most the first line of treatment for ovulation induction.
patients and may be continued for up to six cycles. How- • IUI/IVF-CC is now being used as an important drug for
ever, studies show that CC should not be given for more mild stimulation IVF.
• Enclomiphene levels rise rapidly after administration rise; falling again after the typical 5-day course of ther- than six cycles because the chance of pregnancy is very • Endometriosis—CC is the choice of drug for ovulation
and fall to undetectable concentrations soon. apy is completed. low and alternative treatments should be considered. induction in young women with stage 1 or 2 endome-
• Zuclomiphene is cleared far more slowly. Levels of this • In successful treatment cycles, one or more dominant • CC is easy to use and results in ovulation in most triosis.
less active isomer remain detectable in the circulation follicles emerge and mature, generating a rising tide of patients (60–90%), but the pregnancy rates are dis-
for more than a month after treatment and may accu- estradiol that ultimately triggers the midcycle LH surge
mulate over consecutive cycles of treatment, but there and ovulation.
appointing (10–40%). This has been attributed to its Side Effects
peripheral antiestrogenic effects, mainly on the endo-
is no evidence of any important clinical consequence • Metabolism: ~85% of drug is metabolized through the CC is generally very well tolerated. Some side effects
metrium and the cervical mucus (Fig. 27.3).
(Table 27.1). liver. are relatively common, but rarely are they persistent or
• The multiple pregnancy rate associated with CC: 10–20%.
• Excretion: Through feces. severe enough to threaten completion of the usual 5-day
Mechanism of Action Indications course or next cycle of treatment. Although CC treatment
Dosage does have intrinsic risks, they are typically modest and
Structural similarity to estrogen allows CC to bind to estro-
• Given orally • Anovulation/oligoovulation—CC is the initial treat- manageable.
gen receptors (ERs) throughout the reproductive system
• Start Day 3–5 after spontaneous/medication-induced ment of choice for most anovulatory or oligo-ovulatory
(Fig. 27.2).
menses infertile women. However, given its hypothalamic site • Vasomotor flushes (hot flushes) occur in approxi-
• In ovulatory women, CC treatment increases gonado- • DOSE: 50–250 mg of action, CC is ineffective in women with hypogonad- mately 10% of CC-treated women, but typically abate
tropin-releasing hormone (GnRH) pulse frequency. • If ovulation does not occur at the 50 mg dose, CC is otropic hypogonadism (hypothalamic amenorrhea). soon after treatment ends.
• In anovulatory women with polycystic ovary syndrome increased by 50 mg increments in immediate or subse- • Luteal-phase deficiency—Progesterone levels are typi- • Mood swings are also common.
(PCOS) in whom the GnRH pulse frequency is already quent cycles until ovulation happens. cally higher after CC treatment than in spontaneous • Visual disturbances, including blurred or double
abnormally high, CC treatment increases pulse ampli- • More than 200 mg each day for 5 days is usually not cycles, reflecting improved preovulatory follicle and vision, scotoma, and light sensitivity, are generally
tude, but not frequency. helpful, and patients who do not ovulate on a clo- corpus luteum development. uncommon (2% prevalence) and reversible, although
• During CC treatment, levels of both luteinizing hor- miphene dosage of 200 mg tend to respond better to • Unexplained infertility—In couples whose infertil- there are isolated reports of persistent symptoms and
mone (LH) and follicle-stimulating hormone (FSH) other drugs such as gonadotropins. ity remains unexplained after careful and thorough more severe complications such as optic neuropathy.
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232 The Infertility Manual Drugs Used in Ovarian Stimulation 233
Whenever visual disturbances are identified, it is pru- • Functional ovarian cysts
dent to stop treatment and consider alternative meth- • Visual disorders
ods of ovulation induction.
• Less specific side effects include breast tenderness, pelvic Predictors of Clomiphene Citrate Failure
discomfort, and nausea in 2–5% of CC-treated women.
• Age
• In addition to the desirable central actions respon-
• Obesity
sible for its efficacy as anovulation-inducing agent,
• Hyperandrogenemia—FAI (free androgen index = tes-
CC exerts undesirable adverse antiestrogenic effects
tosterone/SHBG).
in the periphery (endocervix, endometrium, ovary,
• Cycle history, amenorrhea
ovum, and embryo) that explain the “discrepancy”
• Higher fasting insulin, leptin
between the ovulation and conception rates observed
• Lower IGFBP-1
in CC-treated patients. The quality and quantity of cer-
vical mucus production in CC treatment cycles may
sometimes be reduced, but rarely to an extent that
AROMATASE INHIBITORS
risks interference with the effective captures, survival, Aromatase, a cytochrome P450-dependent enzyme, acts
or transport of sperm. Limited endometrial prolifera- as the ultimate step in the synthesis of estrogen, catalyzing
tion has been observed in some CC-treated patients, the conversion of androgens to estrogens. The conversion
but the effect is minor or not at all evident in the large of androgens to estrogens occurs also at peripheral sites,
majority of women. Taken together, available evidence such as in muscle, fat, and the liver.
and accumulated clinical experience suggest that A group of new, highly selective aromatase inhibitors
any adverse antiestrogenic effects of CC present no has been approved to suppress estrogen production in
significant obstacle in the majority of treated women. postmenopausal women with breast cancer.
Third-generation aromatase inhibitors are commonly
Risks and Complications used. These include:
• Multiple gestation • Steroidal derivative: Exemestane
Multifollicular development is relatively common dur- • Nonsteroidal triazole derivatives: Fig. 27.4: Mechanism of action of aromatase inhibitors. FSH, follicle-stimulating hormone.
ing CC treatment, and the risk of multiple gestation is Anastrazole
increased to approximately 8% (10–20%) overall. The over- Letrozole
whelming majority of multiple pregnancies that result Both these drugs are approved in USA for the treat-
from CC treatment are twin gestations; triplet and higher- ment of breast cancer. They offer increased potency, speci- Dosage • In the recent times, another third-generation non-
steroidal aromatase inhibitor, named anastrazole is
order pregnancies are rare but may occur. ficity and better tolerability. • 2.5–5 mg/day for 5 days starting from day 2 or 3 of
being used in a dose of 1 mg twice daily for 5 days.
Letrozole, an orally active aromatase inhibitor, may menses. Letrozole is completely and rapidly absorbed
• Ovarian hyperstimulation syndrome (OHSS) However, RCTs are not available with regard to the
have potential as an ovulation-inducing agent. In con- from gastrointestinal tract (GIT), metabolized in the
Whereas mild OHSS (moderate ovarian enlargement) optimal dosage and results in comparison with other
trast to the central actions of CC and tamoxifen, letrozole liver and excreted through the kidneys.
is relatively common, severe OHSS (massive ovarian ovulation induction agents.
acts in the periphery to inhibit ovarian follicular estradiol • Elimination t1/2 = 2 days/45 hours.
enlargement, progressive weight gain, severe abdominal production, but the end result is similar—a decrease in • Ovulation induction with letrozole is associated with
pain, nausea and vomiting, hypovolemia, ascites, and central estrogen feedback action that stimulates a com- an ovulation rate of 70–84% and a pregnancy rate of Indications
oliguria) rarely occurs. pensatory increase in pituitary gonadotropin secretion. 20–27% per cycle.
• Ovarian cancer Higher rate of success is noted with letrozole than with • The use of letrozole for superovulation is associated Aromatase inhibitors are as effective, but not more effec-
anastrozole. with a pregnancy rate higher than with the use of CC tive than CC as a first-line agent for OI.
Patients with concerns should be counseled that no causal
relationship between ovulation inducing drugs and ovarian (16.7 vs. 5.6%).
cancer has yet been established and no change in prescrib- Mechanism of Action • The addition of letrozole to FSH treatment leads to a
ing practices is warranted.2 In any case, prolonged treatment • Release of H-P axis from E negative feedback → ↑FSH. decreased FSH requirement. The pregnancy rate for
Uses
with CC is generally futile and should therefore be avoided. • Transient (45 hours) ↑ in intraovarian androgenic treatment with letrozole and FSH has been similar to • Clomiphene failure.
environment augments follicular sensitivity to FSH. that for FSH alone in studies. • Poor endometrium with CC.
Contraindications • No inhibition of E receptors in brain → intact feed- • Aromatase inhibitors are as effective as or superior • Letrozole in dose of 5 mg from day 2 to day 6 of the
• Pregnancy back central mechanisms → limits FSH response → to CC in ovulation induction. Unlike CC, they do not menstrual cycle has excellent pregnancy rates com-
• Chronic liver disease monoovulatory cycles (Fig. 27.4). carry an antiestrogenic effect on the endometrium. pared to clomiphene in patients with PCOS.3
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234 The Infertility Manual Drugs Used in Ovarian Stimulation 235
• Letrozole has also been used for ovarian stimulation in Raloxifene
women with breast cancer, as it does not cause high-
estrogen levels yet gives good number of oocytes, which Existing studies suggest that this SERM has a favorable
can be retrieved and preserved for future fertility. antiestrogenic profile for endometrium and functions
similar to CC to increase serum FSH levels.
Advantages of Letrozole
• No adverse antiestrogenic effect on the endometrium
METFORMIN4
or cervical mucus • Metformin is a second-generation biguanide that was
• Absence of ER depletion introduced in 1995.
• Rapid elimination from the body (half-life of 45 hours) • The drug activates glucose transporters to facilitate
• Better uterine blood flow passage of glucose into hepatic and muscle cells,
• Limited number of mature follicles thereby decreasing peripheral insulin resistance and
• Decrease OHSS and multiple pregnancy. lowering serum glucose levels.
• Does not stimulate insulin release and, when adminis-
Fig. 27.5: Mechanism of action of metformin.
Side Effects tered alone, does not cause hypoglycemia.
• Clinical studies have shown that metformin treatment
Hot flashes, nausea, fatigue, alopecia, vaginal bleeding
(500 mg three times per day or 850 mg twice daily with
(long use in Ca breast).
meals) increases the frequency of spontaneous ovula-
Mechanism of Action in Preventing OHSS The first use of gonadotropins extracted from human
Although letrozole causes fetal abnormalities in pituitaries to induce ovulation in women in 1959 repre-
tion, menstrual cyclicity, and ovulatory response to CC A very highly significant decrease of OHSS has been sented a major advance in reproductive medicine. Since
animals, such an effect has not been demonstrated in
in women with PCOS. seen with metformin in women with PCOS undergoing then, gonadotropins of urinary origin (hMG) or more
humans when the drug is stopped 10–12 days before
• Observed to lower fasting insulin levels and to reduce IVF. The elevated levels of insulin in PCOS, aside from recently recombinant FSH and LH have been used suc-
embryo transfer. It became a controversial drug after
but not eliminate insulin resistance. the increase expected with the commonly associated cessfully to restore reproductive function in hypogonado-
the studies suggested letrozole to have a higher risk of
• Beneficial in improving ovulation and pregnancy obesity in this syndrome, are reduced by metformin, tropic men and women. Over the past 40 years, injectable
fetal cardiovascular system (CVS) and musculo-skeletal
rates. However, metformin does not improve live birth and that is one of the principal factors that stimulates gonadotropins have established a leading role in the treat-
malformations. On account of studies showing abnor-
whether it is used alone or in combination with clomi- the production of vascular endothelial growth factor by ment of both male and female infertility.
malities in the newborn in the cardiovascular and skel-
phene.5 luteinized granulosa cells (GCs). Also, because andro-
etal system, letrozole was banned in India. However,
• Appears to have benefits in women with PCOS through- gens stimulate GCs FSH receptors, metformin may Milestones of development in infertility treatment
recently the ban has been lifted and letrozole can be
out ovulation induction treatments and particularly reduce OHSS by decreasing the ovarian response to COS
used now for ovulation induction. 1927 The discovery of pituitary hormone controlling
during IVF cycles by reducing OHSS. The OHSS is the (Fig. 27.5). ovarian function
most serious complication of IVF in women with PCOS 1959 Purification and clinical use of pituitary and urine
OTHER SERMs and may lead to catastrophic complications and even Side Effects gonadotropins
The SERMs are structurally diverse nonsteroidal com- death. In IVF cycles, metformin exerts no clinical effect 1960 Clinical use of clomiphene citrate
pounds that bind to ERs and have tissue-dependent ago- on pregnancy rates or live birth rates, but it reduces the • Gastrointestinal symptoms (e.g. nausea, vomiting, and
diarrhea) that are dose dependent and generally tend 1966 Use of clomiphene citrate and gonadotropins
nistic and antagonistic effects. risk of OHSS.6
becomes common practice
to resolve after several weeks of treatment; a gradual
increase in dose also helps to improve tolerance. 1970 Development of radioimmunoassay for measuring
Tamoxifen Mechanism of Action in COS hormone levels
• Lactic acidosis has rarely been reported.
A recent prospective randomized controlled trial com- • Addition of metformin to FSH treatment for CC- • Liver and kidney disease should be excluded before 1978 Ultrasound imaging of ovarian follicles
pared the efficacy of tamoxifen with that of CC for ovula- resistant patients with PCOS has been reported to use and metformin should be stopped during acute ill- 1984 Use of gonadotropin-releasing hormone (GnRH)
tion induction in anovulatory women. The overall rates reduce the number of preovulatory follicles and the nesses and when radiologic dye is to be used. agonists in infertility treatment
of ovulation and pregnancy were similar in both groups.3 peak estradiol levels. 1985 Further purification of urinary gonadotropins
Other studies have suggested that tamoxifen may be supe- • Ovulation induced with metformin has been associ- 1990 Use of recombinant gonadotropins
rior to CC in that it does not appear to have an adverse ated with decreased testosterone levels and marked
GONADOTROPINS7,8
effect on the endometrium. increases of glycodelin levels during the luteal phase The aim of recruiting and developing multiple follicles is
Tamoxifen has been shown to be successful in the in women with PCOS. readily accomplished with the use of either hMG alone,
Structure
treatment of anovulation, even in patients in whom • Metformin increases uterine blood flow. with FSH alone or by using FSH in combination with hMG. The glycoprotein hormones FSH, LH, human chorionic
CC treatment has failed, but it has yet to be tested for • Use of metformin in PCOS improves altered blood Gonadotropins can be used alone or combined with clo- gonadotropin (hCG), and thyroid-stimulating hormone
superovulation. lipids and may reduce later cardiovascular disease. miphene and with GnRH agonists or antagonists. (TSH) are composed of two noncovalently linked protein
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236 The Infertility Manual Drugs Used in Ovarian Stimulation 237
Table 27.2. Isoforms of FSH.
Follicle-stimulating hormone (FSH) isoforms.
Type of FSH isoform E2 level Sialic acid content Receptor affinity Half-life in vivo
Acidic Low High Low Long
Basic High Low High Short
LH and hCG (Figs. 27.7 and 27.8)
• Although the α subunits of LH and hCG are identical to
that of FSH, the β subunits are different.
• LH has a β subunit containing 121 amino acids that
confers its specific biologic action and is responsible
for its interaction with the LH receptor. This β subunit
Fig. 27.6: Structure of follicle-stimulating hormone (FSH). Fig. 27.8: Structure of human chorionic gonadotropin (hCG). of LH contains the same amino acids in sequence as
FSH is a glycoprotein composed of two subunits, the α subunit (red) hCG has structural similarities to LH except the presence of a long
the β subunit of hCG, but the hCG β subunit contains
and the β subunit (blue). There are four carbohydrate attachment sites, carboxy-terminal segment that is O-glycosylated (O-linked CHO), con-
two in each subunit. ferring a longer half-life to hCG. an additional 23 amino acids.
• The two hormones differ in the composition of their
carbohydrate moieties, which, in turn, affects bioactiv-
The subunits alone have no known biologic activity. ity and half-life.
It is the formation of the heterodimer that provides the • The half-life of LH is 20 minutes and that for hCG is
hormonal activity through attachment of the carbohy- 24 hours.
drate moieties, and the extent of glycosylation, especially
sialylation, that conveys the spectrum of differences in DEVELOPMENT OF GONADOTROPIN
charge, bioactivities, and elimination half-lives. PREPARATIONS FOR FOLLICULAR
STIMULATION
FSH
Research underlying production of the current gonadotro- Fig. 27.9: Gonadotropin products available in the markets.
• FSH plays an important role in gametogenesis in both pin preparations has spanned at least five decades. Gonad-
men and women. otropin manufacture has evolved from extraction of urine
• In men it is required for the establishment and prob- to application of recombinant techniques to yield a variety • Improvements in purification techniques resulted in
ably maintenance of spermatogenesis. of FSH preparations for ovarian stimulation. The result is standardization of the FSH and LH activity to 75 IU for
• In women it stimulates the growth of ovarian follicles a broad choice of effective drugs with similar clinical effi- each type of gonadotropin, although extraneous uri-
and fluctuations in its secretion are involved in the cacy. Because both urinary and recombinant purification nary proteins still existed and are present even in the
Fig. 27.7: Structure of luteinizing hormone (LH).
LH is a glycoprotein with two subunits, the α subunit (red), which is selection of the ovulatory follicle. processes involve exposure to impurities, preparations hMG products available today (Table 27.3).
similar to FSH and hCG, with two carbohydrate attachment sites, and • The β subunit of FSH is composed of 111 amino acids. devoid of extraneous biological materials cannot be guar- • The LH activity in hMG derives primarily from the hCG
the β subunit (blue) with only one carbohydrate attachment site. • Each subunit is attached to two carbohydrate moieties anteed. However, all products appear to be safe (Fig. 27.9). component, which preferentially is concentrated during
with variable compositions that, in turn, create differ- the purification process and sometimes was added to
ent isoforms.
subunits, the α and β subunits, to which carbohydrate
Human Menopausal Gonadotropin achieve the desired amount of LH-like biological activity.
• These multiple forms of FSH differ in their plasma half-
moieties are attached. The α and β subunits are repre- lives (range: 3–4 hours) due to variations in their bind- • hMG, or menotropin, is derived from postmenopausal
sented by red and blue strands, respectively, whereas the ing potentials. urine.
Table 27.3. Differences between activity and purity
carbohydrate chains are represented by light blue balls in • The distribution of isoform types is under endocrine • The urine originally was obtained from a single nun- of hMG and FSH.
(Figs. 27.6 to 27.8). control, and the amount of sialic acid present is influ- nery in Italy but later collection was expanded to
Gonadotropin Purity (%) LH activity (%)
The α subunit composed of 92 amino acids is identical, enced mainly by estradiol (E2) levels—the higher the numerous centers in a wide variety of other countries.
hMG <4 50
whereas the β subunits are distinct and confer the unique E2 levels, the less glycosylated the FSH, the shorter • The early preparations were originally only about 5%
pFSH 5 <1
biological and immunological properties and the receptor the half-life, but the greater the receptor affinity pure and contained varying amounts of FSH, LH, and
specificity of each of these glycoproteins. (Table 27.2). hCG. HP-FSH 96 <0.1
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238 The Infertility Manual Drugs Used in Ovarian Stimulation 239
• Clinical use of hMG began in 1950, but clinical trials the quality, standardization and availability of gonado- Advantages of Recombinant FSH therapeutic actions, thereby reducing the number of
were not started until the early 1960s. tropins. injections required to achieve optimal follicular growth.
• Urinary FSH has a longer half-life (~24 hours) than that
Over-purified FSH To this end, novel FSH preparations with modifications
Urinary FSH obtained by the recombinant technique so significant • No prion scare in FSH glycosylation or replacements of the carboxy-
concentrations of FSH persist in the blood for several • Traceability terminal peptide (CTP) of FSH are undergoing clinical
• Removal of the LH with polyclonal antibodies resulted days after a single intramuscular injection. • High purity, specific activity trials. A single dose of long-acting FSH, named corifolli-
in a biologically pure urinary FSH preparation (uro- • Now possible with recombinant technology to design • Unlimited supply with batch to batch consistency tropin alfa or FSH-CTP is able to keep the circulating FSH
follitropin), but one that still contained urinary pro- FSH preparations with shorter or longer half-lives. • Absolute monohormonal products level above the threshold necessary to support multifolli-
teins. • Circumvention of immune reactions cular growth for an entire week. The optimal dose of long-
• Use of monoclonal antibodies specific to FSH resulted
in further refinements in manufacture, and the pro-
Recombinant FSH Preparations • S/C self-administration acting FSH is still being determined. A single injection of
• Ready to use pen devices long-acting FSH can replace seven daily FSH injections
duction of highly purified (HP) urinary FSH. Such The two recombinant FSH preparations currently avail- • Superior quality control. during the first week of COS and can make assisted repro-
preparations contain less than 0.1 IU LH and less than able are marketed as follitropin α and follitropin β. Both duction more patients friendly. A Cochrane database
5% of unidentified urinary proteins. are structurally identical to native FSH and, despite being RECOMBINANT LH PREPARATION 2012 review states that the use of a medium dose of long-
• Specific activity of the FSH is increased from 100 to named follitropin α and follitropin β, each comprises one acting FSH is a safe treatment option and equally effec-
150 IU/mg protein in purified urinary FSH prepara- α and one β glycoprotein chain. These dissimilar glyco- Recombinant LH has been available for clinical use
since1993 in vials with syringes that together are designed tive compared to daily FSH, but that further research is
tions to approximately 10,000 IU/mg protein in the HP protein chains are noncovalently linked, being conjoined needed to determine if long-acting FSH is safe and effec-
product. by electrostatic and hydrophobic forces, attached to two to deliver 75 IU. The product may enhance follicular devel-
opment when used in conjunction with FSH in patients tive for use in hyper- or poor responders and in women
• The enhanced purity and increasing specific activity complex carbohydrate structures. The post glycosylation with all causes of subfertility.10
of HP-FSH enable subcutaneous delivery in very small process and purification procedures of the two recombi- suffering from hypogonadotropic hypogonadism who
volumes, in addition to virtual elimination of batch-to- nant FSH preparations are not identical, resulting in dif- have profound LH deficiency.
batch variation. ferent sialic acid residue compositions and thus different Indications for Usage
isoelectric coefficients. The subtle differences in structure CHORIONIC GONADOTROPIN • Hypogonadotropic hypogonadism
have not resulted in any proven differences in clinical PREPARATIONS • Clomiphene-resistant anovulation
Recombinant Gonadotropins performance.
Chorionic gonadotropin is used to promote the final stages of • Unexplained infertility
• The source of the natural GnRH being the urine of post- In contrast to the FSH content of urinary-derived FSH, • Intentional superovulation in older women.
follicular maturation and progression of the immature oocyte
menopausal women, the rapid increase in demand led that of recombinant FSH preparations can be quantified
at prophase I (the germinal vesicle stage) through meiotic
to worldwide shortages of the product. by protein content (mass in mg) rather than by biological FUTURE DEVELOPMENTS IN
maturation to reach metaphase II. Approximately 36 hours
• The advent of recombinant DNA technology opened activity (Table 27.4).
the door for production of recombinant FSH prepara- Delivery systems for recombinant FSH products
is required for completion of the meiotic process, and, in the GONADOTROPIN PREPARATIONS
absence of follicular aspiration at oocyte retrieval, ovulation
tions. include pen-shaped administration devices that either are Additional future developments may employ screening of
will ensue approximately 4 hours later. Chorionic gonado-
• Recombinant preparations are manufactured by prefilled or can be adjusted to deliver variable amounts large chemical libraries to identify orally active small mol-
tropin can be human derived from urine of pregnant women
inserting the genes encoding for the α and β subunits of gonadotropin contained in vials. All preparations are ecule agonists of human FSH or LH receptors that might
(hCG) or manufactured using recombinant technology.
of FSH into expression vectors that are transfected into packaged as lyophilized powder with the exception of the obviate the need to inject gonadotropins (Fig. 27.10).
Preparations of hCG are marketed in vials of 5,000
Chinese hamster ovary cell lines. liquid formulations found in cartridges or pens. Neither
or10,000 IU. Recombinant chorionic gonadotropin
• The introduction in 1996 of the first recombinant the packaging nor the method of delivery has translated
human FSH, followed a few years later by recombinant into superior pregnancy rates for either urinary or recom-
syringes contain 250 mg of product, which is equivalent to NEWER FSH PREPARATION
5,000–6,000 IU of hCG.
hCG and then LH, brought about a radical change in binant FSH preparations. • Highly purified urofollitrophin (FSH) tablets (VHB Life
According to a 2011 Cochrane review, there is no evi-
Sciences Limited) Ovufol HP. Tablets are available in
dence of difference between recombinant CG or recom-
75 I.U.X 1`s and 150 I.U.X 1`s package options.
binant LH and urinary hCG in achieving final follicular
Table 27.4. Differences between FSH activity and purity of hMG and FSH. • Long-acting FSH formulation, hyperglycosylated FSH
maturation in IVF, with equivalent pregnancy rates and
Mean Specific FSH activity Injected Protein (AS900672), displays an extended half-life.
Purity (FSH Content) (%) (IU/mg Protein) per 75 IU (μg)
OHSS incidence. According to this review, urinary hCG is
still the best choice for final oocyte maturation triggering
hMG <5 ~100 ~750 Contraindications
in IVF and ICSI treatment cycles.9
hMG-HP <70 2000–2500 ~33
• Hypersensitivity
r-hFSH LONG-ACTING FSH • Tumors of hypothalamus and pituitary gland
Follitropin beta – 7000–10,000 8.1
Molecular engineering provides the technology to • Ovarian cyst
Follitropin alpha >99 13,645 6.1 modify FSH preparations to prolong their half-lives and • Abnormal uterine bleeding
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240 The Infertility Manual Drugs Used in Ovarian Stimulation 241
feedback system involving the pituitary, the ovary, the More than 25 years ago, leuprolide acetate, a GnRH
environment and the rest of the nervous system. agonist, was found to effectively block the premature LH
• It is the key hormone for regulation of the hypotha- surge and hence premature ovulation, which otherwise
lamic pituitary ovarian (HPO) axis with receptors in resulted in the cancellation of about 20% of IVF cycles.
both the pituitary and extra pituitary tissues.
• The receptors in the pituitary, when occupied by the Dosage
GnRH, activate the MAPK (Mitogen activated protein
kinase) cascade. • Leuprolide acetate of 500 µg daily dose is started on
• The main G proteins, mediating pituitary actions are D21 of the previous cycle after ensuring absence of
Gq/G11.Gs and Gi have also been implicated. ovarian cysts. This dosage is continued till the onset of
• The MAP kinase system in turn activates the FSH beta menses, when the dose is reduced to 200 µg daily and
and LH β gene transcriptions. However, difference in continued along with the gonadotropins till the crite-
the GnRH pulse frequencies stimulate preferentially ria for ovulation trigger are reached. The drug is with-
the FSH B /LH B gene transcriptions. held on the day of hCG. This is called “Long protocol”
• The GnRH agonist (GnRHa) molecule by virtue of its Fig. 27.11: Mechanism of action of GnRH agonist and antagonist. and is still the most popular mode of administration
Fig. 27.10: New FSH tablet structure.
structural similarity to the GnRH occupies the GnRH as is reported by the IVF worldwide survey on GnRH
receptors in the pituitary gland.11,12 agonist usage in COS.13
stimulation can be controlled for the timing of the • The depot preparation of 3.75 mg is given intramuscu-
• Ovarian, uterine, and breast cancer larly as a single dosage in the luteal phase of the previ-
Structure and Action of GnRH Agonist oocyte retrieval in the long and short protocols of COS
ous cycle. The downregulation lasts for at least 4 weeks.
• Premature ovarian failure in assisted reproductive techniques (ART).
• Primary testicular failure. The GnRH agonist molecule is derived from the native • In addition to ovulation induction, synchrony of folli- The gonadotropins are begun on the second day of the
GnRH by amino acid substitution in the sixth position. cular growth may be an advantage. menstrual cycle that follows. The convenience of a sin-
Side Effects • Its administration leads to a marked increase of • The initial flare effect is used as a trigger in the antago- gle dose as well as the pretreatment need as in endo-
nist protocol or in the modified natural cycle to initiate metriosis make it the route of choice in about 10% of
• The side effects of the gonadotropins are illustrated follicle-stimulating hormone (FSH) and the luteinizing
the natural LH surge at follicular maturity. the cycles.
below (Table 27.5). hormone (LH).
• Enables endogenous, “physiological” release of FSH • Nasal sprays of GnRH agonists are administered in six
• Continuous administration of the agonist leads to a
and as a consequence, subsequent events closely sim- divided doses between 6 am and 11 pm. This method
suppression of the HPO axis due to the receptor down-
GnRH ANALOGUES regulation and desensitization, causing a decrease in ulate those of a normal ovulatory cycle (Fig. 27.11). ensures a uniform dosage through the day, mimicking
the pulsatile release of GnRH. The doses are reduced to
• Gonadotropin-releasing hormone (GnRH) is structur- the circulating levels of gonadotropins and sex steroids.
two times a day once the gonadotropin injections are
ally a peptide chain secreted by the neuroendocrine • The suppression of endogenous LH is used to pre- GnRH AGONIST PREPARATIONS initiated.
cells of the hypothalamus in response to a complex vent the natural midcycle LH surge so that the ovarian
The GnRH Agonist preparations are different due to the • In case of a missed period, after starting the drug, a
number of peptides (Table 27.6). pregnancy should be ruled out and the gonadotropin
Table 27.5. Various side effects of gonadotropins.
Table 27.6. Different GnRH agonist preparations.
System involved Frequency Manifestation
Nona Peptides
Mild erythema, rash
Immune <1/10,000 Generic name Trade name Half life Relative potency Route of admn Dose
Severe anaphylaxis
50–80 s.c, 500–1000 μg/d
Nervous >1/10 Head ache Leuprolide Lupron 90 minute
20–30 i.m. depot 3.75–7.5 mg/month
Respiratory <1/10,000 Exacerbation of asthma s.c. 200–500 μg/d
Buserelin Suprefact 80 minute 20–40
Vascular <1/10,000 Thromboembolism i.n. 300–400 μg × 3–4/d
GI >1/100 Nausea, vomiting, diarrhea, abdominal cramps, bloating Histrelin Supprelin <60 minute 100 s.c. 100 μg/d
>1/10 Ovarian cysts Goserelin Zoladex 4.5 hours 50–100 s.c. implant 3.6 mg/month
>1/100 Mild to moderate ovarian hyperstimulation syndrome (OHSS) Deca Peptides
Reproductive
<1/100 Severe OHSS Generic name Trade name Half life Relative potency Route of admn Dose
<1/1,000 Ovarian torsion Nafarelin Synarel 3–4 hours 200 i.n. 200–400 μg – 2/d
Administration site >1/10 Pain, redness, bruising, swelling, irritation Triptorelin Decapeptyl 3–4.2 hours 36–144 s.c. i.m. depot 100–500 μg/d 3.75 mg/month
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242 The Infertility Manual Drugs Used in Ovarian Stimulation 243
started after ensuring that the E2 levels are below These are also used to avoid the spontaneous LH surge, in Table 27.7. Various GnRH antagonists. friendly to the patient with lesser dose and duration of
50 pg/mL and the endometrium is less than 4 mm.13 contrast to the long-acting agonists, which first stimulate stimulation (and hence the lesser cost).
Generation Name of antagonist
• In case of pregnancy, there is no indication for termi- and later inhibit pituitary gonadotropin secretion by desen-
First generation 4F Ant
nation. Several reports of children born after such inci- sitizing gonadotropes to GnRH via receptor downregula- Dosage
Second generation Nal Arg
dents have shown no abnormalities in them. tion, the antagonists block the GnRH receptor in a dose-
Detrirelix • Cetrorelix can be used in two different dosages—
• The various protocols are discussed in detail in the dependent competitive fashion and have no similar flare
0.25 mg daily dose injections or 3 mg depot single dose
chapter on stimulation protocols. effect. Gonadotropin suppression is almost immediate. Third generation Cetrorelix
injection (effect lasting up to 96 hours).
At the beginning of the stimulation cycle endogenous Ganirelix
• Ganirelix can be used as 0.25 mg daily dose injections
gonadotropins initiate the ovarian stimulation with a nor- Org30850
Indications in ART only. These dosages represent the minimal effective doses
mal early follicular phase recruitment of a cohort of fol- Ramorelix found in dose-finding studies to prevent LH surges.16,17
• To prevent premature LH surge licles, without any pituitary block. Thus, the endogenous Nal Glu • Premature LH surges have been observed when using
• To trigger final oocyte maturation intercycle FSH rise is utilized rather than suppressed,
Antide lesser doses.
• Luteal phase support resulting in a reduction in the amount of medication
Azaline B • No adjustment in the dose for obese women is required
• To prevent OHSS needed and in the total length of the treatment.
Antarelix for cetrorelix.
• Endometriosis.
A-75998
• Tailoring of the dose to body weight may actually be
Structure required for ganirelix.18
Abarelix
Side Effects • Aminoacids at positions 1, 2, 3, 6, and 10 play impor- • Plasma concentrations of LH fall by 70% (range
• The side effects of hot flushes, vaginal dryness, and tant roles in the structure and function of native GnRH 52–91%) and plasma FSH concentrations by 30%
transient frontal headaches are reported in the first molecule (decapeptide). receptors remain blocked. So a higher dose of antag- (range 23–61%) within 6 hours (range 4–24 hours) of
2 weeks of treatment. • Amino acid at position 6 is involved in enzymatic onists compared to agonists is required for effective administration of GnRH antagonist. The amount and
• Arthralgia, myalgia, vaginal dryness, insomnia, head- cleavage. pituitary suppression (Fig. 27.12). duration of this suppression are dose-dependent.
aches, loss of libido, and emotional lability are less • Positions 2 and 3 are important in gonadotropin • The conventional antagonist protocol starts with ovar- • The half-life of cetrorelix 3 mg single dose and 0.25 mg
reported effects. release and positions 1, 6, and 10 are important for the ian stimulation from day 2 or 3 of a spontaneous men- daily dose has been reported to be 62.8 and 20.6 hours,
• The hypoestrogenic state causes a significant bone loss three-dimensional structure of the molecule. strual cycle and adding antagonist when there is a respectively.
after 6 months of therapy. Add back therapy with estro- • In GnRH agonists there are important changes at posi- threat of LH surge. This permits optimal use of endog- • The half-life of ganirelix 0.25 mg daily dose has been
gens and progestins has been suggested to counteract tions 6 and 10 only.14 enous gonadotropins in the initial part of stimulation found to be 16.2 hours. Complete reversal occurs in
the side effects. • However, there are more complex changes in GnRH without undue suppression of pituitary and at the same 24–72 hours.
• Excessive suppression may be seen in women with antagonists. His at position 2 and Trp at position 3 are time preventing premature LH surge in the mid- or • The incidence of LH surge with antagonists var-
poor ovarian reserve as the endogenous basal LH replaced and D-amino acids are substituted at position late-follicular phase. Hence, the stimulation remains ies between 1 and 35% in various studies.19-22 Hence,
drops to levels when the ovarian steroidogenesis. 6 by neutral D-ureidoalkyl amino acids.15 With these close to the normal cycle and more convenient and patient’s compliance is important, as there is a risk of
Large doses of gonadotropins may be needed to initi- modifications, three-generation antagonists were premature LH surge even if a single injection is missed
ate the folliculogenesis in these situations. developed, of which two are commercially available (Table 27.8).
• The effect on the mature oocyte and the embryo has since 1999: Cetrorelix (Cetrotide, Serono International
been studied and no adverse effects have been noted. SA, Geneva, Switzerland) and Ganirelix (Orgalutran, Advantages of GnRH Antagonist Regimen
• The luteal phase of agonist protocol is deficient and Organon, Oss, the Netherlands) (Table 27.7). • Short, simple, safe, well tolerated, and convenient for
has been intensively investigated. Luteal support in the patient
the form of progesterone or hCG is mandatory. The LH Mechanism of Action • Improved safety: No initial flare-up, no estrogen dep-
secretion in the luteal phase is affected as the GnRH rivation symptoms, less frequent local reactions, less
receptors in the pituitary have to regenerate in order to • GnRH antagonists act as competitive blockers for the
risk for OHSS
reset the HPO axis. native GnRH receptors, and hence block the ability of
• Reduced GT use and duration of treatment
• The data on follow-up of the pregnancies after agonist native GnRH to initiate dimer formation and signal
• Immediately reversible
treatment have shown no increase in untoward outcome transduction (release of FSH and LH from pituitary).
• Advantage of using GnRH agonist for final ovulation
as well as no increase in congenital malformations. • The competitive blockade of GnRH receptors leads to
trigger.
an immediate but reversible arrest of gonadotropin
secretion. Hence, the action of GnRH antagonists is
GnRH Antagonists highly dose-dependent in contrast to the agonists.
Side Effects
Antagonist analogs of GnRH have a direct inhibitory, • Since there is no receptor loss, a constant supply Fig. 27.12: Differences in mechanism of action of GnRH agonist and • The introduction of GnRH antagonists in clinical
antagonist. use was delayed owing to the property of the first
reversible suppressive effect on gonadotropin secretion. of antagonists is required to ensure that all GnRH
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244 The Infertility Manual Drugs Used in Ovarian Stimulation 245
Table 27.8. Differences between GnRH agonists and Oral Contraceptives and Estrogen Mechanism of Action phenomenon. It is not known how long ovarian blood
antagonists. flow must be increased to potentially influence ovar-
• Oral contraceptives pretreatment reduces the amount • Although the major increase of androgens during ian response. The most important time for maximal
Agonists Antagonists
of gonadotropin required for COS and therefore ovarian stimulation results from FSH stimulation, blood flow may be between hCG and egg retrieval,
Initial flare up Immediate gonadotropin appears to improve synchronization of the follicular suppression of adrenal production of androgens by
suppression during which meiosis resumes.34
cohort as seen in the GnRH agonist alone cycles, sug- dexamethasone may contribute to maximizing uter- • Ancillary benefits: Ovarian stimulation is accom-
Pituitary desensitization Competitive blockade of
gesting it as a useful adjunct for scheduling cycles and ine receptivity by lowering total androgen levels and panied by increases of clotting factors. The platelet-
GnRH receptors
improving IVF outcome, in both GnRH agonist and resulting in improved IVF results. inhibiting effect of aspirin may reduce the chance of a
Slow reversal Rapid reversal
GnRH antagonist cycles.24 • Risks: Minimal. thrombotic event with COS and OHSS.
Longer duration of stimula- Lesser duration of stimulation • The number of premature LH rises remains low. • Glucocorticoids should not be used with peptic ulcer
tion • Risks: The anticoagulant effect could increase the
• Luteal estrogen was suggested for scheduling of COS, disease, infection, diabetes, or latent tuberculosis. chance of bleeding with egg retrieval.
More ampoules of gonado- Lesser amount of gonadotro- and that adjunct has subsequently been reported to
tropins required pins required
synchronize the follicles and improve the response to Human Chorionic Gonadotropin
More risk of OHSS Significantly lesser risk OHSS
COS especially in poor responders.25,26 Dopamine Agonists
Only hCG or rLH as trigger GnRH agonist, hCG or rLH • Use of 10–30 IU daily of hCG may be logically used as
an alternative to substituting 75–225 IU of hMG for the The pathophysiology of OHSS and the benefit of caber-
as trigger
More estrogen deprivation Lesser estrogen deprivation Mechanism of Action same dose of pure FSH when addition of LH activity is goline have been extensively worked out, and it has been
desired.23 reported that it successfully reduced vascular perme-
symtoms symptoms • The FSH and follicular growth are suppressed by either ability, hemoconcentration, and ascites. With careful
Gold standard in normo- Particularly advantageous in oral contraceptives or estrogen.
responders hyper and poor responders
• Ancillary benefits are that cyst formation resulting
Mechanism of Action informed consent, the benefits warrant use in certain
high-risk situations.35
from GnRH agonists is also reduced by oral contracep- • Human chorionic gonadotropin has been routinely
tives pretreatment.23 used and is approved as an LH surrogate to induce
generation of antagonists to induce systemic hista- ovulation. Mechanism of Action
mine release and a subsequent general edematogenic
Growth Hormone • With the increasing use of recombinant FSH for ovar- • Cabergoline and other dopamine agonists decrease
state. ian stimulation, it was proposed that small doses of expression of the receptor for vascular endothelial
• The potential of third-generation antagonists to release • Growth hormone has been reported to increase suc- hCG would be effective adjuncts for ovarian stimula- growth factor and therefore the actions of vascular
histamine is negligible since the dose required to do so cessful IVF outcome like pregnancy rates and birth tion, therefore the use of hMG would be unnecessary.32 endothelial growth factor in causing OHSS.36
is more than 1,000 times more than the effective plasma rates in low-responding women.27,28 • Most of the LH activity in hMG is from the approxi- • Cabergoline is used in women with hyperprolactine-
concentration. Thousands of infertile patients have • Intrafollicular estradiol levels have been significantly mately 10 units of hCG in each 75 IU vial, but the mia at up to 1.0 mg twice weekly.
been treated with third-generation antagonists without increased. amount of hCG and LH bioactivity varies from batch to • Risks: The most common side effects are headache,
evidence of systemic or major local skin reactions. • Not effective in increasing ovarian response. batch and among suppliers. Dilute hCG has the advan- nausea, and dizziness. With long-term treatment val-
• Common side effects include minor injection-site tage of providing a consistent LH-like effect, as long as vular heart disease has rarely been observed (3/1,000),
reactions, nausea, headache, fatigue, and malaise. Mechanism of Action a sufficient volume is used. generally with doses much higher than 0.5 mg. No
• Local skin reactions seem to occur less frequently with cases were observed with less than 6 months of use. It
antagonists than GnRH agonists. Increased apoptosis has been reported in the GCs of Low-dose Aspirin is difficult to ascribe any significant risk to administra-
• No significant changes have been noted in serum older women having IVF. Growth hormone and its inter-
• In a large trial, aspirin 100 mg begun with the onset of tion for 8 days at this very low dose.
chemistry or hematological indices. mediary, insulin-like growth factor I (IGF-I), are two of
the most well-characterized factors known to reduce midluteal agonist and continued through early preg-
• No significant drug interactions have been noted.
• The follow-up of children born so far with the use of apoptosis and improve the health and proliferation of nancy was found to increase ovarian response, preg- Androgens and Androgenic Drugs
GCs, which are crucial to the nourishment of the matur- nancy outcome, and ovarian and uterine blood flow
antagonists is reassuring. The data available dem- • Androgens and drugs that increase ovarian androgens,
ing oocyte. compared with placebo.33
onstrates good safety profile for the antagonists. No such as letrozole, may become important adjuncts for
increase in malformations or abnormal development patients with low prognosis IVF.37
has been observed.
• Risks: Minimal. Mechanism of Action • The use of transdermal testosterone for 5 days preced-
• Use in a diabetic could adversely influence blood sugar
control. • Low-dose aspirin is thought to increase blood flow ing ovarian stimulation in poor responders has been
ADJUNCTS FOR OVARIAN by changing the balance of vasoconstricting throm- reported with a marked increase in the number of fol-
boxane relative to vasodilating prostacyclin. Ovar- licles and peak estradiol, and an increase of circulating
STIMULATION Dexamethasone ian blood flow has been reported to correlate with IGF-I.38
There are a number of medications that are important • Dexamethasone has been reported to be a highly effec- ovarian response, and uterine blood flow has been • DHEA (a precursor for testosterone in the ovary)
adjuncts to COS for optimizing the outcomes of COS.23 tive adjunct to CC.29-31 implicated in implantation, which is a highly vascular administration before and during IVF in poor
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246 The Infertility Manual Drugs Used in Ovarian Stimulation 247
responders has been associated with an increase in • There are no confirmed differences in safety, purity, or 6. Palomba S, Falbo A. Effects of metformin in women with (LHRH)-antagonist cetrorelix and the LHRH-agonist
the number of oocytes, embryos, and the rate of clini- clinical efficacy among the various available urinary or PCOS treated with gonadotrophins for IVF and ICSI cycles. buserelin. Hum Reprod. 2000;15:526-31.
A systematic review and meta-analysis of randomized 20. European and Middle East Orgalutran Study Group.
cal pregnancy compared with retrospective controls.39 recombinant gonadotropin products.
controlled trials. BJOG. 2013;120(3):267-76. Comparable clinical outcome using the GnRH antagonist
• New, longer acting gonadotropin preparations are 7. Practice Committee of American Society for Reproductive ganirelix of a long protocol of the GnRH agonist triptorelin
Mechanism of Action under development and hold promise for improving Medicine, Birmingham, Alabama. Gonadotropin prepara- for the prevention of premature LH surges in women
• Androgens increase FSH receptor activity and increase patient satisfaction while maintaining efficacy. tions: past, present, and future perspectives. Fertil Steril. undergoing ovarian stimulation. Hum Reprod. 200116:6
• The role of hCG trigger in ovulation induction cycles 2008;90(5 Suppl):S13-20. 44-51.
IGF-I and therefore may act on GCs in a way similar to
has not been established. 8. Ledger W. Clinical pharmacology of gonadotrophin 21. Engel JB, Ludwig M, Felberbaum R, Albano C, Devroey
growth hormone. preparations. Reprod Biomed. 2005;10:19-24. P, Diedrich K. Use of cetrorelix in combination with
• Risks: Although letrozole causes fetal abnormalities in • GnRH agonist trigger should be considered in donor
9. Youssef MA, Abou-Setta AM, Lam WS. Recombinant versus clomiphene citrate and gonadotrophins: a suitable
animals, such an effect has not been demonstrated in oocyte ART cycles to eliminate the chance of OHSS. urinary human chorionic gonadotrophin for final oocyte approach to “friendly IVF”. Hum Reprod. 2002;17:2022-6.
humans when the drug is stopped 10–12 days before • GnRH antagonists represent a more physiological and maturation triggering in IVF and ICSI cycles. Cochrane 22. Messinis IE, Loutradis D, Domali E, Kotsovassilis CP,
embryo transfer. patient-friendly way to ovarian stimulation with equiv- Database Syst Rev. 2011;13(4):CD003719. Papastergiopoulou L, Kallitsaris A, et al. Alternate day
alent pregnancy rates compared to agonist protocols. 10. Pouwer AW, Farquhar C, Kremer JA. Long-acting FSH versus and daily administration of GnRH antagonist may prevent
KEY NOTES • GnRH antagonists are particularly advantageous in daily FSH for women undergoing assisted reproduction.
Cochrane Database Syst Rev. 2012;13;6:CD009577.
premature luteinization to a similar extent during FSH
treatment. Hum Reprod. 2005;20:3192-7.
special groups of patients, i.e. hyper-responders and 11. Bonfil D,Chuderland D, Kraus S, Shahbazian D, Friedberg 23. Meldrum DR, Chang J, de Ziegler D, Schoolcraft WB, Scott
• Clomiphene is the best initial treatment for the large
poor responders, where they are associated with less I, Seger R, Naor Z. Extracellular signal regulated kinase, RT Jr, Pellicer A. Adjuncts for ovarian stimulation: when do
majority of anovulatory infertile women.
complications (OHSS) and acceptable pregnancy rates Jun N terminal N kinase, p. 38 and cSrc are involved in we adopt ‘‘orphan indications’’ for approved drugs? Fertil
• Enclomiphene is the more potent isomer and the
in a more economical approach, respectively. GnRh stimulated activity of FSH beta sub unit promoter. Steril. 2009;92(1).
one primarily responsible for the ovulation inducing Endocrinology 2004;145;2228-44. 24. Biljan MM, Mahutte NG, Dean N, Hemmings R, Bissonnette
actions of CC. 12. Liu F, Usui I, Evans LG, Austin DA, Mellon PL, Olefsky JM, F, Tan SL. Effects of pretreatment with an oral contraceptive
• CC will induce ovulation in about 80% of properly PROBABLE QUESTIONS et al. Involvement of both G(q/11) and G(s) proteins in on the time required to achieve pituitary suppression
selected patients. GnRH receptor mediated signaling in L beta T2 cells. J Biol with gonadotropin-releasing hormone analogues and on
1. Enumerate the drugs commonly used in ovulation Chem. 2002;277:32099-108. subsequent implantation and pregnancy rates. Fertil Steril.
• CC is ineffective in women with hypogonadotropic
induction. 13. B. Fauser, IT Kaspa. The use of GnRH agonist in IVF 1998;70:1063-9.
hypogonadism (hypothalamic amenorrhea). protocols. IVF Worldwide Online. Survey; 2012. 25. Fanchin R, Salomon L, Castelo-Branco A, Olivennes F,
2. What are the differences between clomiphene and
• Third-generation aromatase inhibitors are commonly 14. Karten MJ, Rivier JE. Gonadotropin-releasing hormone Frydman N, Frydman R. Luteal estradiol pre-treatment
aromatase inhibitors? Which of the two drugs is now
used in ovulation induction. analog design. Structure–function studies towards the coordinates follicular growth during controlled ovarian
preferred and why? development of agonists and antagonists: rationale and hyperstimulation with GnRH antagonists. Hum Reprod.
• The use of letrozole for superovulation is associated
3. Discuss about the various gonadotropins. perspective. Endocrinol Rev. 1986;7:44-66. 2003;18:2698-703.
with a pregnancy rate higher than with the use of
4. Discuss the different GnRH analogues in detail. 15. Nestor JJ Jr, Tahilramani R, Ho TL, Goodpasture JC, Vickery 26. Frattarelli JL, Hill MJ, McWilliams GD, Miller KA, Bergh
CCCC (16.7 vs. 5.6%).
5. Discuss about the evidence-based usage of adjuvants BH, Ferrandon P. Potent gonadotropin releasing hormone PA, Scott RT. A luteal estradiol protocol for expected poor-
• Letrozole is associated with an ovulation rate of 70–84% antagonists with low histamine-releasing activity. J Med responders improves embryo number and quality. Fertil
in ovarian stimulation.
and a pregnancy rate of 20–27% per cycle. Chem. 1992;35:3942-8. Steril. 2008;89:1118-22.
6. Recent advances in gonadotropins.
• On account of studies showing abnormalities in the 16. Olivennes F, Alvarez S, Bouchard P, Fanchin R, Salat-Baroux 27. Harper K, Proctor M, Hughes E. Growth hormone for in
7. Role of metformin in ART. J, Frydman R. The use of a GnRH antagonist (Cetrorelix) in a vitro fertilization. Cochrane Database Syst Rev. 2003;3:1-33.
new born in the cardiovascular and skeletal system,
single dose protocol in IVF-embryo transfer: a dose finding 28. Tesarik J, Hazout A, Mendoza C. Improvement of delivery
letrozole is not used.
study of 3 versus 2 mg. Hum Reprod. 1998;13:2411-14. and live birth rates after ICSI in women aged >40 years
• Tamoxifen has been shown to be successful in the REFERENCES 17. The Ganirelix Dose-finding Study Group. A double-blind, by ovarian co-stimulation with growth hormone. Hum
treatment of anovulation, even in patients in whom CC 1. The Practice Committee of the American Society for randomized, dose-finding study to assess the efficacy of Reprod. 2005;20:2536-41.
treatment has failed. Reproductive Medicine. Use of clomiphene citrate in the gonadotrophin-releasing hormone antagonist ganirelix 29. Trott EA, Plouffe L, Hansen K, Hines R, Brann DW,
• Metformin lowers fasting insulin levels and reduces women. Fertil Steril. 2003;80:1302-8. (Org 37462) to prevent premature luteinizing hormone Mahesh VB. Ovulation induction in clomiphene-resistant
but does not eliminate insulin resistance. 2. Impicciatore GG, Tiboni GM. Ovulation inducing agents surges in women undergoing ovarian stimulation with anovulatory women with normal dehydroepiandrosterone
and cancer risk: review of literature. Curr Drug Saf. recombinant follicle stimulating hormone (Puregon). Hum sulfate levels: beneficial effects of the addition of
• Metformin is beneficial in improving ovulation and
2011;6(4):250-8. Reprod. 1998;13:3023-31. dexamethasone during the follicular phase. Fertil Steril.
pregnancy rates but does not improve live birth whether 3. Kar. S. Clomiphene citrate or letrozole as first line of 18. Ludwig M, Katalinic A, Banz C, Schröder AK, Löning M, 1996;66:484-6.
it is used alone or in combination with clomiphene. ovulation induction drug in infertilie PCOS women. Weiss JM, et al. Tailoring the GnRH antagonist cetrorelix 30. Parsanezhad ME, Alborzi S, Motazedian S, Omrani G. Use
• Metformin appears to have benefits in women with A prospective randomized trial. J Human Reprod Sci. acetate to individual patients’ needs in ovarian stimulation of dexamethasone and clomiphene citrate in the treatment
PCOS throughout ovulation induction treatments and 2012;5(3):262-5. for IVF: results of a prospective, randomized study. Hum of clomiphene citrate-resistant patients with polycystic
4. Practice Committee of the American Society for Reprod. 2002;17:2842-5. ovary syndrome and normal dehydroepiandrosterone
particularly during IVF cycles by reducing OHSS.
Reproductive Medicine. Use of insulin-sensitizing agents 19. Albano C, Felberbaum RE, Smitz J, Riethmüller-Winzen sulfate levels: a prospective, double-blind, placebo-
• Compared with earlier crude animal extracts, modern H, Engel J, Diedrich K, et al. European Cetrorelix controlled trial. Fertil Steril. 2002;78:1001-4.
in the treatment of polycystic ovary syndrome. Fertil Steril.
highly purified urinary and recombinant gonadotro- 2008;90(5):S69-S73. Study Group. Ovarian stimulation with HMG: results 31. Elnashar A, Abdelmageed E, Fayed M, Sharaf M.
pin products have clearly superior quality, specific 5. Tanq T, Lord JM. Withdrawn: insulin sensitizing drugs for of a prospective randomized phase III European study Clomiphene citrate and dexamethasone in treatment of
activity, and performance. PCOS. Cochraine Database Syst. 2009;8(3):31-5. comparing the luteinizing hormone-releasing hormone clomiphene citrate-resistant polycystic ovary syndrome:
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C HA PTE R
248 The Infertility Manual
a prospective placebo-controlled study. Hum Reprod.
2006;21:1805-8.
32. Thompson KA, La Polt PS, Rivier J, Henderson G, Dahl
KD, Meldrum DR. Gonadotropin requirements of the
developing follicle. Fertil Steril. 1995;63:273-6.
33. Rubinstein M, Marazzi A, Polak de Fried E. Low-dose
36. Soares SR, Gomez R, Simon C, García-Velasco JA, Pellicer
A. Targeting the vascular endothelial growth factor system
to prevent ovarian hyperstimulation syndrome. Hum
Reprod Update. 2008;14:321-33.
37. Garcia-Velasco JA, Moreno L, Pacheco A, Guillén A,
Duque L, Requena A, et al. The aromatase inhibitor
Ovulation Induction and
Superovulation
28
Mekhala Dwarakanath
aspirin treatment improves ovarian responsiveness, letrozole increases the concentration of ovarian andro-
uterine and ovarian blood flow velocity, implantation, gens and improves in vitro fertilization outcome in low
and pregnancy rates in patients undergoing in vitro responder patients: a pilot study. Fertil Steril. 2005; Chapter Outline
fertilization: a prospective, randomized, double-blind 84:82-7. • Brief Overview of Menstrual Cycle • WHO Class 3 Category—Hypergonadotropic Hypogonadism
placebo-controlled assay. Fertil Steril. 1999;71:825-9. 38. Balasch J, Frabregues F, Penarrubia J, Carmona F, • Ovulation Induction and Superovulation • Hyperprolactinemia
• Ovulation Induction in Anovulation • Superovulation
34. Ruopp MD, Collins TC, Whitcomb BW, Schisterman EF. Casamitjana R, Creus M, et al. Pretreatment with
• Drugs Used in Ovulation Induction in PCOS • Mild or Moderate Oligoasthenospermia
Evidence of absence or absence of evidence? A reanalysis transdermal testosterone may improve ovarian response • Aromatase Inhibitors • Mild Endometriosis
of the effects of low dose aspirin in in vitro fertilization. to gonadotrophins in poor-responder IVF patients with • Other SERMs • Unexplained Infertility
Fertil Steril. 2008;90:71-6. normal basal concentrations of FSH. Hum Reprod. • Gonadotropin Therapy • Problems Encountered with Ovulation Induction and Super-
35. Alverez C, Marti-Bonmati L, Novella-Maestre E, Sanz R, 2006;21:1884-93. • Combined Approach of Sequential Step-up and Step-down ovulation
Gómez R, Fernández-Sánchez M, et al. Dopamine agonist 39. Barad D, Brill H, Gleicher N. Update on the use of Regimen
cabergoline reduces hemoconcentration and ascites in dehydroepiandrosterone supplementation among women
hyperstimulated women undergoing assisted reproduction. with diminished ovarian function. J Assist Reprod Genet.
J Clin Endocrinol Metab. 2007;92:2931-7. 2007;24:629-34. INTRODUCTION The normal menstrual cycle under the control of the
neuroendocrine system and the hypothalamic-pituitary-
Fertility issues have been there since the ancient times and ovarian (HPO) axis, is the cycle of changes that occur in the
are now escalating in this modern era. ovary and uterus resulting in the production of eggs and
The most popular treatment for infertility is the use of preparation of the uterine endometrium, that is essential
ovulation induction agents intended for either ovulation for reproduction (Fig. 28.1).
induction or superovulation.
Superovulation or controlled ovarian stimulation BRIEF OVERVIEW OF
wherein normally ovulating women are given ovulation-
induction agents to increase the number of follicles and
MENSTRUAL CYCLE
thereby oocytes are thought to increase the probability of Each menstrual cycle has two components: the ovarian
pregnancy by increasing the number of oocytes available cycle and the uterine cycle (Table 28.1).
for fertilization.1,2 Here we will be focusing only on the ovarian cycle:
The goal is to prevail over a possible flaw in ovulatory • Follicular phase is the first half of the menstrual cycle.
function and luteal phase deficiencies that are not uncov- It represents the time taken from the start of menstrual
ered by conformist testing.1 cycle to ovulation.
• Rising levels of follicle-stimulating hormone (FSH)
that starts in the luteo-follicular transition phase and
continues during menstruation brings about follicular
recruitment.
• This rise in FSH is transient and spans for a few days.
• The critical level of FSH required to recruit follicles
is called as FSH threshold and the duration of expo-
sure to FSH above this level is called as FSH window
(Fig. 28.2).
• A short FSH window enables the development of a
single dominant follicle.
• Widening the FSH window enables growth of two or
more follicles at the same time, which is the under-
lying principle behind superovulation strategies
Fig. 28.1: Life cycle of ovarian folliculogenesis. (Fig. 28.3).3
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250 The Infertility Manual Ovulation Induction and Superovulation 251
Table 28.1. Overview of menstrual cycle. OVULATION INDUCTION IN •• Multiple pregnancy rate of about 17.4% is
observed
Ovarian cycle Uterine cycle ANOVULATION
Disadvantages:
Follicular phase Proliferative phase The clinical approach of management of patients with ovu- a. Pump needs to be connected to the body at all
Ovulatory phase Secretory phase latory disorders requires an understanding of its causes. times
Luteal phase Menstruation The WHO Classification of Anovulation is as follows: b. Development of antibodies to GnRH
c. Allergic skin reactions
Luteal–follicular transition phase • WHO Class 1: Hypogonadotropic Hypogonadal Ano-
2. Substitution Therapy
vulation
Characterized by the following: This is achieved by the administration of urinary or
recombinant gonadotropins (Table 28.2). Human chori-
FSH low or low normal
onic gonadotropin (hCG) trigger is required for ovulation
Estradiol (E2) low
in either modality of treatment. Occurrence of ovarian
This is on account of reduced hypothalamic secre-
hyperstimulation syndrome (OHSS) was about 1% in both
tion of GnRH or the pituitary is not responding to
modalities of treatment.1
GnRH.
Fig. 28.3: FSH threshold and window concepts in a stimulated cycle. • WHO Class 2: Normogonadotropic Normoestrogenic
(FSH, follicle-stimulating hormone; LH, luteinizing hormone.) Anovulation (Pituitary Ovarian Dysfunction) Ovulation Induction in WHO Class 2
Characterized by the following: Category—PCOS
feedback by luteal steroids and inhibin A and change FSH normal • This is the commonest type of anovulation comprising
in gonadotropin-releasing hormone (GnRH) secre- Estradiol normal 60–80% of ovulatory disorders.
tion toward an increasing pulse frequency favoring Polycystic ovarian syndrome (PCOS) and other • Hyperinsulinemia and/or insulin resistance results in
FSH secretion. similar hyperandrogenic anovulatory disorders are antral follicle dysfunction and anovulation in PCOS.6,8
seen in this group. • The follicular dynamics in women with PCOS is differ-
Any alterations in the normal ovarian cycle result in • WHO Class 3: Hypergonadotropic Hypoestrogenic ent compared to regularly ovulating women. They can
failure to release ovum, thus resulting in anovulatory cycle. Anovulation
Fig. 28.2: FSH threshold and window concepts in a spontaneous cycle. have periods of amenorrhea with interspersed nor-
During the luteo-follicular transition, due to the demise of corpus luteum
Anovulatory disorders account for 30–40% of all causes of
Characterized by the following: mal cycles, which can be understood by the Lacker’s
and the subsequent decreased estrogen production, the FSH serum con- female infertility. It constitutes one of the most easily diag-
FSH high model.
centration rises (perimenstrual rise), maintaining a plateau in the first days nosed problems and is also the most easily treatable cause
of the follicular phase. FSH threshold: a critical level of FSH is required to
E2 low
of infertility.6
start the process of follicular development. The maturing follicle becomes Suggesting ovarian failure.
less dependent on FSH by acquiring LH receptors and LH responsive- • Hyperprolactinemic Anovulation Table 28.2. Urinary human menopausal gonadotropin
ness.4 (FSH, follicle-stimulating hormone; LH, luteinizing hormone.) OVULATION INDUCTION AND Characterized by normal FSH, low E2, and high (hMG) vs. recombinant gonadotropins.
SUPEROVULATION prolactin levels. Recombinant FSH
In ovarian stimulation cycles daily administration of Intervention Urinary hMG and Rec LH
Though often ovulation induction and superovulation Ovulation Induction in WHO Class 1 Dose 75–150 IU rFSH 150 plus
exogenous FSH brings about widening of the FSH window
are used interchangeably, they are actually different and Category of Anovulation rLH 75
(above the threshold level), thus increasing the number of
should not be used synonymously. Duration For the 1st 5 days and For the 1st 5 days
follicles. 1. Pulsatile GnRH Therapy
Ovulation induction is defined as development of a reassess on scan to and reassess on
• Exposure of dominant follicle to the ideal “LH window” follicle in an anovulatory patient.1,2,7 Women with intact pituitary gland, idiopathic hypog- step up or step down scan to step up or
essential for final follicular maturation.5 Superovulation is defined as enhanced follicular onadotropic hypogonadism, and weight loss-related amen- the dose step down the dose
• Positive feedback of estrogen also accompanied by development in an already ovulating patient. In simple orrhea are eligible candidates for pulsatile GnRH therapy. When to Once the dominant Once the
a small rise is progesterone brings about LH surge The GnRH infusion is given by means of a computer- stop admin- follicle reaches a size of dominant follicle
words, anything more than one follicle is referred to as
ized minipump. istration 16 mm reaches a size of
resulting in ovulation, a complex cascade of events in superovulation.1,2,7 16 mm
association with other paracrine influences. Superovulation is useful in many conditions of male Pulse intervals are maintained between 60 and
180 minutes. Success Cumulative pregnancy Pregnancy rate of
• Luteal phase-corpus luteum production of estrogen and female subfertility such as transient anovulation, mild rate rate of 89% at the end 18% per cycle
and progesterone. to moderate oligoasthenoteratospermia (OAT), cervical Results: of six cycles
• Luteal-follicular transition phase: selective increase factor, mild endometriosis, and unexplained infertility on •• >90% achieve ovulation Multiple 30% 26%
in FSH 2 days prior to menstruation on account of the basis that superovulation could probably correct sub- •• Cumulative pregnancy rate up to 96% are seen pregnancy
demise of corpus luteum, decrease in the negative tle ovulatory defects. after six cycles rate
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252 The Infertility Manual Ovulation Induction and Superovulation 253
• Lacker’s model—according to this model each follicle cycle. The dose can be increased to a maximum of • Having a short half-life, there is no accumulation of • Mechanism of action—It binds to hypothalamic estro-
possesses two properties: (i) its initial maturity as it 150 mg/day depending on response to previous cycle drug and its metabolites with repeated use. gen receptors, blocks the negative feedback mecha-
enters the terminal phase and (ii) its sensitivity to gon- and weight of the person. • Its use is associated with significantly higher number nism thus increasing the FSH secretion.
adotropins for any given level of maturity.9 • Administration of hCG does not improve the outcome. of cycles with monofollicular growth compared to CC, • Dose: Oral form of 40 mg per day for 5 days starting
• In a normal ovulatory cycle, one or more sufficiently • Efficacy—75–80% will ovulate with conception rates of thereby reducing the multiple pregnancy rates. from the 2nd day of menstrual cycle is to be given.
mature “low sensitivity” follicles are recruited, where 22% per cycle. • Mechanism of action—aromatase inhibitors suppress • A prospective randomized controlled trial compared
one of these will dominate suppressing all others • Duration of treatment—treatment with CC can be biosynthesis of estrogen leading to reduction in the the efficacy of tamoxifen with clomiphene citrate for
which will go into atresia. offered for a maximum of six cycles beyond which lap- negative feedback effect on the hypothalamic–pituitary ovulation induction in anovulatory women showed
• In anovulatory cycles one or more follicles with aroscopic ovarian drilling or addition OG gonadotro- system, thereby resulting in increased secretion of FSH. that the overall ovulation rates and pregnancy rates
higher sensitivity are relatively more mature enter the pins should be considered. • Efficacy were similar in both groups. Tamoxifen may be supe-
beginning of the selection phase resulting in arrest of • Use of insulin sensitizers should be considered only Ovulation rate of 70–84% rior to clomiphene citrate in that it does not appear
all follicles. when the glucose tolerance test is abnormal. Pregnancy rate of 20–27% per cycle.11 to have an antiestrogenic effect on the endometrium.
• The only way to obtain a model that can both exhibit • Combination therapy with metformin and dexameth- • Dosage—Tab letrozole 2.5–5 mg/day (from day 3 to day Tamoxifen has been shown to be successfully used
ovulation of a single follicle and arrest of a number asone has no beneficial effect in ovulation induction. 7 of the cycle). Additional use of FSH (usually 100 IU/ for ovulation induction in clomiphene citrate failure
of follicles is to have a population of follicles with a day, although doses can vary depending on the char- cases. However, its role in superovulation is yet to be
• CC failure—it is defined as failure to conceive after acteristics of the patients) is considered in intrauterine
mixture of different sensitivities. ascertained.
three treatment cycles with CC with documented ovu- insemination (IUI) cycles for unexplained infertility.
• Uniformly “low sensitivity” follicles give rise to nor- lation.
mal ovulatory cycles and uniformly “high sensitivity” • In cases of CC failure, other factors contributing to
This lowers the dose of FSH with achieves moderate Raloxifene
ovarian responses are achieved.12
results in follicular arrest leading to anovulation. subfertility/infertility have to be evaluated. • It is a SERM.
• It is also used in in vitro fertilization (IVF) cycles, to
• A combination of follicles of above said in the ovary • Tubes have to be evaluated. • Mechanism of action is similar to CC leading to
reduce the dose of gonadotropins, thereby resulting in
presents with periods of arrest interspersed with nor- • Addition of gonadotropins (sequential therapy) to be increase in serum FSH levels.16
cost reduction in IVF.
mal cycles. considered. • It is more favorable on the endometrium as far as the
• It is recommended as the drug of choice for stimula-
• In anovulatory patients, some follicles would have already • Alternative drugs of ovulation induction/superovula- antiestrogenic profile is concerned.
tion in cancer patients who wish to preserve their
under gone premature arrest. The oocytes in such folli- tion to be considered. oocytes prior to chemoradiation as it decreases the E2
cles do not have normal developmental potential. Super- • CC resistance—failure to achieve ovulation with the levels, which is of help in hormone dependent tumors GONADOTROPIN THERAPY
ovulation helps in these circumstances as these ovaries highest permissible dose. without affecting oocyte quality, fecundation rate, and The underlying principle of ovulation induction with
also contain sufficient number of healthy follicles.7 • In cases of CC resistance, other alternative drugs of number of embryos obtained.13 gonadotropin therapy relies on the fact that initiation
ovulation induction such as letrozole, anastrozole, • On account of studies (Dr Bijjan et al., 2005) showing and maintenance of follicle growth is brought about by a
DRUGS USED IN OVULATION tamoxifen, or gonadotropins should be used. abnormalities in the new born in the cardiovascular transient increase in the duration of FSH level above the
INDUCTION IN PCOS • Life style modification, weight loss to be considered in and skeletal system, letrozole was not used for the last threshold level to recruit a limited number of developing
obese individuals. 5 years.13 follicles.18
Clomifene Citrate10 • Laparoscopic ovarian drilling to be considered in lean • The Government of India has revoked ban on letro-
• Clomifence citrate (CC) is the preferred first choice
PCOS and in those subjects where there is no facility zole as per the notification dated 17/2/2017 following Regimens Using Gonadotropins
for regular ultrasound monitoring during use of GT for studies reassuring the safety and efficacy of letrozole in
drug for ovulation induction in anovulatory women
ovulation induction. ovulation induction.14 Step-up Protocols
with PCOS.
• Adjuncts like metformin to be used in individuals with • There are two types of step-up protocols, one is the
• Mechanism of action—CC exhibits both agonistic and
high insulin resistance. low-dose step-up protocol and the other is chronic low
antagonistic actions at the level of estrogen receptors. Anastrazole
Dexona could also be used in certain cases. dose step-up protocol, these are the available types of
However, the antiestrogenic action dominates at the In the recent times, another third-generation nonsteroidal step-up protocols.
hypothalamus and pituitary thereby blocking the neg- aromatase inhibitor, named anastrazole is being used in a
ative feedback mechanism and thus increasing the AROMATASE INHIBITORS dose of 1 mg twice daily for 5 days. However, RCTs are not
• Stepwise increase in FSH dose to determine the FSH
threshold for follicular development.
FSH secretion.
• It is patient friendly with no requirement of intense
Letrozole available with regard to the optimal dosage and results in • Starting doses of 37.5–75 IU are employees.
comparison with other ovulation induction agents.15 • In the low-dose step-up protocol, the selected dose of
monitoring. • Letrozole is an aromatase inhibitor that has similar
GT is given for week and then assessed for follicular
• However, obesity, hyperandrogenemia, and age do results comparable to clomifene citrate with regard to OTHER SERMS growth. If follicular growth is not satisfactory, then the
affect the outcome. ovulation induction and superovulation.
dose is increased preferably by 75 IU. Once the growth
• Dose—the initial starting dose would be 50 mg/day for • Additionally, they do not have the antiestrogenic effect Tamoxifen is seen the same dose is maintained till follicular selec-
5 days starting from the 2nd to 5th day of menstrual on the endometrium that is seen in CC. tion is achieved (Fig. 28.4).1,16,17,21
• It is a selective estrogen receptor modulator (SERM).
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254 The Infertility Manual Ovulation Induction and Superovulation 255
GnRH antagonist can be used for suppression of LH • The first of reported studies (Donesky and Adashi, Mild or moderate OAT
during ovulation induction with gonadotropins. However, 1996, consisting of 947 patients) showed that 82% Cervical factor
evidence does not show any significant benefit with its use of patients ovulated following procedure and 63% Mild endometriosis
in PCOS. conceived either spontaneously or with treatment Unexplained infertility.
with medications to which they were resistant previ-
Appropriate treatment for hyperprolactinemia, hypo-
Efficacy of Treatment ously.19
thyroidism, correction of very low BMI, or obesity should
• However, 50% of women undergo length of stay (LOS)
• Low-dose protocol with good monitoring gives mono- be considered first in transient anovulation and then ovu-
will still require the use of ovulation induction agents.
follicular ovulation rate of about 70%. lation induction has to be started.
• CC is added if anovulation persists at the end of
Fig. 28.4: Step-up regime. • Pregnancy rate—20%.
• Multiple pregnancy rate—5.7%.16
12 weeks. MILD OR MODERATE
• Addition of gonadotropins is warranted only if anovu-
• OHSS—1%.
latory cycles persist after 6 months of procedure.
OLIGOASTHENOSPERMIA
Laparoscopic Ovarian Drilling19 • LOS significantly reduces the multiple pregnancy rates • Superovulation with intention of generating 2–3 folli-
in comparison with the use of gonadotropins as wit- cles coupled with IUI gives better results than expect-
Multiple ovarian puncture performed either by diathermy
nessed in five studies. ant management.22
or by laser is known as “ovarian drilling.”
• Miscarriage rates in LOS are comparable with seen in • Agents employed—CC, sequential treatment with CC
Indications for laparoscopic ovarian drilling (LOD):
other modes of ovarian stimulation. and gonadotropins, gonadotropins with the use of
• CC resistance
GnRH analog.
• LH hypersecretion WHO CLASS 3 CATEGORY—HYPERGONA-
• PCOS patients who require assessment of pelvis
DOTROPIC HYPOGONADISM MILD ENDOMETRIOSIS
• Anovulatory PCOS who stay far away from hospitals
and cannot afford regular monitoring. • This category deals with patients who have primary • Superovulation with gonadotropins combined with
Fig. 28.5: Step-down regime.
ovarian insufficiency. IUI significantly improves the chances of conception
Methods and Doses • A number of treatment strategies have been tried, like in mild endometriosis when compared to expectant
• Methods employed are monopolar electrocautery the use of CC+ GT, only GT, GnRH analogs with GT management.20
• In chronic low-dose step-up protocol, the initial (diathermy) and laser. have been tried with limited success of 5–10%.
selected dose is given for 14 days and then follicular21 • The number of punctures and power required are • The only reliable treatment is the use of donor eggs. UNEXPLAINED INFERTILITY
growth is assessed. If the growth is not satisfactory, determined by parameters seen on scan as the volume
weekly increment of 37.5 IU are made till follicular and stromal thickness.
HYPERPROLACTINEMIA • Use of clomiphene citrate is not found to superior to use
of placebo or no treatment (OR 0.79, 95% CI 0.45–1.38)
growth is seen and thereafter the same dose is main- • Evidence does not indicate any one methodology to • Prolactin (PRL) is secreted by the lactotrope cells of the
for clinical pregnancy/woman randomized with IUI
tained till follicular selection. This regimen is intended be superior to another. In clinical practice, four to ten anterior pituitary.
(OR 2.40, 95% CI 0.70–8.19), without IUI (OR 1.03, 95%
at reducing the risk of hyper response to stimulation. punctures are done per ovary as decided by the param- • It is also present in the decidualized endometrium,
CI 0.64–1.66) using human chorionic gonadotropin.23,25
eters such as volume of ovary, body mass index (BMI), myometrium, follicular fluid, and amniotic fluid.
• A meta-analysis and systematic review were per-
Step-down Protocol nature of cycles, baseline LH levels, previous response • Raised PRL level induces galactorrhea and HH because
formed by Polyzos et al. (2008) comparing the efficacy
to ovarian stimulation, features of hyperandrogenism. it leads to reactive elevation of hypothalamic dopa-
• This protocol is designed to be more physiological. An of aromatase inhibitors (letrozole, anastrozole) versus
Over enthusiastic drilling can lead to premature ovar- mine secretion and subsequent suppression of GnRH
initial higher loading dose of FSH is given intended to clomiphene citrate for unexplained infertility stated
ian failure. pulsatile secretion.
cross the FSH threshold level followed by a step-wise that pregnancy rates are comparable with use of clo-
• Four punctuates with 4 mm depth with 4 PW energy is • Patients present with secondary oligo/amenorrhea
reduction in the dose following follicular development miphene citrate.27
ideal to follow. with anovulatory infertility.
(Fig. 28.5).1 • According to the Cochrane review, the use of gonado-
• Repeat procedures are not associated with results and • It can be coexistent in PCOS in 3% of patients.
• Vigilant monitoring is mandatory to avoid hyperstimu- tropins is associated with a higher pregnancy rate than
hence are not encouraged. • Restoration of normal levels of PRL restores ovulation.
lation. oral treatment (8 vs. 25%), but cost is the limiting factor.
However, there are no comparative studies and this is
Efficacy SUPEROVULATION
COMBINED APPROACH OF SEQUENTIAL only an observation. Even among the GTs, clinical preg-
• The best results of LOD are seen in the first 6–9 months • It refers to enhanced folliculogenesis in an already nancy rate was significantly higher with use of hMG.
STEP-UP AND STEP-DOWN REGIMEN post procedure. ovulating patient.1,20 • Evidence reflects that ovarian stimulation with gonad-
The concomitant use of GnRH agonist, though theoretically • The cumulative ongoing pregnancy rates after • Superovulation is applied to “multifactorial subfertility.” otropins combined with IUI increased the pregnancy
reduces the LH levels and promotes folliculogenesis, is asso- 6–12 months post-LOD is comparable with 3–6 cycles • Indications: rate per cycle from 8 to 18% when compared to empiri-
ciated with the risk of OHSS and thus not recommended. of gonadotropin therapy. Transient anovulation cal treatment.26,28
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256 The Infertility Manual Ovulation Induction and Superovulation 257
PROBLEMS ENCOUNTERED WITH to cause the long-term harm of childlessness.24 Therefore, 14. The Gazette of India No 118, New Delhi, Friday, February 17, 21. Lan VTN, Norman RJ, Nhu GH, Tuan PH, Tuong HM.
we are left to weigh beneficence versus autonomy. In the 2017/MAGHA 28, 1938. Ovulation induction using low-dose step-up rFSH in
OVULATION INDUCTION AND 15. Tourgeman DE. Ovulation induction is not the same as Vietnamese women with polycystic ovary syndrome.
future, ovulation induction regimens should concentrate
SUPEROVULATION on the quality output and aim not to exceed three follicles
superovulation: the effect of selective estrogen receptor Reprod Biomed Online. 2009;18(4):516-21.
modulators and aromatase inhibitors. Fertil Steril. 22. The ESHRE Capri Workshop Group. Intrauterine
in all induction programs. 2003;80(6). insemination. Human Reprod Update. 2009;15(3):265-77.
1. CC failure: 16. de Paula Guedes Neto E, Savaris RF, von Eye Corleta H, de 23. Tummon IS, Asher LJ, Martin JS, Tulandi T. Randomized
• Patients must be counseled regarding laparo-
scopic evaluation if not done earlier
PROBABLE QUESTIONS Moraes GS, do Amaral Cristovam R, Lessey BA. Prospective,
randomized comparison between raloxifene and
controlled trial of superovulation and insemination for
infertility associated with minimal or mild endometriosis.
• Adjuncts such as insulin sensitizers may be used 1. Ovulation induction in PCOS clomiphene citrate for ovulation induction in polycystic Fertil Steril. 1997;68(1):8-12.
• Life style modification/weight loss 2. CC vs. letrozole ovary syndrome. Fertil Steril. 2011;96(3):769-73. 24. Ray A, Shah A, Gudi A, Homburg R. Unexplained infertility:
17. ESHRE Capri Workshop Group. Mono-ovulatory cycles: an update and review of practice. Reprod Biomed Online.
• Use of gonadotropins to be considered 3. Role of superovulation in unexplained Infertility—
a key goal in profertility programmes. Human Reprod 2012;24:591-602.
2. Thin endometrium: evidence based Update. 2003;9(3):263-74. 25. Reindollar RH, Regan MM, Neumann PJ, Levine BS,
• Use of ecosprin 4. Ovulation induction in HH 18. Birch Petersen K, Pedersen NG, Pederse AT, Lauritsen Thornton KL, Alper MM, et al. A randomized clinical trial
• Use of estradiol valerate MP, Cour Freiesleben NL. Mono-ovulation in women with to evaluate optimal treatment for unexplained infertility:
polycystic ovary syndrome: a clinical review on ovulation the fast track and standard treatment (FASTT) trial. Fertil
• Starting of CC earlier, i.e. from day 2/3 instead of REFERENCES induction. Reprod Biomed Online. 2016;32:563-83. Steril. 2010;94(3):888-99.
day 5
1. Homburg R, Insler V. Ovulation induction in perspective. 19. The Thessaloniki ESHRE/ASRM-Sponsored PCOS Consensus 26. Sarhan A, Beydoun H, Jones HW, Bocca S, Oehninger S,
3. Asynchrony: Human Reprod Update. 2002;8(5):449-62. Workshop Group. Consensus on infertility treatment related Stadtmauer L. Gonadotrophin ovulation induction and
• While it is desired to get more than a follicle, there 2. Messinis IE. Ovulation induction: a mini review. Human to polycystic ovary syndrome Thessaloniki, Greece. Human enhancement outcomes: analysis of more than 1400 cycles
may be synchronicity which again leads to failure Reprod. 2005;20(10):2688-97. Reprod. 2008;23(3):462-477. Reprod Biomed Online. 2011;(23):220-6.
of therapy 3. Ludwig M, Felberbaum RE, Diedrich K, Lunenfeld B. Ovarian 20. Nahuis MJ, Kose N, Bayram N, van Dessel HJHM, Braat 27. Gunn DD, Bates W. Evidence-based approach to
• This can be overcome by starting CC earlier and stimulation: from basic science to clinical application. DDM, Hamilton CJCM, et al. Long-term outcomes unexplained infertility: a systematic review. Fertil Steril.
Reprod BioMed Online. 2002;5(Suppl. 1):73-86. in women with polycystic ovary syndrome initially 2016;105(6):1566-74.
delaying hCG trigger randomized to receive laparoscopic electrocautery of 28. Beall SA, DeCherney A. History and challenges surrounding
4. Vegetti W. FSH and folliculogenesis: from physiology to ovarian
4. Hyper-response to stimulation in a patient not willing stimulation. Reprod Biomed Online. 2006;12(6):684-94. the ovaries or ovulation induction with gonadotrophins. ovarian stimulation in the treatment of infertility. Fertili
for conversion to IVF: 5. Balasch J, Fábregues F. LH in the follicular phase: Human Reprod. 2011;26(70):1899-904. Steril. 2012;97(4):795-801.
• Use of antagonist neither too high nor too low. Reprod Biomed Online.
• Aspiration of excess oocytes combined with IUI at 2006;12(4):406-15.
the same time can save the cycle from being can- 6. Oktem O, Urman B. Understanding follicle growth in vivo.
Human Reprod. 2010;25(12):2944-54.
celled 7. Holzer H, Casper R, Tulandi T. A new era in ovulation
5. OHSS: induction. Fertil Steril. 2006;85(2):277-84.
• It is more common with the use of gonadotropins 8. Baerwald AR, Adams GP, Pierson RA. Ovarian antral
though it can happen with CC alone. folliculogenesis during the human menstrual cycle: a
• If it is primary OHSS, patient can be counse- review. Human Reprod Update. 2012;18(1):73-91.
9. Franks S, Stark J, Hardy K. Follicle dynamics and
led regarding options of cycle cancellation, use
anovulation in polycystic ovary syndrome. Human Reprod
of GnRH analog for trigger with modified LPS Update. 2008;14(4):367-78.
with low-dose hCG or aspiration of excessive 10. The Practice Committee of the American Society for
oocytes. Reproductive Medicine Fertility and Sterility®. Use of
• In cases of secondary OHSS, supportive care with clomiphene citrate in infertile women: a committee
symptomatic treatment is offered. In cases of opinion. 2013;100(2):0015-0282.
11. Requena A, Herrero J, Landeras J, Navarro E, Neyro
severe OHSS, paracentesis needs to be offered. JL, Salvador C, et al. Use of letrozole in assisted
reproduction: a systematic review and meta-analysis.
CONCLUSION Reproductive Endocrinology Interest Group of the Spanish
Society of Fertility. Human Reprod Update. 2008;14(6):
With freedom and choice of many agents of ovulation 571-82.
induction/superovulation comes great responsibility.18 12. Badawy A, Metwally M, Fawzy M. Randomized controlled
First and foremost in our care of patients is the dictum trial of three doses of letrozole for ovulation induction
“primum non nocere,” or “first do no harm.” It is well in patients with unexplained infertility. Reprod Biomed
Online. 2007;14(5):559-62.
known that high-order multifetal pregnancies represent 13. Casper RF, Mitwally MFM. A historical perspective of
the largest single cause of poor obstetric and perinatal out- aromatase inhibitors for ovulation induction. Fertil Steril.
come. However, infertility as a diagnosis has the potential 2012;98:1352-5.
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C HA PT E R
Controlled Ovarian Stimulation and
Individualization of Stimulation (iCOS)
29 Controlled Ovarian Stimulation and Individualization of Stimulation (iCOS) 259
This prediction of response also helps clinicians give
women more precise information on possible prolonged
treatment, cycle cancellation, ovarian hyperstimulation
syndrome (OHSS), treatment burden, and decreased suc-
cess.2 As stated above if individualization of stimulation is
Table 29.1. Markers of ovarian reserve.
Clinical
Age
Smoking status
Investigations
Basal FSH
Ovarian volume
Mamta Dighe
established on exact prediction of ovarian response, then Menstrual cycle characteristics Antimüllerian hormone
the prediction of ovarian response should be based on the Body mass index (BMI) Antral follicle count
Chapter Outline
• iCOS • Assessment of AMH and AFC
most precise markers of ovarian reserve. Though there is a Previous response to stimulation
• Predictors of Ovarian Response definite decrease in fertility after the age of 35 and the inci-
Features of PCOS
dence of poor response increases with age, chronological
age does not always collate to biological age.
INTRODUCTION (rFSH) and recombinant luteinizing hormone The clinical and laboratory criteria used by most clini- all detectable antral follicles of 2–10 min diameter would
(rLH). cians to select a protocol are outlined in Table 29.1. provide the most rational method for assessment of AFC
The introduction of controlled ovarian stimulation for b. Availability of gonadotropin-releasing hormone Out of all the markers, antimüllerian hormone (AMH) in clinical practice.12 Advancement in ultrasound imaging
multiple follicular development helped in generating mul- (GnRH) antagonists. and antral follicle count (AFC) are the best prognosticators has considerably decreased the intra- and inter-observer
tiple embryos and significantly increased pregnancy rates. c. The option of making small changes in dose of even for response to stimulation.4-6 These can be used to predict variability.13
These stimulation protocols were designed in order to
12.5 or 25 IU helps in designing the right dose for responses at either end.7
obtain multiple oocytes, and thereby increase the number
each patient.
of embryos. Identification of Response Based
d. Better embryo culture techniques and newer meth- ASSESSMENT OF AMH AND AFC
It is important to achieve good response to controlled
ods of embryo selection give a better clinical o
utcome on AMH and AFC
ovarian stimulation (COS) as the number of oocytes is
even after transferring fewer embryos. AMH Using predictors for ovarian response, women are divided
directly correlated to ongoing pregnancy rate.1 However,
e. Better freezing techniques of embryos and oocytes. into the following sub groups:
every patient undergoing stimulation with the same pro- AMH assays have been modified and gone through
tocol may not always respond in the same manner and this Factors deciding individualization are as follows: versions. a. Expected poor responders
may result in suboptimal results. • The endocrine and follicular reaction to stimulation is b. Expected normal responders
Diagnostic System Immunotech Generation II assay
the ovarian response. c. Expected hyper-responders.
Lab(DSL) (IOT) (Beckman Coulter)
iCOS • The goal of individualization is to produce optimal
number of oocytes for each patient and evade cycle Webstar, TX France Brea, CA The modification of the ovarian stimulation protocol
Tailoring stimulation taking into consideration individual and the gonadotropin dose is made depending on which
cancellation or adverse events related to under- or Sensitivity-0.006 Sensitivity-0.05 Sensitivity-0.08
variability and deciding protocols and gonadotropin doses group the patient is categorized (Table 29.2).
overstimulation. Lower values Higher values Similar to IOT
suited for individual response is known as individualiza-
• There is a lot of intra- and inter-individual variability in Value Curve- Value Curve- Value Curve-
tion of ovarian stimulation or iCOS.
ovarian response to stimulation. 0.05–15 ng/mL 0.1–24.5 ng/mL 0.16–22.5 ng/mL Expected Poor Responders
• To predict the response to stimulation we need good
Objective of iCOS2 markers telling us about the ovarian reserve. Values found with the Diagnostic Systems Lab (DSL)
A poor response has been defined as the retrieval of four or
less oocytes following a standard IVF protocol, i.e. follow-
• To tailor make the protocol for every single woman • The tests to ascertain these markers should be easy for assay can be transformed to the Immunotech Beckman
ing maximal stimulation.14
with no compromise on quality. clinical application and universally available. Coulter (IBC) assay by multiplying by 1.39,8 while the new
Incidence—10–20%. Prevalence is less among women
• Maximize the chances of pregnancy. • Individualization of treatment should not just be Generation II assay should give values identical to old IBC
aged less than 34 years and increasing markedly with
• Eliminate the risk of OHHS, which is purely iatrogenic. looked at in terms of adjusting the FSH or gonadotro- assay.9 Latest, fully automated AMH assays are available
advancing age, embracing 50% in women aged 43–44 years.14
• Minimize the risk of cycle cancellation. pin doses but also in terms of deciding the strategic now (access assay).
AMH values—The two largest prospective studies pub-
• Create a better endometrial environment for implan- approach for the different GnRH analogs and deter-
lished to date have included 340 and 356 women, respec-
tation. mining the correct stimulation protocol. Antral Follicle Count tively.15,16 The first study15 treated the best cutoff value for
• Give a more appropriate dose to expected poor
The total number of antral follicles present in the ovaries AMH as 5 pmol/L (0.7 ng/mL) (DSL assay), which was
responders. PREDICTORS OF OVARIAN RESPONSE and appreciable by transvaginal ultrasound scan is the correlated with a sensitivity of 75% and specificity of 91%.
• Reduce costs and dropout rates.
The goal is to choose the correct protocol yielding opti- AFC. AFC is firmly correlated to circulating AMH levels The second study16 found an AMH value of 1.36 ng/mL (9.7
Individualization is also possible today, due to the fol- mal oocytes based on this prediction. Analysis of a large since the hormone is formed by small antral follicles them- pmol/L) (IBC assay) to be correlated with 75.5% sensitiv-
lowing reasons: number of in vitro fertilization (IVF) cycles in the UK sug- selves.10 The number of smaller antral follicles (2–5 mm) ity and 74.8% specificity. A cutoff value of AMH ranging
a. Advancements made in the gonadotropin prepara- gested that obtaining 15 oocytes may be the optimal num- is highly collated with the number of larger antral follicles between 0.7 and 1.3 ng/mL may be treated acceptable for
tions like recombinant follicle-stimulating h
ormone ber to aim for, in order to maximize treatment success.3 (2–10 mm).11 It was therefore recommended that counting prognosticating poor response in IVF.
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260 The Infertility Manual Controlled Ovarian Stimulation and Individualization of Stimulation (iCOS) 261
AFC cutoff values used for predicting poor response vary • Deciding the appropriate stimulation protocol involv- • These patients would respond well to both the GnRH • Leads to a smaller number of growing follicles when
largely between studies. Remaining focused on the most ing the right GnRH analog, i.e. deciding whether to use agonist downregulation protocol as well as the antago- compared with the standard long GnRH agonist
recent papers, the most regularly reported cutoff values of GnRH agonist or the GnRH antagonist-based protocol. nist protocol. protocol.34
AFC for prediction of poor response ranged between 5 to 7. • Deciding the correct dose of gonadotropin. • The aim would be to procure about 8–14 oocytes. • Required lesser days of stimulation than the GnRH
While AFC and AMH are the best predictive markers of • Incorporating the use of adjuvants to optimize stimula- • The decision can be made depending on the patient agonist protocol (9 days vs. 13 days).
ovarian reserve accessible, neither is completely depend- tion results, which is discussed at the end of the chapter. compliance, time available for stimulation, and the • Was associated with elimination of the need for
able, with a false positive rate of 10–20%. Moreover, even • Using minimal and mild stimulation protocols when comfort of the physician with the particular proto- cryopreservation of embryos due to excess response
though the possibility of pregnancy is decreased, preg- required. col. and decreased hospitalization for OHSS (13.9% in the
nancy rates in younger poor responders are still treated agonist group versus 0.0% in the antagonist group).34,35
acceptable.17,18 Decision Regarding the Stimulation Protocol Expected High Responders • Induction of an LH surge to trigger ovulation could be
obtained by administering a single bolus of GnRH ago-
Expected Poor Responders AMH levels greater than 3.5 and AFC greater than 15. nist.36 This regimen may prove highly effective in terms
Expected Hyper-responders
a. AMH level of less than or equal to 0.1 and negligible • The antagonist protocol in high responders was also of OHSS prevention.21
Hyper response refers to the retrieval of 1519,20 or 2015 antral follicles seen. correlated with significantly higher clinical pregnancy To summarize, a modified therapeutic protocol with
oocytes following a standard COS protocol. • No pregnancy was observed irrespective of the rates (61.7 vs. 31.8%, P = 0.05).31 low gonadotrophin doses and GnRH antagonist seems
Prevalence—7% and varies with the woman’s age: it is stimulation protocol used. • Use of antagonists is correlated with a decline in the to be optimal for women at a high risk of OHSS. Conse-
around 15% in women aged less than or equal to 30 years, • These patients should be counseled regarding poor occurrence of a high response and a significant reduc- quently, identification of high responders on the base of
decreasing with advancing age. It is the main risk factor for chances of success and using a modified natural tion in the incidence of OHSS or of cycle cancellation ovarian reserve markers must be considered as invaluable
OHSS.21,22 cycle stimulation or minimal stimulation should because of the risk of OHSS.32,33 in women undergoing IVF.
AMH—Studies based on the IBC assay have recorded be the goal in order to decrease the cost burden, if
AMH cutoff levels between 2.6 and 4.83 ng/mL while for the patient decides to go ahead with the cycle.
“old” DSL assay values ranging from 1.59 to 5 ng/mL have b. AMH levels between 0.5 and 1.3 ng/mL and antral Parameters Protocol Principle Pros and Cons
been reported. The two studies based on the AMH Gen follicles between 5 and 7. AMH ≤0.1 Protocol Low reserve Lower the cost burden
II23,24 found 3.9 and 3.52 ng/mL, respectively, as acceptable • Antagonist protocols were associated with a con- AFC 0–2 Minimal/Mild Poor pool of oocytes. Get better quality vs. quantity.
cutoff values for the prediction of hyper response. Gonadotropin Dose Unlikely to bring up follicles Inherently poor chances of success
siderable drop in cycle cancellation and showed 150–225 with any stimulation Better patient compliance
AFC—The largest prospective study published to date a trend toward higher pregnancy rates.2
was based on 159 women undergoing IVF.25 reported an AMH 0.7–1.3 Protocol Can see cohort on D2 Higher success rates with antagonist
• The short GnRH agonist protocol or the micro- AFC 5–7 Antagonist Downregulation may cause protocol reported
AFC value of 16, with a supposed sensitivity of 89% and a dose flare protocol is also good options in these Gonadotropin Dose suppression and reduced Substantial drop in cycle cancellation.
specificity of 92%, for the prediction of high response. patients. 300 IU response Ability to use GnRHa trigger
• The long downregulation protocol could have Self follicular phase FSH and May increase yield/quality
LH can be utilized Patient more likely to repeat cycle if required
Expected Normal Responders an adverse effect in poor responders because it
AMH 1.4–3.5 Protocol Good follicle reserve. Good yield in terms of quality and quantity
Patients falling in the zone between poor and hyper- may generate an excessive ovarian suppression
AFC 8–14 GnRH analog Will respond well to either of oocytes expected with both
responders are the expected normal responders. that could lead to a decreased or absent follicular downregulation. stimulation Decision based on patient compliance,
AMH cutoff values in them are generally between 1.5 response.26,27 OR time available for stimulation, and physician
Antagonist comfort
and 3.4 ng/mL. AFC between 8 and 14 in this subgroup. With Antagonist protocols it is possible to check AFC
Gonadotropin Dose
prior to starting stimulation and choose a cycle where 225
Deciding/Individualizing Ovarian favorable outcome is expected as poor responders are AMH >3.5 Protocol Excessive number of folic. Fewer days of stimulation Higher pregnancy
known to have significant cycle to cycle variation.28 AFC >15 Antagonist Higher chances of rates
Stimulation With Antagonist cycles we can use the new long act- Gonadotropin Dose hyper-response Decreased chances of OHSS
Personalizing the stimulation for each patient involves: ing gonadotropin that supports growth of the cohort for 150 Analog protocol will require Allows use of GnRH analog for trigger
human chorionic gonadotropin
seven days29 Long acting gonadotropins cause a rapid
(hCG) trigger
and sustained rise of FSH levels which exposes small
Table 29.2. Prediction of response as per AMH and AFC antral follicles to constant high levels of FSH, helping poor
Patient Category AMH (ng/mL) AFC responders.30 Individualizing the Gonadotropin Dose starting dose for individual women derives from the
observations that different variables may independently
Poor responders 0.7–1.36 5–7
It is clear that the outcome of response is based on various contribute to its prediction. Combining two or more of
Normal responders 1.5–3.4 8–14 Expected Normal Responders
markers of ovarian reserve. The theory of using multivari- these markers to predict the appropriate starting dose of
Hyper-responders >3.5 >15 AMH levels between 1.3 and 3.4 and AFC between 8 and 10. ate models to identify the most appropriate gonadotropin gonadotropin have been tried.
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262 The Infertility Manual Controlled Ovarian Stimulation and Individualization of Stimulation (iCOS) 263
Many models have been tried combining different A model incorporating all the variables, namely, AMH, Growth Hormone GnRH antagonist declines the risk of cycle cancellation
markers of ovarian reserve, both clinical and hormone AFC, age, BMI, and smoking habits has been designed by and increases the chance of clinical pregnancy in poor
and ultrasound based. Some examples are the models cre- the PIVET Medical Centre. This algorithm suggests even Addition of growth hormone during stimulation in poor responder patients.44 The biological rationale might be
ated by Popovic-Todorovic et al, which incorporated age, minute changes in dosing to allow the exact response and responders may improve the yield of oocytes. Two recent that luteal estradiol priming could promote synchroniza-
ovarian stromal blood flow, AFC, and smoking status.37 has greatly helped in decreasing the chances of ovarian meta-analyses of six randomized trials (128 patients tion of the pool of follicles available to controlled ovarian
Another predictive model by Howles et al used basal FSH, hyperstimulation. Extremely small increments or changes in total)42,43 suggested that the addition of GH signifi- stimulation.45
body mass index (BMI), age, and AFC.38 While appear- in dosing have been suggested in this model. cantly increased the possibility of live birth in poor res-
ing to be useful, both models were rather complicated An algorithm designed by Nelson and Yates suggests ponders. Metformin
and have not had a wide clinical application. Also, both the stimulation protocol and the ideal gonadotropin dose
these models did not incorporate AMH levels as a predic- Metformin pretreatment in women with PCOS signifi-
based on the predictors of response. Estradiol Addition in Luteal Phase
tive tool. However, we have seen that AMH and AFC are Figure 29.1 is a good indicator for using ovarian reserve cantly reduces the risk of OHSS and should be considered
the two most reliable markers for prediction of ovarian markers to use the protocol and gonadotropin dose. In a recent meta-analysis of eight selected studies from for these patients starting prior to stimulation.
response and models incorporating both these markers 1,227 initially searched, the addition of estradiol in the These options can be utilized as per the requirement of
would be ideal. La Marca et al used age, AFC, and AMH to Individualizing Therapy Using Adjuvants luteal phase with or without the simultaneous use of the specific patient.
predict the response to gonadotropin dose in their model. Androgens
The primary end point considered the number of
oocytes obtained per unit of starting dose of Recombinant Using androgen pretreatment in poor responders is said to Type of response Pretreatment therapies Stimulation protocol Gonadotropin dose & type
FSH. This normogram was quite accurately able to pre- increase the ovarian sensitivity to gonadotropins. Andro- Poor Testosterone Gel Antagonist/Flare/Mild rFSH with LH, hMG,
dict the ovarian response to the dose.39,40 However, AMH gens such as dehydroepiandrosterone (DHEA) or testos- DHEA GH supplementation High dose
and AFC were used separately and not incorporated into terone have been used pre-IVF for this. A recent meta- (LH supplementation helps)
the same predictive model. Both models are easy to use analysis of four randomized controlled trials of adjuvant Normal – Agonist or Antagonist FSH or hMG
and utilize markers that are routinely done by every prac- androgens (DHEA and testosterone) in poor responder Both work well standard dose
titioner and hence can be used as a baseline guidance to patients exhibited a significantly higher o ngoing preg-
High Metformin Antagonist rFSH
decide gonadotropin dose. nancy rate in the androgen supplementation group.41 Small adjustments in doses
possible
Analog trigger
Individualizing Addition of LH and Natural Cycle IVF, Mild Protocols,
the Ovulation Trigger and Random Start Protocols
The addition of LH helps in patients expected to have a poor Natural cycle IVF and mild ovarian stimulation proto-
response, older patients and in those where the response cols are generally used in patients with expected poor
plateaus a few days after using rFSH. A very recent meta- response. The use of clomiphene in these protocols usu-
analysis of 40 randomized controlled studies46 showed that ally creates the need to freeze all embryos and transfer
significantly more oocytes were retrieved and significantly them in the next cycle.
higher clinical pregnancy rates were observed with recom- Random start protocols are used in women who are
binant human follicle-stimulating hormone (r-hFSH) plus going to start treatment for malignancy and here stimu-
recombinant human LH (r-hLH) versus r-hFSH treatment lation is started on any day of the menstrual cycle. The
in poor responders, implying that there is a relative boost availability of GnRH antagonists has made these protocols
in the clinical pregnancy rates of 30% in poor responders possible.
and that the addition of r-hLH to r-hFSH may be helpful for
women with poor ovarian response.
Ovulation trigger can be modified in patients with
CONCLUSION
hyper-response in order to avoid the possibility of OHSS. • After decennial practice using IVF, it is now very clear
Using a GnRH trigger in these patients nearly eliminates that the “one size fits all” approach may no longer exist.
the risk of OHSS. Reducing hCG dose for the trigger has • This is an era of personalized medicine and treatments
Fig. 29.1: Individualizing ovarian stimulation as per predicted response. also been described. should be customized to individual characteristics.
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264 The Infertility Manual Controlled Ovarian Stimulation and Individualization of Stimulation (iCOS) 265
• The availability of new markers of ovarian reserve, the 5. La Marca A, Sighinolfi G, Radi D, Argento C, Baraldi E, 18. Oudendijk JF, Yarde F, Eijkemans MJ, Broekmans FJ, 31. Nelson SM, Yates RW, Lyall H, Jamieson M, Traynor I,
advancement in methodology for their measurement, ArtenisioAC, et al. Anti-Mullerian hormone (AMH) as Broer SL. The poor responder in IVF: is the prognosis Gaudoin M, et al. Anti-Müllerian hormone-based approach
a predictive marker in assisted reproductive technology always poor? A systematic review. Hum Reprod Update. to controlled ovarian stimulation for assisted conception.
and the huge amount of studies providing clinical data
(ART). Hum Reprod Update. 2010;16:113-130. 2012;18:1-11. Hum Reprod Update. 2009;24:867-75.
have backed the view that individualization in IVF is 6. La Marca A, Grisendi V, Giulini S, Argento C, Tirelli A, 19. La Marca A, Sighinolfi G, Radi D, Argento C, Baraldi E, 32. Al-Inany HG, Abou-Setta AM, Aboulghar M. Gonadotrophin-
the way forward. Dondi G, et al. Individualization of the FSH starting dose Artenisio AC, et al. Anti-Mullerian hormone (AMH) as releasing hormone antagonists for assisted conception: a
• The key is having good markers of ovarian reserve, in IVF/ICSI cycles using the antral follicle count. J Ovarian a predictive marker in assisted reproductive technology cochrane review. Reprod Biomed Online. 2007;14:640-9.
which are easily measurable. Res. 2013;6:11. (ART). Hum Reprod Update. 2010;16:113-30. 33. Al-Inany HG, Youssef MA, Aboulghar M, Broekmans F,
• Important role for both AFC and AMH in the predic- 7. Broer SL, Do´lleman M, Opmeer BC, Fauser BC, Mol BW, 20. Broer SL, Do´lleman M, Opmeer BC, Fauser BC, Mol BW, Sterrenburg M, Smit J, et al. Gonadotrophin-releasing
Broekmans FJ. AMH and AFC as predictors of excessive Broekmans FJ. AMH and AFC as predictors of excessive hormone antagonists for assisted reproductive technology.
tion of the extremes of ovarian response and for ena-
response in controlled ovarian hyperstimulation: a meta- response in controlled ovarian hyperstimulation: a meta- Cochrane Database Syst Rev. 2011;CD001750.
bling the consecutive individualization of a therapeu- analysis. Hum Reprod Update. 2011;17:46-54. 34. Nelson SM, Yates RW, Lyall H, Jamieson M, Traynor I,
analysis. Hum Reprod Update. 2011;17:46-54.
tic strategy. 8. Wallace AM, Faye SA, Fleming R, Nelson SM. A multicentre 21. Humaidan P, Ejdrup Bredkjaer H, Westergaard LG, Yding Gaudoin M, et al. Anti-Müllerian hormone-based approach
• Multivariate models consisting of all important mark- evaluation of the new Beckman Coulter anti-Mullerian Andersen C. 1,500 IU human chorionic gonadotropin to controlled ovarian stimulation for assisted conception.
ers are an important step toward individualization. hormone immunoassay (AMH Gen II). Ann Clin Biochem. administered at oocyte retrieval rescues the luteal phase Hum Reprod Update. 2009;24:867-75.
• The ultimate goal would be to choose an effective pro- 2011;48:370-3. when gonadotropin-releasing hormone agonist is used for 35. Lainas TG, Sfontouris IA, Papanikolaou EG, Zorzovilis JZ,
9. Kumar A, Kalra B, Patel A, McDavid L, Roudebush WE. ovulation induction: a prospective, randomized, controlled Petsas GK, Lainas GT, et al. Flexible GnRH antagonist
tocol for ovarian stimulation which has to be well bal- study. Fertil Steril. 2010a;93:847-54. versus flare-up GnRH agonist protocol in poor responders
J Immunol Methods. 2010;362:51-9.
anced between the risk of maximal and suboptimal 22. Humaidan P, Quartarolo J, Papanikolaou EG. Preventing treated by IVF: a randomized controlled trial. Hum Reprod
10. Weenen C, Laven JS, Von Bergh AR, Cranfield M, Groome NP,
ovarian response, optimize results and decrease costs Visser JA, et al. Anti-Müllerian hormone expression pattern ovarian hyperstimulation syndrome: guidance for the Update. 2008;23:1355-8.
and stress. in the human ovary: potential implications for initial and clinician. Fertil Steril. 2010b;94:389-400. 36. Griesinger G, Diedrich K, Devroey P, Kolibianakis EM.
• Better predictive models which incorporate genetic cyclic follicle recruitment. Mol Hum Reprod. 2004;10:77-83. 23. Arce JC, La Marca A, Mirner Klein B, Nyboe GnRH agonist for triggering final oocyte maturation in
Andersen A, Fleming R. Antimüllerian hormone in the GnRH antagonist ovarian hyperstimulation protocol: a
markers will further enhance personalization. 11. Jayaprakasan K, Deb S, Batcha M, Hopkisson J, Johnson I,
gonadotropin releasing-hormone antagonist cycles: systematic review and meta-analysis. Hum Reprod Update.
• Further research into predictive models where pri- Campbell B, et al. The cohort of antral follicles measuring
prediction of ovarian response and cumulative treatment 2006;12:159-68.
mary outcome is clinical pregnancy will significantly 2–6 mm reflects the quantitative status of ovarian reserve
outcome in good-prognosis patients. Fertil Steril. 37. Popovic-Todorovic B, Loft A, Lindhard A, Bangsbøll S,
as assessed by serum levels of anti-Müllerian hormone and
add to our knowledge. response to controlled ovarian stimulation. Fertil Steril.
2013;99:1644-53. Andersson AM, Andersen AN. A prospective study of
24. Polyzos NP, Tournaye H, Guzman L, Camus M, Nelson predictive factors of ovarian response in ‘standard’ IVF/
2010b;94:1775-81.
PROBABLE QUESTIONS 12. Broekmans FJ, de Ziegler D, Howles CM, Gougeon A,
SM. Predictors of ovarian response in women treated with
corifollitropin alfa for in vitro fertilization/intracytoplasmic
ICSI patients treated with recombinant FSH. A suggestion
for a recombinant FSH dosage normogram. Hum Reprod
Trew G, Olivennes F. The antral follicle count: practical
1. Write an essay on iCOS. sperm injection. Fertil Steril. 2013. Update. 2003a;18:781-7.
recommendations for better standardization. Fertil Steril.
2. Role of ovarian reserve tests in controlled ovarian 25. Aflatoonian A, Oskouian H, Ahmadi S, Oskouian L. 38. Howles CM, Saunders H, Alam V, Engrand P; FSH
2010;94:1044-51.
Prediction of high ovarian response to controlled ovarian Treatment Guidelines Clinical Panel. Predictive factors and
stimulation. 13. Deb S, Campbell BK, Clewes JS, Pincott-Allen C, Raine- a corresponding treatment algorithm for controlled ovarian
hyperstimulation: anti-Müllerian hormone versus small
3. Various models to determine gonadotropin starting Fenning NJ. Intracycle variation in number of antral follicles antral follicle count (2–6 mm). J Assist Reprod Genet. stimulation in patients treated with recombinant human
dose in COS. stratified by size and in endocrine markers of ovarian 2009;26:319-25. follicle stimulating hormone (follitropin alfa) during assisted
4. Critically evaluate various stimulation protocols in reserve in women with normal ovulatory menstrual cycles. 26. Yoshimura Y, Nakamura Y, Ando M, Shiokawa S, Koyama N, reproduction technology (ART) procedures. An analysis of
Ultrasound Obstet Gynecol. 2013;41:216-2. Nanno T. Direct effect of gonadotropin-releasing hormone 1378 patients. Curr Med Res Opin. 2006;22:907-18.
poor responders. 14. Ferraretti AP, La Marca A, Fauser BC, Tarlatzis B, agonists on the rabbit ovarian follicle. Fertil Steril. 39. La Marca A, Grisendi V, Giulini S, Argento C, Tirelli A,
Nargund G, Gianaroli L; ESHRE working group on Poor 1992;57(5):1091-7. Dondi G, et al. Individualization of the FSH starting dose
REFERENCES Ovarian Response Definition. ESHRE consensus on the 27. Kowalik A, Barmat L, Damario M, Liu H, Davis O, in IVF/ICSI cycles using the antral follicle count. J Ovarian
definition of ‘poor response’ to ovarian stimulation for Rosenwaks Z. Ovarian estradiol production in vivo. Res. 2013;6:11.
1. Verberg MF, Eijkemans MJ, Macklon NS, Heijnen EM,
in vitro fertilization: the Bologna criteria. Hum Reprod Inhibitory effect of leuprolide acetate. J Reprod Med 40. La Marca A, Papaleo E, Grisendi V, Argento C, Giulini S,
Baart EB, Hohmann FP, et al. The clinical significance of
Update. 2011;26:1616-24. Obstetr Gynecol. 1998;43(5):413-7. Volpe A. Development of a nomogram based on markers
the retrieval of a low number of oocytes following mild
ovarian stimulation for IVF: a meta-analysis. Hum Reprod 15. Nelson SM, YatesRW, Fleming R. Serum anti-Müllerian 28. Bancsi LFJMM, Broekmans FJM, Looman CWN, Habbema of ovarian reserve for the individualisation of the follicle-
Update. 2009;15:5-12. hormone and FSH: prediction of live birth and extremes JDF, te Velde ER. Predicting poor ovarian response in IVF: stimulating hormone starting dose in in vitro fertilisation
2. Marca AL, Kamal Sunkara S. Individualization of controlled of response in stimulated cycles—implications for use of repeat basal FSH measurement. J Reprod Med cycles. BJOG. 2012b;119:1171-9.
ovarian stimulation in IVF using ovarian reserve markers: individualization of therapy. Hum Reprod Update. Obstetr Gynecol. 2004;49(3):187-94. 41. Sunkara SK, Coomarasamy A. Androgen pretreatment in poor
from theory to practice. Hum Reprod Update. 2013;1-17. 2007;22:2414-21. 29. van Schanke A, van De Wetering-Krebbers SFM, Bos E, Sloot responders undergoing controlled ovarian stimulation and in
3. Sunkara SK, Rittenberg V, Raine-Fenning N, Bhattacharya S, 16. Al-Azemi M, Killick SR, Duffy S, Pye C, Refaat B, Hill N, WN. Absorption, distribution, metabolism and excretion vitro fertilization treatment. Fertil Steril. 2011;95(8):e73-e75.
Zamora J, Coomarasamy A. Association between the num- et al. Multi-marker assessment of ovarian reserve predicts of corifollitropin alfa, a recombinant hormone with a 42. Kyrou D, Kolibianakis EM, Venetis A, Papanikolaou EG,
ber of eggs and live birth in IVF treatment: an analy- oocyte yield after ovulation induction. Hum Reprod sustained follicle-stimulating activity. Pharmacology. Bontis J, Tarlatzis BC. How to improve the probability
sis of 400135 treatment cycles. Hum Reprod Update. Update. 2011;26:414-22. 2010;85(2):77-87. of pregnancy in poor responders undergoing in vitro
2011;26(7):1768-74. 17. Klinkert ER, Broekmans FJ, Looman CW, Habbema JD, te 30. Polyzos NP, Devos M, Humaidan P, Stoop D, Ortega- fertilization: a systematic review and meta-analysis. Fertil
4. van Rooij IA, Broekmans FJ, te Velde ER, Fauser BC, Velde ER. Expected poor responders on the basis of an Hrepich C, Devroey P, et al. Corifollitropin alfa followed Steril. 2009;91(3):749-66.
Bancsi LF, de Jong FH, et al. Serum anti-Müllerian hormone antral follicle count do not benefit from a higher starting by rFSH in a GnRH antagonist protocol for poor ovarian 43. Kolibianakis EM, Venetis CA, Diedrich K, Tarlatzis BC,
levels: a novel measure of ovarian reserve. Hum Reprod. dose of gonadotrophins in IVF treatment: a randomized responder patients: an observational pilot study. Fertil Griesinger G. Addition of growth hormone to gonad-
2002;17:3065-71. controlled trial. Hum Reprod Update. 2005;20:611-5. Steril. 2013;99(2):422-6. otrophins in ovarian stimulation of poor responders
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C HA PTE R
266 The Infertility Manual
treated by in-vitro fertilization: a systematic review
and meta-
613-22.
analysis. Hum Reprod Update. 2009;15(6):
44. Reynolds KA, Omurtag KR, Jimenez PT, Rhee JS, Tuuli MG,
Jungheim ES. Cycle cancellation and pregnancy after luteal
estradiol priming in women defined as poor responders: a
coordinates follicular growth during controlled ovarian
hyperstimulation with GnRH antagonists. Hum Reprod
Update. 2003;18(12):2698-703.
46. Lehert P, Kolibianakis EM, Venetis CA, Schertz J,
Saunders H, Arriagada P, et al. Recombinant human
follicle-stimulating hormone (r-hFSH) plus recombinant
Monitoring of Ovarian Stimulation
30
Sandhya Krishnan
systematic review and meta-analysis. Hum Reprod Update. luteinizing hormone versus r-hFSH alone for ovarian
2013;28(11):2981-9. stimulation during assisted reproductive technology:
45. Fanchin R, Salomon L, Castelo-Branco A, Olivennes F, systematic review and meta-analysis. Reprod Biol Chapter Outline
Frydman N, Frydman R. Luteal estradiol pre-treatment Endocrinol. 2014;12:17. • What is Monitoring of Ovarian Stimulation? • Clinical Monitoring
• Why to Monitor? • Methodology of Monitoring Follicular Dynamics and Timing Trigger
• When and How to Monitor? • Minimal Monitoring
• Where and Who should Monitor? • Prevention of Ovarian Hyperstimulation Syndrome
• Home Monitoring • Current Guidelines
“I Have six honest serving men.
They taught me all I know
I call them what and why and when
And how and where and who.”
Rudyard Kipling
INTRODUCTION WHY TO MONITOR?
Since the inception of assisted reproductive technique, Advantages of Monitoring
knowing the moment of ovulation has always been a pri-
ority. The unfertilized ovum survives only 12–24 hours Advantages include:
after ovulation whereas the sperm survives in the cer- • Predicting ovarian response—from antral follicle
vical mucus till 5 days. Identifying the time of ovula- count by ultrasound
tion determines the window of treatment wherein the • Monitoring pituitary downregulation
sperm and egg can be brought at proximity at the right • Checking adequate dosage of gonadotropins
time. • Avoiding complications—OHSS severity and prevention
The ovulation time needs to be determined for deter- • Deciding the timing of trigger, time intrauterine insem-
mining the fertile period, to plan timed intercourse or ination, and oocyte retrieval.
timed insemination or to choose the time of ovulation
trigger, for oocyte retrieval to get mature oocyte. Disadvantages of Monitoring
Monitoring in non-IVF cycle provides the possibility to
• Needs special training
decide and plan treatment especially in induced cycle.
• Time consuming
• Need not always be accurate
WHAT IS MONITORING OF OVARIAN • Increases anxiety of couple.
STIMULATION?1,2
In simple words, this is just examining or monitoring an Ideal Monitoring Method
induced or natural cycle to assess the follicle size, matu- • Should be user friendly
rity, timing of ovulation and timing intercourse, intrau- • Reduce time needed
terine insemination, and oocyte retrieval for in vitro fer- • Easily learnable
tilization (IVF). This is necessary to improve chance of • Accurate
pregnancy and also to assure patient safety by prevent- • Economic
ing ovarian hyperstimulation and preventing multiple • Fast results
pregnancy. • Cost worthy.
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268 The Infertility Manual Monitoring of Ovarian Stimulation 269
Table 30.1. Different types of monitoring. • Even with daily testing, LH surge may be missed in
Treatment planned Monitoring method
some, especially women with irregular cycles.
Timed intercourse only Home monitoring
Timed intercourse with Home monitoring
CLINICAL MONITORING
ovulation induction • Ultrasound (Fig. 30.2)
IUI without ovulation Transvaginal ultrasound— To assess size and number of ovarian follicles
induction minimal monitoring
Endometrial thickness and pattern.
IUI with ovulation Transvaginal ultrasound— • Hormonal assays
induction frequent monitoring
Estradiol
IVF-ART Serial transvaginal ultrasound
Progesterone
and hormonal assay
Luteinizing hormone
IUI, intrauterine insemination; IVF-ART, in vitro fertilization-assisted
reproductive technique. Combining ultrasound to monitor growth of fol-
licles and estradiol levels to monitor functional
activity of follicles
WHEN AND HOW TO MONITOR? Fig. 30.1: Basal body (BB) temperature and karyopyknotic index of
Serial transvaginal ultrasound monitoring.
Fig. 30.2: Ultrasoung picture of mature ovarian follicle ready for
vaginal cytology throughout the menstrual cycle in 10 ovulatory trigger.
This depends upon type of treatment planned (Table 30.1). women. Day 0 = day of luteinizing hormone surge (dotted line). Verti-
cal bars represent one standard error of the mean.4 Advantages5-7
WHERE AND WHO SHOULD MONITOR? • Noninvasive
Table 30.2. Protocol for ultrasound monitoring in
in vitro fertilization (IVF) cycle.
This again depends upon type of treatment planned. Cervical Mucus3 • Simple and reliable
Ultrasound monitoring of IVF cycle
For timed intercourse, home monitoring alone • Includes evaluation of ovary
Immediately after ovulation the volume and viscoelastic- •• Day 21
would suffice. If planning intrauterine insemination, • No known harmful effect on endometrium or oocyte
ity of the cervicovaginal fluid increase. For choice of optimal protocol (long, short, ultrashort)
hospital monitoring with transvaginal ultrasound and • Assessment of both structural (number, size, growth,
• Advantage: low cost and ease of application. To rule out ovarian cyst
if for IVF, hospital transvaginal monitoring and labora- location of follicle) and functional aspect of ovar-
• Disadvantage: all women may not be able to interpret •• Day 2
tory hormonal monitoring will be needed by a trained ian stimulation possible. Estradiol production can be
the results. To assess antral follicle count and ovarian volume
doctor. estimated from measuring endometrial thickness and
For dosage and type of gonadotropin
pattern.
Salivary Ferning Kit3 •• Day 6
• Doppler can be used to monitor ovarian follicular vas-
HOME MONITORING An increase in salivary NaCl concentration surrounding cularity and blood flow.
For dosage and type of gonadotropin-increase or
decrease dose
ovulation period results in crystallization and ferning on
Basal Body Temperature (Fig. 30.1) 3,4 For adding antagonist
slide preparation.
Disadvantages •• Day 10
• Most simple method of monitoring ovulation • Advantage: low cost and ease of application. Decision of timing hCG/agonist
(Fig. 30.1). • Disadvantage: • Cost of ultrasound and infrastructure for ultrasound
• Woman records temperature (oral, vaginal, or rectal), All women may not be able to interpret the results maintenance.
daily morning before getting up from bed. Poor accuracy, high degree of pattern variation • Doctor dependant. Transvaginal Scan Repeatedly Done from
• Ovulatory cycle will have biphasic chart. Air bubbles or excess saliva may invalidate the
Day 6–10 (Table 30.2)30,32
• Anovulatory cycle will have monophasic chart. results. METHODOLOGY OF MONITORING
• A shift of BBT to hyperthermia phase occurs within • Follicle growth beyond 10 mm is apparent by this
Luteinizing Hormone Testing3,4 FOLLICULAR DYNAMICS AND TIMING
48 hours of ovulation (WHO definition). time
• Three consecutive daily BBT of at least 0.36°F (0.2°C)
TRIGGER (TABLE 30.2) • Follicles destined to ovulate will normally increase in
• The luteinizing hormone (LH) kit requires women to
higher than previous six daily temperatures confirms size at the rate of 1–3 mm per day up to 18–24 mm. The
test in their urine from day 6 onward for 5–9 days until Baseline Ultrasound Scan on
ovulation. a positive result is obtained. follicle may even enlarge to 3–4 cm and still ovulate.
• Advantages: home monitoring possible and low cost
Day 2 or 38 • Follicular diameter is measured by taking the mean of
• Ovulation follows 12–48 hours after urinary detection
• Disadvantages: of LH surge peak. • Done to assess antral follicle count (number of follicles two perpendicular diameters.
Clear temperature shift not seen in all women— • Most LH kits detect LH levels at more than between 5 and 10 mm). • Human chorionic gonadotropin (hCG) is usually
Inaccurate 20–40 mIU/mL. • To predict patient response to stimulation—hyper, administered when there is minimum one follicle of
Daily reading to be recorded, hence burdensome • Accuracy is 90–100%. normal, or poor responder and to rule out ovarian cyst. 16–18 mm size.
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270 The Infertility Manual Monitoring of Ovarian Stimulation 271
• The number should be three or more of this size in case Monitoring Endometrium (Fig. 30.3)6,7,34 • Also better reproducibility than classical 3D. This Table 30.3. Protocol for hormonal monitoring in IVF cycle.
of IVF, along with an endometrial thickness of at least would also reduce the need for systematic hormone
• Endometrial thickness is measured from outside to Hormone assay for monitoring IVF cycle
7 mm. If co-assessed with hormone assays, the estra- testing (Fig. 30.4).
inside in an anteroposterior view at the widest point. •• Day 2—Estradiol,FSH, LH, progesterone
diol level should be 200–300 pgm/mL per large follicle
Assessment of endometrium also plays a role in plan- To decide dose and type of gonadotropin
(Fig. 30.2). Disadvantages
ning stimulation protocol, monitoring cycles, and pre- •• Day 6—Estradiol, LH, progesterone
dicting clinical outcome (Fig. 30.3). To decide dose and type of gonadotropin
Predicting Ovulation • Needs special training.
To add antagonist
Different echogenic patterns of endometrium are: • Costly equipment
• Follicle rupture can happen from 13 to 33 mm (mean •• Day 10—Estradiol, LH, progesterone
diameter being 21 mm). Hence size is a poor indica- • Triple line pattern—hyperechogenic outer and central Deciding time for ovulation trigger and embryo
tor of imminent ovulation. Ultrasound evidence of
Serial Serum Hormone Levels (Table 30.3)12 transfer
line—this indicates best prognosis if seen before oocyte
impending ovulation include—double contour sign— retrieval and if endometrial thickness is more than 7 mm. Usually done in women undergoing IVF-ET (in vitro
a line of decreased reflectivity around the follicle which • An intermediate isoechogenic pattern with a non- fertilization-embryo transfer). Ovarian stimulation is
Estradiol26-28
is due to separation of granulosa cell layer from theca prominent central line. monitored by serial measurement of estradiol, LH, and
cell layer prior to rupture and this suggests impending • An entirely homogenous endometrium. progesterone to monitor follicular growth, evaluate pro- Used for adjusting the gonadotropin dosage and predict-
ovulation within 24 hours. gression of stimulation, adjust daily gonadotropin dosage, ing risk of ovarian hyperstimulation syndrome (OHSS).
• Irregular follicle lining 6–10 hours prior to ovulation Automated Monitoring (Fig. 30.4)9-11 and predict optimal day for trigger. Also to decide the type of trigger to be given. In long pro-
because of folding and separation of granulosa cell tocol downregulation is indicated by serum E2 level of less
layer. The development of three-dimensional (3D) ultrasound Disadvantages13,14 than 50 pg/mL. Day 6/7 E2 level is repeated again and if
• Echoes seen in the follicle due to the expanded cumu- in late 1980s enabled acquisition and analysis of volume
• Need for frequent blood sampling E2 has not increased by 50% per day, gonadotropin dose
lus oophorus just before ovulation. data. The software is called sonography based automated
• Need for a reliable laboratory is increased. The amount of estrogen produced by domi-
volume calculation (SonoAVC, GE medical systems). After
• Costs incurred. nant follicle increases as it grows and there is a linear cor-
Confirming Ovulation5,25 capturing 3D image of the ovary, SonoAVC automatically
relation between follicular diameter and E2 levels. An E2
analyses the dataset, identifying the boundaries of hypo-
window of 1,000–1,500 pgm/mL is optimum. Risk of hyper-
• Follicles decrease in size echoic follicles and provides estimates of their absolute Follicle-stimulating Hormone13 stimulation is increased when E2 level is more than 3,000
• Follicles disappear completely dimensions.
• The follicular contour becomes irregular Not commonly done in stimulation cycle. During coast- pg/mL.23
• The follicle gets filled with echo-dense structures ing to prevent OHSS, a decline in serum FSH to 5 IU/L
Advantages
• There is fluid in the cul de sac can help to predict a decline in serum estrogen (E2) to Estradiol Levels on Day of hCG31
• There is a corresponding hyperechogenic secretory • Faster, precise, and more efficient. safe levels of lesser than 2,725 pg/mL within 24 hours.
A value of more than 200–300 pg/mL per follicle indicates
endometrium. This threshold value together with a 25% daily decline of
adequate dosage of gonadotropin.
follicle-stimulating hormone (FSH) levels can be com-
Supraphysiological or high E2 might adversely affect
bined to predict safe timing of hCG trigger.11
endometrial receptivity.
The pre-hCG E2 levels are correlated with ultrasound
Luteinizing Hormone15-22 findings to decide time of trigger.
A high concentration with LH/FSH ratio more than 2 sug- Estradiol level is an important marker to assess patient
gests a hyper-responder and a very low concentration on at risk of OHSS. Most studies selected an E2 value of less
d2 of less than 1 mIU/mL predicts ovarian failure. than 3,000 pgm/mL as a safe value for hCG administ-
ration.
Progesterone23,24
Periovulatory serum progesterone levels if elevated on day
Serum Inhibin B29
of hCG is associated with reduced pregnancy rate espe- An early indicator of follicle recruitment once gonado-
cially when the level was more than 1.5 ng/mL, irrespec- tropin injections are started. Patients with day 2 inhibin
tive of GnRH analog used for IVF.21 Also hypothesizing that B more than 100 pgm/mL may be reassured to continue
when serum progesterone level is above 1 ng/mL during the same FSH dosage while those with value less than
Fig. 30.4: Ultrasound picture of automated 3-dimentional ultrasound ovarian stimulation, it might mimic LH surge in natural 100 pgm/mL advised to increase the dosage or cancel the
Fig. 30.3: Ultrasound picture of tripple line endometrium. monitoring. cycle and induce a change in implantation window. cycle if very low.27
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272 The Infertility Manual Monitoring of Ovarian Stimulation 273
MINIMAL MONITORING CONCLUSION
• Minimal monitoring implies reduced number of ultra- • Aim of monitoring ovarian stimulation to identify the
sound and hormone assays. stage at which oocyte is best matured.
• Estradiol only monitoring was used in early days of • Ultrasound monitoring is the method of choice, but in
IVF, not recommended now. women with risk of OHSS combining ultrasonography
• Ultrasound has the advantage of assessment of both (USG) and serum E2 should be practiced as a precau-
structural (number, size, growth, location of follicles) tionary measure for prevention of OHSS.
and functional aspects (endometrial thickness and • The rationale of monitoring a stimulation cycle is to
pattern suggest the level of estradiol production) of ensure an optimal number of mature oocytes on the
one hand and to prevent OHSS on the other. Serial
ovarian stimulation.
ultrasound scan and estradiol measurement afford
• Hence ultrasound as a single monitoring is reliable,
us a reliable and noninvasive method of doing so
safe, patient friendly, and reduces treatment expense.
(Tables 30.2 to 30.4).
PREVENTION OF OVARIAN PROBABLE QUESTIONS Fig. 30.5: Ovarian reserve testing before the first IVF cycle would permit to categorize patients as expected poor-, normal- or hyper-responders.
HYPERSTIMULATION SYNDROME33,35
1. What is the aim of monitoring ovarian stimulation? In hyper-responder patients, one of the most important objectives of medical counseling is to prevent OHSS. Hence, the first-line protocol would
One of the main reasons for tight monitoring of ovar- What are the advantages and disadvantages? be based on administration of low doses of FSH in a GnRH-antagonist-based scheme. AFC, antral follicle count; AMH, antimüllerian hormone.36
ian stimulation with serial E2 levels and scans is to pre- 2. Elaborate the different methods of monitoring ovar-
vent OHSS, the most common serious complication. It is ian stimulation.
characterized by multiple luteal cysts, ovarian enlarge- 3. Can monitoring ovarian stimulation help in prevent-
ing OHSS? Elaborate.
REFERENCES controlled ovarian stimulation for IVF. Hum Reprod.
ment and fluid shifts leading to ascites, pleural effu- 2011;26(1):27-33.
sion. Torsion, rupture or hemorrhage of ovary may rarely 4. Compare 3D monitoring with 2D USG for ovarian 1. Roman GS, Long CA, Reshef E, Michael J. Monitoring 11. Salama S, Torre A, Paillusson B, Thomin A, Ben Brahim F,
stimulation. Can it replace 2D USG in future? the ovulation induction cycle. Am I Obstet Gynecol. Muratorio C, et al. Ovarian stimulation monitoring past,
occur.
1995;172(2):785-8. present and perspectives. Gynecol Obstet Fertil.
A level of E2 of 500 pg/mL or more per follicle or a total 5. What is the role of monitoring hormones in ovarian 2. Kwan I, Bhattacharya S, McNeil A, van Rumste MM. 2011;39(4):245-54.
of 3,000 pg/mL or more irrespective of the number of fol- stimulation? Monitoring of stimulated cycles in assisted reproduction (IVF 12. Taieb J, Benattar C, Pous C. Hormone determination in
licles is suggested as the level to withhold hCG administra- 6. What is meant by minimal monitoring of ovarian and ICSI). Cochrane Database Syst Rev. 2008;(2):CD005289. the management and monitoring of cycles of medically
tion in order to prevent OHSS. stimulation? Elaborate. 3. Brezina PR, Haberl E, Wallach E. At home testing assisted reproductive technology, value and difficulties of
optimizing management for the infertility physician. Fertil use. Ann Biol Clin (Paris). 2003;61(5):533-40.
Ultrasound findings in OHSS include:
Steril. 2011;95(6):1867-78. 13. Al-Shawaf T, Zosmer A, Tozer A. Value of measuring
• Markedly enlarged ovary 5–10 cm in diameter Table 30.4. Protocol for combined ultrasound and
4. Moghissi KS, Syner FN, Evans TN. A composite picture of the serum FSH in addition to serum estradiol in a coasting
hormonal monitoring in IVF cycle.
• Rapid enlargement of follicle menstrual cycle. Am J Obstet Gynecol. 1972;114(3):405-18. programme to prevent severe OHSS. Hum Reprod Update.
• Hemorrhage or rupture of follicle •• Ultrasound and hormone assay combined 5. Moghissi K, Puscheck E, et al. Glob Libr Women’s Med. 2008;14(1):1-14.
•• Starting dose of gonadotropin depends on Documentation of ovulation (ISSN:1756-2228). 2015. 14. Fauser BCIM, Diedrich K, Devroey P. Predictors of ovarian
• Ascites and pleural effusion.
Ultrasound—Antral follicle count and ovarian volume 6. Cohen BM, Berry L, Roethemeyer V, Smith D. Sonographic response, progress towards individualized treatment in
Hormone assay—Estradiol, FSH, LH assessment of late proliferative phase endometrium during ovulation induction and ovarian stimulation. Hum Reprod
CURRENT GUIDELINES25,34,36 Others—Age, past history of ovulation induction ovulation induction. I Reprod Med. 1992;37:685-90. Update. 2008;14(1):1-14.
response. 7. Grunfeld L1, Walker B, Bergh PA, Sandler B, Hofmann G, 15. Kolibinakis EM, Collins J, Tarlatzis B, Papanikolaou E,
There is no evidence from randomized trials to suggest Navot D. High-resolution endovaginal ultrasonography Devroey P. Are endogenous LH levels during ovarian
•• Continuous gonadotropin administration
that combined monitoring by transvaginal ultrasonogra- of the endometrium: a noninvasive test for endometrial stimulation for IVF using GnRH analogues associated with
Ultrasound—Follicle diameter and number
phy (TVUS) and serum estradiol is more efficacious than adequacy. Obstet Gynecol. 1991;78(2):200-4. the probability of ongoing pregnancy? A systematic review.
■■ Rate and synchronism of follicle growth 8. Shoham Z, Di Carlo C, Patel A, Conway GS, Jacobs HS.
monitoring by TVUS alone with regard to clinical preg- Hum Reprod Update. 2006;12(1):3-12.
Hormone assay—Estradiol levels Is it possible to run a successful ovulation induction 16. Penarrubia J, Fabregues F, Creus M, Manau D, Casamitjana R,
nancy rates and the incidence of OHSS (Fig. 30.5). The •• Timing of hCG administration program based solely on ultrasound monitoring? The Guimerá M, et al. LH serum levels during ovarian stimulation
number of oocytes retrieved appears similar for both mon- Ultrasound—Follicle diameter more than 18 mm importance of endometrial measurements. Fertil Steril. as predictors of ovarian response and assisted reproduction
itoring protocols. Both these monitoring methods are safe Endometrial thickness more than 7 mm 1991;56(5):836-41. outcome in downregulated women stimulated with
and reliable. A combined monitoring protocol including •• Hormone assay 9. Ata B, Tulandi T. Ultrasound automated volume recombinant FSH. Hum Reprod. 2003;18(12):2689-97.
both TVUS and serum estradiol may need to be retained Estradiol levels coincide with follicle diameter and calculation in reproduction and in pregnancy. Fertil Steril. 17. Kol S. To add or not to add LH, consideration of LH
number 2011;95(7):2163-70. concentration changes in individual patients. Reprod
as precautionary good clinical practice and as a confirma- 10. Ata B, Seyhan A, Reinblatt SL, Shalom-Paz E, Krishnamurthy. Biomed Online. 2005;11:664-6.
tory test in a subset of women to identify those at high risk LH—to assess LH control
Comparison of automated and manual follicle monitoring 18. Lahoud R, Al-Jefout M, Tyler J, Ryan J, Driscoll G. A relative
of OHSS (Table 30.4). Progesterone—to postpone embryo transfer if high in an unrestricted population of 100 women undergoing reduction in mid-follicular LH concentrations during
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C HA PTE R
31
274 The Infertility Manual
GnRH agonist IVF/ICSI cycles leads to lower live birth
rates. Hum Reprod. 2006;12:2645-9.
27. Kyrou D, Popovic Todorovic B, Fatemi HM, Bourgain C.
Does the estradiol level on the day of human chorionic
Ovarian Hyperstimulation
19. Esposito MA, Barnhart KT, Coutifaris C, Patrizio P. Role
of previously luteinizing hormone concentrations during
gonadotrophin administration have an impact on
pregnancy rates in patients treated with rec-FSH/GnRH Syndrome
assisted reproductive technology cycles stimulated antagonist? Hum Reprod. 2009;24(11):2902-9.
exclusively with recombinant follicle-stimulating hormone. 28. Aboulghat M. Prediction of ovarian hyperstimulation
Divya Sardana
Fertil Steril. 2001;75(3):519-24. syndrome of OHSS. Estradiol level has an important role
20. Ye H, Zeng PH, Pei L, Hedges LV. Recombinant human in the prediction of OHSS. Hum Reprod. 2003;18(6):1140-1.
follicle stimulating hormone (r-FSH) versus urinary– 29. Eldar–Geva T, Margalioth EJ, Ben–Chetrit A, Gal M, Chapter Outline
derived human menopausal gonadotropin (hMG) in Robertson DM. Serum inhibin B levels measured early • Pathophysiology • Incidence
patients with different serum LH levels in the mid- during FSH administration for IVF may be of value in • Early and Late Forms • High Risk Factors
• Classification • Prevention
follicular phase of stimulation during IVF/ICSI. Fertil predicting the number of oocytes to be retrieved in normal
• Clinical Symptoms and Signs • Treatment of Established Ovarian Hyperstimulation Syndrome
Steril. 2006;86(3):S420-1. and how responders. Hum Reprod. 2002;17(9):2331-7.
21. Humaidan P. To add or not to add LH: comments on a 30. Wiser A, Gonen O, Ghetler Y, Shavit T, Berkovitz A,
recent commentary. Reprod Biomed Online. 2006;12:284-5. Shulman A. Monitoring stimulated cycles during in vitro
22. Tavaniotou A, Albano C, Smitz, I, Camus M. Comparison of fertilization treatment with ultrasound only–preliminary INTRODUCTION angiotensin II, insulin-like growth factor-1 (IGF-
LH concentrations in the early and mid-luteal phase in IVF results. Gynecol Endocrinol. 2012;28(6):429-31. 1), epidermal growth factor (EGF), transforming
cycles after treatment with HMG alone or in association 31. Strawn EY, Roesler M, Granlund A, Bohling S. In vitro
• Ovarian hyperstimulation syndrome (OHSS) is an iat- growth factor α and β, fibroblastic growth factor,
with the GnRH antagonist Cetrorelix. Hum Reprod. fertilization can be successfully accomplished without rogenic hCG dependent complication of ovarian stim- platelet derived growth factor, and interleukins (IL-
2001;16(4):663-7. routine estradiol monitoring: a randomized pilot study. ulation. 1β, IL-6).4-7
23. Bosch E, Labarta E, Crespo I, Simón C, Remohí J, Fertil Steril. 2007;88:S147. • Various classification systems have been developed to
• In particular, VEGF is a potent angiogenic cytokine
Jenkins J, et al. Circulating progesterone levels and ongoing 32. Hurst BS, Tucker KE, Schlaff WD. A minimally monitored categorise patients according to severity of OHSS and
pregnancy rates in controlled ovarian stimulation cycles assisted reproduction stimulation protocol reduces cost that stimulates follicular growth, corpus luteum func-
provide guidelines for their management.
for in vitro fertilization. Analysis of over 4000 cycles. Hum without compromising success. Fertil Steril. 2002;77(1):98-100. tion, and ovarian angiogenesis.
Reprod. 2010;25(8):2092-100. 33. Aboulghar M. Ovarian hyperstimulation syndrome and
• Certain patient groups are at high risk of developing
• hCG increases VEGF secretion by granulosa cells and its
24. Chu-Chun Huang, Yih-Ron Lien, Hsin-Fu Chen, Shieh CJ, ultrasound. Reprod Biomed Online. 2011;22:595. OHSS. Recognition of these risk factors and individu-
levels correlate with the severity of OHSS.8-10
Yao YL, Chang CH, et al. The duration of pre-ovulatory 34. Lass A. Monitoring of in vitro fertilization–embryo transfer alization of stimulation protocols can prevent OHSS.
• These substances can cause a fluid shift from intravas-
serum progesterone elevation before hCG administration cycles by ultrasound versus by ultrasound and hormonal • Patients with developed OHSS can be managed on out-
affects the outcome of IVF/ICSI cycles. Hum Reprod. levels: a prospective, multicentre, randomized study. Fertil cular space to the third space due to capillary hyper-
patient basis or admitted in hospital setting depending
2012;27(7):2036-45. Steril. 2003;80(1):80-5. permeability and ovarian neoangiogenesis.
on the severity of OHSS.
25. Kalyan B. Monitoring of ovulation induction. In: Kamini 35. Kwan I, Bhattacharya S, Kang A, Woolner. Monitoring of • This can result in a wide spectrum of clinical symptoms
A Rao (Ed). The Infertility Manual, 3rd edition. New stimulated cycles in fertility treatment involving in vitro
• Ovarian hyperstimulation syndrome is an iatrogenic
and laboratory signs and can also be life threatening in
Delhi: Jaypee Brothers Medical Publishers (P) Ltd; 2009. fertilisation (IVF) and intra-cytoplasmic sperm injection complication of luteal phase or/and early pregnancy
severe cases.11
pp. 340-8. (ICSI). Cochrane. 2014. after ovulation induction or ovarian stimulation.
26. Kosmas IP, Kolibianakis EM, Devroey P. Association of 36. Antonio La Marca1, Sunkara SK. Individualization of • OHSS is generally associated with exogenous gonado-
estradiol levels on the day of hCG administration and controlled ovarian stimulation in IVF using ovarian reserve tropin stimulation. Occasionally, it is seen in clomi-
EARLY AND LATE FORMS
pregnancy achievement in IVF: a system review. Hum markers: from theory to practice. Hum Reprod Update. phene induced cycles. The OHSS is an hCG dependent complication.
Reprod. 2004;19(11):2446-53. 2013;0(0):1-17.
• Rarely, it is seen in spontaneous pregnancies when
• The early form of OHSS occurs:
there are supraphysiologic concentrations of human
In response to hCG trigger
chorionic gonadotropin (hCG), e.g. in multiple
In a woman with exaggerated ovarian response to
pregnancy and molar pregnancy or when there is
ovulation induction
follicle-stimulating hormone (FSH) receptor mutation
Is seen within 7–9 days of hCG injection.12-14
increasing its sensitivity to trophoblastic hCG.1-3
• Late form of OHSS:
• It is a self-limiting disorder that generally resolves
Seen in conception cycles
spontaneously in few days but can persist longer in
hCG from the trophoblast trigger granulosa cells to
conception cycles.
produce vasoactive substances
This form occurs more than 10 days after hCG
PATHOPHYSIOLOGY trigger.13
• The syndrome essentially consists of: It is more likely to be more severe and long-lasting.14
Cystic ovarian enlargement This form can also be seen when hCG is used for
Overproduction of ovarian hormones luteal phase support.
Other vasoactive substances, e.g. vascular end- • In both forms, hCG is the underlying causative agent
othelial growth factor (VEGF), prorenin, rennin, for OHSS.
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276 The Infertility Manual Ovarian Hyperstimulation Syndrome 277
CLASSIFICATION CLINICAL SYMPTOMS AND SIGNS Contd...
Table 31.2. Proposed new clinical grading system for ovarian hyperstimulation syndrome.
• Various classification systems have been proposed to • Mild OHSS:
categorize OHSS according to severity and guidelines Lower abdominal distension Mild Moderate Severe
for management, e.g. whether the patient can be man- Heaviness and pain lower abdomen Pleural effusion ±c
aged on outpatient basis or needs hospitalization and Nausea and vomiting Subjective criteria
intensive management. The most commonly used Diarrhea.
Abdominal distention
classification system is shown in Table 31.1.15 • More severe signs and symptoms:
• A new OHSS severity classification scheme incorpo- Rapid weight gain Pelvic discomfort
rating vaginal ultrasound and laboratory parameters Tense ascites Breathing disorder ±b ±b
has been proposed by Humaidan et al.,16 and is more Hemodynamic instability (orthostatic hypoten- Acute pain ±b ±b ±b
objectively related to symptoms than previous classifi- sion, tachycardia)
Nausea/vomiting ± ± ±
cations (Table 31.2). Respiratory difficulty (tachypnea)
Ovarian enlargement
Pregnancy occurrence ± ±
Table 31.1. Commonly used classification system for ovarian hyperstimulation syndrome.
Note: The ± sign means may or may not be present.
Grade Mild Moderate Severe a
If two of these are present, consider hospitalization.
1. Abdominal distention and b
If present, consider hospitalization.
discomfort c
If present, consider intersive care.
2. Criteria of grade 1+ nausea,
Humaidan. Prevention strategies for OHSS. Fertil Steril 2010.
vomiting and/or diarrhea
Ovaries enlarged 5–12 cm
3. Criteria for mild
OHSS+ ascites on Progressive oliguria HIGH-RISK FACTORS5-7,19-28
sonography Laboratory findings:5,17
■■ Electrolyte disorders (hyponatremia, hyper- •• Young age
4. Criteria for moderate OHSS + clinical signs of ascites
and/or hydrothorax and respiratory distress kalemia) •• Low body mass index (BMI)
5. All of the above + changes in blood volume and viscos- ■■ Hypovolemia •• Polycystic ovarian syndrome (PCOS)
ity, hemoconcentration, coagulation disorders, and ■■ Hemoconcentration (hematocrit >45%) •• High serum anti-Müllerian hormone (AMH) level
decreased renal output and function ■■ Leukocytosis more than 15,000/mm3 •• Previous history of OHSS
6. Life-threatening form ■■ Creatinine clearance less than 50 mL/min;
•• GnRH agonist downregulatory protocol
serum creatinine more than 1.2 mg/dL
•• High doses of gonadotropins
■■ Elevated liver enzymes
Table 31.2. Proposed new clinical grading system for ovarian hyperstimulation syndrome. •• High absolute or rapidly increasing estradiol levels
■■ Hypercoagulability
(>18 follicles and/or E2 ≥5,000 pg/mL
Mild Moderate Severe ■■ Hypoproteinemia and hypoalbuminemia
•• Multiple stimulated follicles (ART>15, ovulation
Objective criteria (<30 g/L).
induction >6)
• Other complications:
Fluid in pouch of douglas •• High or repeated doses of hCG
Ovarian torsion
Fluid around uterus (major pelvis) •• hCG luteal phase supplementation
Ovarian rupture
Fluid around intestinal loops Thromboembolic complications. •• Pregnancy
Hematocrit >45% a
White blood cells >15,000/mm3 ±a
INCIDENCE PREVENTION
Low urine output <600 mL/24 h ±a
• The exact incidence of OHSS is not known due to lack Primary Prevention
of systematic registration.
Creatinine >1.5 mg/dL ±a ± Prevention of OHSS begins with recognition of risk factors
• Mild form of OHSS occurs in about 8–23% of cycles of
Elevated transaminases ±a ± and individualization of stimulation protocols according
ovulation induction.17,18
to the risk of OHSS.
Clotting disorder ±c • The incidence of moderate OHSS is less than 1–7%
and severe form is seen in less than 0.5% of stimulated • Reducing the exposure to gonadotropins: Whenever
Contd... cycles.15-18 possible, all other safer treatments should be tried,
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278 The Infertility Manual Ovarian Hyperstimulation Syndrome 279
e.g. lifestyle changes (diet and exercise), oral ovula- (0.5 mg) to trigger ovulation in antagonist cycles has • Calcium infusion: Intravenous infusion of 10% cal- • Inpatient treatment:
tion induction, use of pulsed GnRH, and laparoscopic been associated with significantly reduced risk of cium gluconate, 10 mL in 200 mL normal saline on Criteria for hospitalization: All patients with severe
ovarian surgery. When mandatory to use gonadotro- OHSS.36-38 However, it was associated with reduced preg- the day of oocyte pick-up and for 3 days thereafter has OHSS or deranged laboratory parameters
pins, reducing the starting dose of FSH or duration of nancy rates39 and hence, freezing of embryos and later been reported to reduce risk of OHSS.54,55 This is due Management of hospitalized patients involves:
FSH primarily reduces the number of growing follicles transfer of frozen-thawed embryos was suggested.40 to inhibition of cAMP synthesis and cAMP dependent ■■ Vitals charting (at least three times a day)
and hence, the risk of OHSS. • Coasting (soft landing): In GnRH agonist protocol, renin secretion from juxtaglomerular cells in kidneys, ■■ Daily weight measurement
• GnRH antagonist protocol: Recent Cochrane review omission of gonadotropins and continued administra- which then reduces angiotensin II production and ■■ Intake/output record
puts GnRH antagonist protocol ahead of agonist tion of GnRH agonist can be used to prevent or reduce hence, reduced VEGF levels.56,57 However, more data is ■■ Daily record of abdominal circumference
downregulatory protocol in terms of safety as the risk the severity of OHSS. needed to validate these results. ■■ Complete physical examination (bimanual
of OHSS is reduced by more than 50%.29 Withdrawal of FSH causes the larger follicles to continue • Dopamine agonists: Cabergoline (dopamine D2 recep- examination of ovaries is avoided due to risk of
• Avoidance of hCG for luteal phase support: Luteal growth while the intermediate and smaller follicles tor agonist) inactivates VEGF receptor 2 and hence, ovarian rupture)
phase support with progesterone is the best option for undergo atresia. This alters the capacity of granulosa reduces vascular permeability.58-60 Starting cabergoline ■■ Ultrasound examination (ascites, ovarian size),
prevention of OHSS. cells to produce VEGF.41 Significant reduction in OHSS 0.5 mg/day from the day of hCG and continuing for repeated as necessary
with coasting was reported when it was continued until 6 days post ovum pick-up has been shown to reduce ■■ Chest X-ray and echocardiography (when pleu-
Prevention in Polycystic Ovarian serum estradiol levels fell below 3,000 pg/mL.42 the incidence of moderate and severe OHSS.61-64 ral or pericardial effusion is suspected)
Syndrome Patients However, some studies have found no benefit in coast- • Continued administration of GnRH agonists following ■■ Daily complete blood count (CBC), electrolytes
ing.43 Other studies have shown diminished oocyte col- oocyte pick-up minimizes the risk of developing OHSS. ■■ Serum creatinine, liver enzymes, repeated as
• In vitro maturation (IVM): Use of IVM in PCOS patients
lection rate, implantation, and pregnancy rates44 when This is accompanied either with cryopreservation of necessary
may drastically reduce the risk of OHSS as the total
coasting is prolonged, particularly more than 3 days.45 embryos, or administration of luteal estradiol and pro- ■■ Pulse oximetry (for patients with breathing dif-
exposure to gonadotropins is drastically reduced.30,31 ficulty)
• Blastocyst culture: In cases with risk to develop OHSS, gesterone support following embryo transfer.65,66
• Metformin: Few studies have shown that addition of
extended culture to blastocyst stage and re-evaluation • Corticosteroids: There is insufficient evidence of ben- • Guidelines for fluid management:53,58,69
metformin in PCOS patients reduces the incidence
of patient before embryo transfer can guide to prevent eficial effect of anti-inflammatory activity of steroids in Renal and pulmonary function must be monitored
of OHSS significantly.32,33 However, larger studies are
severe OHSS.18 In at-risk cases, embryos can be frozen the prevention of OHSS.67,68 carefully.
needed to prove the same.
and transferred in a later cycle. Strict monitoring of fluid input and urine output is
• Low dose gonadotropins: Chronic low dose regimen
for ovulation induction in PCOS patients results in
• Single embryo transfer: This prevents multiple preg- TREATMENT OF ESTABLISHED OVARIAN required.
nancy, which by itself can result in severe OHSS.46,47 Oral fluid intake should be limited to amount nec-
mono or bifollicular response and hence, the risk of HYPERSTIMULATION SYNDROME69 essary for patient’s comfort (1 L/day).
• Cryopreservation of embryos: This can be helpful in
OHSS is significantly reduced.
guarding against severe late-onset OHSS,48 although • Patients with mild OHSS can be managed on outpa- Intravenous (IV) fluid administration should be
• Mild stimulation regimens in IVF: By reducing the
this cannot prevent early onset OHSS.49 tient basis which involves: titrated carefully, in the volume required for ade-
exposure to gonadotropins, the risk of OHSS is consid- quate urine output (>20–30 mL/hour).
• The removal of granulosa cells from follicles during Oral analgesics
erably reduced Ringer lactate solution is avoided for the tendency
ovum pick-up may also help in reducing the severity High protein diet
• Antagonist protocol for hyponatremia (5% dextrose is preferable).
of OHSS. However, present evidence is insufficient to Daily weight measurement
• GnRH agonist trigger IV fluid administration should be reduced and oral
consider this as a standard mode of treatment.49 Oral fluid intake of at least 1 L/day
• Cryopreservation of embryos and later frozen embryo fluid intake encouraged when symptoms appear
Avoidance of strenuous physical activity and inter-
transfer. improving or at the onset of brisk diuresis.
Adjuvants for Ovarian Hyperstimulation course. However, strict bed rest is not recommended.
Daily record of frequency and/or volume of urine Diuretics, e.g. furosemide 20 mg IV can be given
Secondary Prevention Syndrome Prevention output after adequate intravascular volume restoration
These involve the withdrawal, delay, or modification of • Colloid agents: Since OHSS is associated with increased Counseling regarding the signs and symptoms of Plasma expander, e.g. 25% albumin (50–100 g) can
elements of the stimulation protocol with the aim of avert- be given when infusion of IV fluids fail to maintain
capillary permeability, intravascular colloidal agents, progressing illness.
ing OHSS in patients who have progressed to high risk dur- intravascular volume and adequate urine output
e.g. albumin and hydoxyethylstarch (HES) at the time • Treatment of worsening OHSS can still be monitored
ing the treatment.16
of ovum pick-up can increase intravascular oncotic on outpatient basis, but with more careful evaluation. (repeated every 4–12 hours).
• Cycle cancellation: This decision may result in consid- pressure and reduce the risk of developing OHSS.50-52 Antiemetics and more potent analgesics Electrolyte imbalance must be corrected appropri-
erable financial loss and psychological disturbance. Recent Cochrane analysis53 mentioned a marked More frequent physical and ultrasound measure- ately. Hyperkalemia is associated with cardiac arrhyth-
• Low dose hCG: Since the occurrence of OHSS is utterly decrease in the incidence of severe OHSS in high risk ments mias and needs immediate correction with insulin and
hCG dependent, triggering ovulation with lower dose patients with the infusion of HES but the evidence Serial laboratory testing for hematocrit, electro- glucose (drives potassium ions into the cells) or cal-
hCG (between 3,300 and 5,000 IU) is proposed to is limited for albumin. Moreover, albumin infusion lytes, and serum creatinine. cium (protects heart from elevated potassium levels).
decrease the risk of OHSS.34,35 carries risk of allergic reactions and transmission of Such patients are instructed to stay in touch with • Indications of paracentesis:70,71
• GnRH agonist trigger: Use of single subcutane- viruses and prions. their doctor and have easy access to the hospital, Persistent oliguria/anuria after adequate volume
ous injection of triptorelin (0.2 mg) or leuprolide • Metformin: as discussed earlier. whenever required. replacement
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280 The Infertility Manual Ovarian Hyperstimulation Syndrome 281
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3. Montanelli L, Delbaere A, Di Carlo C, Nappi C, Smits and treatment. Fertil Steril. 1992;58:249-61. Steril. 1999;72:282-5.
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G, Vassart G, et al. A mutation in the follicle-stimulating 19. Blankstein J, Shalev J, Saadon T, Kukia EE, Rabinovici J, 33. Khattab S, Fotouh IA, Mohesn IA, Metwally M, Moaz M. Use
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Paracentesis is generally effective in resolving
2000;74:429-38. ovarian hyperstimulation syndrome. Am J Obstet Gynecol. Reprod Biomed Online. 2003;7:43-9.
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• Thromboprophylaxis: 6. Warren RS, Yuan H, Matli MR, Ferrara N, Donner DB. Dhont M, et al. The ovarian hyperstimulation syndrome syndrome. Fertil Steril. 2006;86 (Suppl 2);S182-3; (P-138).
Thromboprophylaxis should be provided for all Induction of vascular endothelial growth factor by insulin- in in-vitro fertilization: a Belgian multicentric study. I. 36. Emperarie J. Triggering ovulation with endogenous
like growth factor I in colorectal carcinoma. J Biol Chem. Clinical and biological features. Hum Reprod. 1993;8: luteinizing hormone may prevent the ovarian
women admitted to hospital with OHSS, those with
1996;271:483-8. 1353-60. hyperstimulation syndrome. Hum Reprod. 1991;6:506-10.
history of thromboembolic disease, clinical signs 7. Ferrara N, Davis-Smyth T. The biology of vascular 22. MacDougall MJ, Tan SL, Jacobs HS. In-vitro fertilization 37. Imoedemhe D, Chan R, Sigue A, Pacpaco E. A new
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SUMMARY HA, et al. Human chorionic gonadotropin-dependent Severe ovarian hyperstimulation syndrome using agonists in high-risk patients undergoing in vitro fertilization
expression of vascular endothelial growth factor/vascular of gonadotropin-releasing hormone for in vitro fertilization: prevents the risk of ovarian hyperstimulation syndrome:
Knowledge of OHSS pathophysiology, various risk factors, permeability factor in human granulosa cells: importance a European series and a proposal for prevention. Fertil a prospective randomized controlled study. Fertil Steril.
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ovulation induction therapy can be helpful in preventing Metab. 1995;80:1967-71. 25. Mizunuma H, Andoh K, Yamada K, Takagi T, Kamijo T, Ibuki 39. Griesinger G, Dietrich K, Devroey P, Kolibianakis EM.
and managing OHSS. 10. Pellicer A, Albert C, Mercader A, Bonilla-Musoles F, Remohí Y. Prediction and prevention of ovarian hyperstimulation GnRH agonist for triggering final oocyte maturation in
Serious illness resulting from OHSS is much less com- J, Simón C. The pathogenesis of ovarian hyperstimulation by monitoring endogenous luteinizing hormone release the GnRH antagonist ovarian hyperstimulation protocol: a
syndrome: in vivo studies investigating the role of during purified follicle-stimulating hormone therapy. Fertil systematic review and meta-analysis. Hum Reprod Update.
mon, but can be life-threatening. interleukin-1β, interleukin-6, and vascular endothelial Steril. 1992;58:46-50. 2006;12:159-68.
OHSS is a preventable condition, and implementation growth factor. Fertil Steril. 1999;71:482-9. 26. Mordel N, Schenker JG. Gonadotrophin-releasing hormone 40. Griesinger G, von Otte S, Schroer A, Ludwig AK, Diedrich
of evidence based prevention strategies should enable us 11. Gerris J, De Sutter P. Ovarian hyperstimulation syndrome. agonist and ovarian hyperstimulation syndrome in assisted K. Elective cryopreservation of all pronuclear oocytes
to reduce its occurrence significantly. Ind J Obstet Gynecol. 2006;59(1):30-6. reproduction. Hum Reprod. 1993;8:2009-14. after GnRH agonist triggering of final oocyte maturation
12. Dahl Lyons CA, Wheeler CA, Frishman GA, Hackett RJ, 27. Haning RV Jr, Austin CW, Carlson IH, Kuzma DL, Shapiro in patients at risk of developing ovarian hyperstimulation
Seifer DB, Haning RV Jr, et al. Early and late presentation SS, Zweibel WJ. Plasma estradiol is superior to ultrasound syndrome: a prospective, observational proof-of-concept
PROBABLE QUESTIONS of the ovarian hyperstimulation syndrome: two distinct and urinary estriol glucuronide as a predictor of ovarian study. Hum Reprod. 2007;22:1348-52.
entities with different risk factors. Hum Reprod. 1994;9: hyperstimulation during induction of ovulation with 41. Tozer AJ, Iles RK, Iammarrone E. The effects of ‘coasting’
1. What are the different forms of OHSS? 792-9. menotropins. Fertil Steril. 1983;40:31-6. on follicular fluid concentrations of vascular endothelial
2. What are the predisposing factors for OHSS? 13. Mathur RS, Akande VA, Keay SD, Hunt LP, Jenkins NM. 28. Enskog A, Henriksson M, Unander M, Nilsson L, growth factor in women at risk for developing ovarian
3. What is the incidence of mild, moderate and severe Distinction between early and late ovarian hyperstimulation Brannstrom M. Prospective study of the clinical and hyperstimulation syndrome. Hum Reprod. 2004;19:522-8.
OHSS after ovarian stimulation? syndrome. Fertil Steril. 2000;73:901-7. laboratory parameters of patients in whom ovarian 42. Levinson-Tavor O, Friedler S, Schachter M, Raziel M, Raziel
14. Papanikolaou EG, Tournaye H, Verpoest W, Camus M, hyperstimulation syndrome developed during controlled A, Strassburger D, et al. Coasting-what is the best formula?
4. How can we prevent OHSS in PCOS patients?
Vernaeve V. Early and late ovarian hyperstimulation ovarian hyperstimulation for in vitro fertilization. Fertil Hum Reprod. 2003;18:937-40.
5. What is the role of fluid management in patients with syndrome: early pregnancy outcome and profile. Hum Steril. 1999;71:808-14. 43. D’Angelo A, Brown J, Amso NN. Coasting (withholding
OHSS? Reprod. 2005;20:636-41. 29. Al-Inany HG, Youssef MA, Aboulghar M, Broekmans F, gonadotrophins) for preventing ovarian hyperstimulation
15. Golan A, Ron-El R, Herman A, Soffer Y, Weinraub Z Sterrenburg M, Smit J, et al. Gonadotrophin-releasing syndrome. Cochrane Database Syst Rev. 2011;6:CD002811.
REFERENCES enCaspiE. Ovarian hyperstimulation syndrome: an update hormone antagonists for assisted reproductive technology. 44. Isaza V, Garcia-Velasco JA, Aragonas M, Remohi J, Simon C.
review. Obstet Gynecol Surv. 1989;44:430-40. Cochrane Database Syst Rev. 2011;5:CD001750. Oocyte and embryo quality after coasting: the experience
1. Smits G, Olatunbosun O, Delbaere A, Pierson R, Vassart G, 16. Humaidan P, Quartarolo J, Papanikolaou EG. Preventing 30. Child TJ, Phillips SJ, Abdul-Jalil AK, Gulekli B, Tan SL. A from oocyte donation. Hum Reprod. 2002;17:1777-82.
Costagliola S. Ovarian hyperstimulation syndrome due to ovarian hyperstimulation syndrome: guidance for the comparison of in vitro maturation and in vitro fertilization 45. Ulug U, Bahceci M, Erden HF, Shalav E, Ben-Shlomo I.
a mutation in the follicle-stimulating hormone receptor. clinician. Fertil Steril. 2010;94:389-400. for women with polycystic ovaries. Obstet Gynecol. The significance of coasting duration during ovarian
N Engl J Med. 2003;349:760-6. 17. Brinsden PR, Wada I, Tan SL, Barlen A en Jacobs HS. 2002;100:665-70. stimulation for conception in assisted fertilization cycles.
2. Vasseur C, Rodien P, Beau I, Desroches A, Gérard C, de Diagnosis, prevention and management of ovarian 31. Tan SL, Child TJ. In vitro maturation of oocytes from Hum Reprod. 2002;17:310-13.
Poncheville L, et al. A chorionic gonadotropin-sensitive hyperstimulation syndrome. Br J Obstet Gynaecol. unstimulated polycystic ovaries. Reprod Biomed Online. 46. Kinget K, Nijs M, Cox AM. A novel approach for patients
mutation in the follicle-stimulating hormone receptor 1995;102:767-72. 2002;4(Suppl 1):18-23. at risk for ovarian hyperstimulation syndrome: elective
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282 The Infertility Manual
transfer of a single zona-free blastocyst on day 5. Reprod altering VEGF receptor 2-dependent luteal angiogenesis
Biomed Online. 2002;4:51-5. in a rat ovarian hyperstimulation model. Endocrinol.
47. Trout SW, Bohrer MK, Deifer DB. Single blastocyst transfer
in women at risk of ovarian hyperstimulation syndrome. 61.
2006;147:5400-11.
Alvarez C, Marti- Bonmati L, Novella-Maestre E, Sanz Ovulatory Triggers
Fertil Steril. 2001;76:1066-7. R, Gómez R. Dopamine agonist cabergoline reduces
48. Garrissi G, Navot D. Cryopreservation of semen, oocytes, hemoconcentration and ascites in hyperstimulated women
Vinay Kumar, Ratnakar Majalekar
and embryos. Curr Opin Obstet Gynecol. 1992;4:726-31. undergoing assisted reproduction. J Clin Endocrinol
49. D’ Angelo A, Amso NN. Embryo freezing for preventing Metab. 2007;92:2931-7.
ovarian hyperstimulation syndrome: a Cochrane review. 62. Spitzer D, Wogatzky J, Murtinger M, Zech MH, Haidbauer Chapter Outline
Hum Reprod. 2002;17:2787-94. R, Zech NH. Dopamine agonist bromocriptine for the • Definition • Dual Trigger
50. Shalev E, Giladi Y, Matilsky M, Ben-Ami M. Decreased prevention of ovarian hyperstimulation syndrome. Fertil • Physiology of Oocyte Maturation and Release
incidence of severe ovarian hyperstimulation syndrome in Steril. 2011;95:2742-4.
high risk in-vitro fertilization patients receiving intravenous 63. Soares S, Gomez R, Simon C, Garcia-Velasco J, Pellicer A.
albumin: a prospective study. Hum Reprod. 1995;10: Targeting the endothelial growth factor system to prevent INTRODUCTION • LH causes luteinization of granulosa cells and produc-
1373-6. ovarian hyperstimulation syndrome. Hum Reprod Update. tion of progesterone.
51. Isik AZ, Gokmen O, Zeyneloglu HB, Kara S, Guleki B. 2008;14:321-33. Until recently human chorionic gonadotropin (hCG) was
• Progesterone causes preovulatory FSH Surge by posi-
Intravenous albumin prevents moderate-severe ovarian 64. Youssef M, van Wely M, Hassan MA, Al-Inany HG, Mochtar the most common therapeutic option to induce oocyte
hyperstimulation in in-vitro fertilization patients: a M, Khattab S, et al. Can dopamine agonists reduce the tive feedback mechanism from pituitary.
maturation in ART cycles. Due to its long half-life and
prospective, randomized and controlled study. Eur J Obstet incidence and severity of OHSS in IVF/ICSI treatment higher affinity for luteinizing hormone (LH) receptors, it is • FSH surge is thought to help in cumulus expansion,
Gynecol Reprod Biol. 1996;70:179-83. cycles? A systematic review and meta-analysis. Hum promotion of nuclear maturation, and induction of LH
notorious in causing ovarian hyperstimulation syndrome
52. Gokmen O, Ugur M, Ekin M, Keles G, Turan C, Oral H. Reprod Update. 2010;16:459-66.
(OHSS). The upsurge of antagonist protocol opened up the receptors on granulosa and cumulus cells.3,4
Intravenous albumin versus hydroxyethyl starch for the 65. Endo T, Honnma H, Hayashi T, Chida M, Yamazaki
prevention of ovarian hyperstimulation in an in-vitro K, Kitajima Y, et al. Continuation of GnRH agonist option of using gonadotropin-releasing hormone agonist • LH, FSH, and progesterone will open a cascade of
fertilization programme: a prospective randomized administration for one week, after HCG injection, prevents (GnRHa) trigger to induce oocyte maturation and almost events resulting in nuclear maturation (resumption
placebo controlled study. Eur J Obstet Gynecol Reprod ovarian hyperstimulation syndrome following elective eliminating the risk of OHSS owing to very short endog- of meiosis), cytoplasmic maturation, and release of a
Biol. 2001;96:187-92. cryopreservation of all pronucleate embryos. Hum Reprod. enous surge induced. Recombinant LH (r-LH) appears a developmentally competent oocyte from follicle.
53. Youssef MA, Al-Inany HG, Evers JL, Aboulghar M. 2002;17:2548-51.
simple and straightforward option, but has its own limita-
Intravenous fluids for the prevention of severe ovarian 66. Rjosk HK, Abendstein BJ, Kreuzer E, Schwartzler P.
tions. Options of using Kisspeptins (KPs) and combining
hyperstimulation syndrome. Cochrane Database Syst Rev. Preliminary experience with steroidal ovarian suppression Resumption of Meiosis
2011;1-37. for prevention of severe ovarian hyperstimulation syndrome small dose of human chorionic gonadotropin (hCG) with
54. Yakovenko SA, Sivozhelezov VS, Zorina IV, Dmitrieva NV, in IVF patients. Hum Fertil. 2001;4:246-8. GnRHa in certain subgroup of patients has also being stud- • Oocyte arrest in prophase I of meiosis is maintained by
Apryshko VP. Prevention of OHSS by intravenous calcium. 67. Lainas T, Petsas G, Stavropoulou G, Alexopoulou E, Iliadis ied. This chapter will focus on various options available for high concentrations of cyclic adenosine monophos-
Hum Reprod. 2009;24 (Suppl 1):i61. G, Minaretzis D. Administration of methylprednisolone triggering final oocyte maturation in assisted reproductive phate (c-AMP) in the oocyte which is transported
55. Gurgan T, Demirol A, Guven S, Benkhalifa M, Girgin B. to prevent severe ovarian hyperstimulation syndrome
technique (ART) cycles. inside oocyte from cumulus/somatic cells via gap-
Intravenous calcium infusion as a novel preventive therapy in patients undergoing in vitro fertilization. Fertil Steril.
of ovarian hyperstimulation syndrome for patients with 2002;78:529-33. junctions.5
polycystic ovarian syndrome. Fertil Steril. 2011;96:53-7. 68. Tan SL, Balen A, el Hussein E, Campbell S, Jacobs HS. The DEFINITION • LH and FSH surge results in cumulus expansion
56. Ortiz-Capisano MC, Ortiz PA, Harding P, Garvin JL, administration of glucocorticoids for the prevention of
A bioactive preparation which will trigger a cascade of and disruption of these gap-junctions, resulting in
Beierwaltes WH. Decreased intracellular calcium ovarian hyperstimulation syndrome in in vitro fertilization:
stimulates rennin release via calcium-inhibitable adenylyl a prospective randomized study. Fertil Steril. 1992;58: events resulting in the maturation and release of develop- decrease in c-AMP concentrations in the oocyte. This
cyclase. Hypertension. 2007;49:162-9. 378-83. mentally competent oocyte from a preovulatory Graafian results in the germinal vessel breakdown and resump-
57. Beierwaltes WH. The role of calcium in the regulation of 69. Borenstein R, Elhalah U, Lunenfeld B, Schwartz ZS. follicle. The physiological trigger in a natural cycle is the tion of meiosis (Fig. 32.1).
rennin secretion. Am J Physiol Renal Physiol. 2010;298: Severe ovarian hyperstimulation syndrome: a reevaluated
F1-11. therapeutic approach. Fertil Steril. 1989;51:791-5.
midcycle surge of gonadotropins, i.e. FSH and LH surge
58. Busso C. Symposium: update on prediction and 70. Abramov Y, Elchalal U, Schenker JG. Pulmonary from the pituitary gland. Follicular Rupture
management of OHSS. Prevention of OHSS-dopamine manifestations of severe ovarian hyperstimulation
Various precise, complex, inter-related, and overlapping
agonists. Reprod Biomed Online. 2009;19:43-51.
59. Garcia-Velasco J. How to avoid ovarian hyperstimulation 71.
syndrome: a multicenter study. Fertil Steril. 1999;71:645-51.
ASRM Educational Bulletin. Ovarian hyperstimulation
PHYSIOLOGY OF OOCYTE MATURATION molecular events like changes in the cumulus and matrix,
syndrome: a new indication for dopamine agonists. Reprod syndrome. Fertil Steril. 2008;90:S188-93. AND RELEASE appearance of more inflammatory cells in the follicular
Biomed Online. 2009;19:71-5. 72. Rinaldi ML, Spirtos NJ. Chest tube drainage of pleural wall, prostaglandin production by cumulus cells, follicular
60. Gomez R, Gonzalez-Izquierdo M, Zimmermann effusion correcting abdominal ascites in a patient with • Positive feedback by sustained high estradiol levels
vascular and membrane remodeling, proteolytic digestion
RC, Novella-Maestre E. Low-dose dopamine agonist severe ovarian hyperstimulation syndrome: a case report (>300 pg/mL for >48 hours) in late follicular phase will of the tunica and basement membrane, apoptosis of the
administration blocks vascular endothelial growth factor [comment. Fertil Steril. 1995;64:1228-9.]. Fertil Steril. result in LH surge from pituitary.1
(VEGF)-mediated vascular hyperpermeability without 1995;63:1114-7. epithelial cells over the apex/stigma, and rise in intrafol-
• Endogenous LH surge has three phases, lasting over a licular pressure and contractions of the myoepithelial cells
period of 48 hours: ascending phase of 14 hours; plateau will result in the rupture of the follicle with extrusion of the
phase of 14 hours; and descending phase of 20 hours.2 mature oocyte6 (Fig. 32.2).
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284 The Infertility Manual Ovulatory Triggers 285
• Recombinant luteinizing hormone (r-LH) Table 32.1. Comparison between rLH and hCG.
• Gonadotropin releasing hormone agonists
Property rLH hCG
• Dual trigger (hCG + agonist)
Half-life Shorter (23 Longer (32–33
• Kisspeptins. hour S/C) hour S/C)
Potency Less potent More potent
Human Chorionic Gonadotropins Equivalency 6–8 IU of LH 1 IU of hCG
Chorionic gonadotropins (CG) are heterodimeric gly- Embryo quality difference Not proven Not proven
coproteins composed of α- and β-subunits. Along with
LH, FSH, and thyroid stimulating hormone (TSH), they
have exactly same α-subunit.8 The unique functions and Table 32.2. Various GnRH analouges.
receptor-binding capacity of each of these hormones stem Decapeptides Nanopeptides
from differences among their β-subunits.
Native GnRH Leuprolide
The amino acid sequences of human LH (hLH) and
Fig. 32.1: Resumption of meiosis. Nafarelin Buserelin
hCG share 82% homology.8
Due to their similarity, the hCG bind to the same LH Fig. 32.3: Difference in duration of LH activity between endogenous Triptorelin Goserelin
receptors and is proven to be an active surrogate for LH. surge and hCG. Histrelin
A mature oocyte is released about 36 hours posto- For long, the hCG has been playing a lead role in ovulation
vulation trigger and may allow to perfectly time natural trigger in infertility management.
intercourse or intrauterine insemination (IUI). The same • Urinary products still remain first choice as it is cheap Gonadotropin Releasing Hormone Agonist
property is used in in vitro fertilization (IVF) cycles where • As a result of structural differences and post-
and easily available. A recent meta-analysis has found
we retrieve mature oocytes artificially around 34–36 hours translational modifications, hCG becomes more sta- The initial “flare effect” following GnRH agonist admin-
no difference in terms of follicular maturation, risk of
after administration of ovulation trigger. Administering ble, longer acting, and greater receptor affinity than istration mimics natural midcycle FSH and LH surge,
OHSS, and pregnancy outcomes between two prepa-
LH, thus making it more biologically active.9 though the duration varies. This property can be utilized
trigger in cases of anovulation that undergo ovulation rations.11
• After subcutaneous (S/C) administration, the hCG to achieve oocyte maturation. The capability of GnRH ago-
induction with oral ovulogens remains doubtful.7 How- • 250 µg r-hCG = 5,000 IU u-hCG12
has significantly longer half-life in comparison to LH10 nist to induce ovulation was first described by Nakano et
ever, in case of IUI, ovulation trigger appears beneficial in al. in 1973.14 Owing to the dominance of the long agonist
timing the IUI and in IVF it is a must as endogenous surge (Fig. 32.3).
• Due to above mentioned pharmacokinetic properties, Recombinant Luteinizing Hormone (rLH) protocol over the years in ART, the agonist trigger did not
is deliberately suppressed by GnRH analogues. receive much attention. With the refinement of antago-
hCG is notorious in producing OHSS. Owing to the disadvantage of carrying high risk of OHSS, nists from first to third generation, the antagonist protocol
• Recombinant preparations are associated with higher rLH was looked at with a great hope, as its half-life is
Various Pharmacological Options serum hCG and progesterone concentrations and shorter with hCG and looks more physiological. In a pro-
has gained a very fast popularity in recent years. Antago-
nist protocol per se is associated with low risk of OHSS but
• Urinary human chorionic gonadotropin (u-hCG) lower incidence of local adverse reactions after admin- spective study, The European Recombinant LH study they provide an additional option to have an agonist trig-
• Recombinant human chorionic gonadotropin (r-hCG) istration. Group found that a single dose of rhLH of at least 15,000 IU ger which almost eliminates the risk of OHSS.
(750 mg rhLH) or 30,000 IU (1,500 mg rhLH) equivalent
to that of 5,000 IU u-hCG with better safety profile than • GnRH agonist is derived from native GnRH by substi-
u-hCG, in terms of incidence of OHSS. Serono Interna- tution of amino acids at 6th and 10th positions. These
tional, however, did not pursue this study further, and modifications result in alteration of enzymatic cleav-
therefore recombinant LH is commercially unavailable as age and potency of the molecule.
a trigger.13 • Native GnRH is a decapeptide (Table 32.2).
• Though hLH looks promising, the research for the • Dose of agonists as trigger: triptorelin 0.2 mg, leupro-
same as trigger in literature is limited. lide 1–1.5 mg, and buserelin 0.5 mg.
• Presently, hLH is not routinely used as ovulation • A short lasting surge is induced by GnRH agonist and
trigger. is characterized of two phases: a very small ascending
• Dose of hLH required to trigger ovulation is way too phase (approximately 4 hours) and a long descending
high as is not cost effective. phase (approximately 20 hours) in a total of 24–36 hours
• A recent Cochrane analyzed three RCTs comparing as compared to 48 hours in a natural cycle (Fig. 32.4).15
hLH with u-hCG. There was no evidence of a difference • GnRHa as an ovulation trigger is as effective as hCG
between rLH and u-hCG in rates of live birth/ongo- and also has an additional theoretical advantage of
ing pregnancy or OHSS (very low quality evidence) inducing FSH surge which along with LH may bring up
Fig. 32.2: Various events concerting in ovulation. (Table 32.1).11 better follicular maturation.
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286 The Infertility Manual Ovulatory Triggers 287
DUAL TRIGGER successfully induced ovulation as well as live births. 6. hCG is notorious to cause OHSS as compared to LH
Such molecules are likely to be of potential use in due to:
Dual trigger involves inducing oocyte maturation by two
humans. a. Longer half-life
different pharmacological agents, i.e. GnRH agonist and
• Exogenous KPs administered during preovulatory b. Higher affinity
low dose hCG (1,000–2,500 IU). It was first described by
Shapiro.21 phase has been shown to increase LH secretion by c. None of the above
three- to fourfold in humans.26 d. Both of the above
• This method gives the advantage in being able to pro- • In a recent break through study in women undergo- 7. Which of the following is trigger of choice in patient at
long the time between ovulation triggering and oocyte ing ART, KPs successfully elicited an effective LH surge risk of OHSS on long agonist protocol of stimulation?
retrieval by giving GnRHa at 40 hours before retrieval that resulted in oocyte maturation and live birth.27 a. u-hCG
and hCG 34 hours before retrieval. Also the simultane- • KPs, therefore, may emerge a new, “natural” option for b. GnRH agonist
ous induction of an FSH surge may help in granulosa induction of ovulation and trigger in ART without the c. rec-LH
cell function, oocyte nuclear maturation, and cumulus risk of OHSS. d. rec-hCG
expansion.22 • The molecule is also under research for the treatment 8. Which of the following is false?
• When compared with hCG trigger, it has been shown for hypothalamic amenorrhea and prostatic cancer.28 a. 1 IU of hCG ≈ 6–8 IU of LH
that there was a significantly higher levels of LH imme- • More research is needed to understand the dose, phar- b. 250 µg r-hCG ≈ 5,000 IU of u-hCG
Fig. 32.4: Difference between duration of endogenous LH surge and
GnRHa induced LH surge. diately after trigger in the dual trigger group along with macokinetics, pharmacodynamics, and effectiveness c. 5,000 IU r-LH ≈ 5,000 IU of u- hCG
FSH surge. No significant difference was found in the of the drug as ovulation trigger before they can be reg- d. 30,000 IU r-LH ≈ 5,000 IU of u-hCG
number of meiosis II (MII) oocytes, or the number of ulated into clinical practice. So far, promising results 9. Kisspeptins act on which level of HPO axis to cause
2PN oocytes.23 have been obtained from human studies. endogenous LH surge?
• No difference in oocyte and embryo quality when
• The OHSS risk was significantly minimized because of a. Hypothalamus
compared to hCG with almost elimination of OHSS.16
GnRH agonist and luteal function was rescued by the b. Pituitary
• After triggering with GnRH agonist, lack of sustained
added hCG, when dual trigger was used. PROBABLE QUESTIONS
endogenous LH in luteal phase results in early corpus c. Direct action on the ovary
• Dual trigger has shown good improvements in patients d. None of the above
luteolysis and inadequate steroidogenesis culminat- 1. Endogenous LH surge happens in response to:
with immature oocyte syndrome and empty follicle 10. Decreased pregnancy rates after GnRH agonist trig-
ing in decreased pregnancy rates and miscarriage a. Positive feedback of estrogen
syndrome in the previous cycles.22 ger are due to:
rates.17 b. Negative feedback of estrogen
• Dual trigger may improve implantation, clinical a. Short duration of endogenous LH surge
• Initial fears of high incidence of empty follicle syn- c. Positive feedback of progesterone
pregnancy, and live birth rates when used in normal b. Poor oocyte yield and oocyte maturity
drome with agonist trigger owing to its short LH surge d. Negative feedback of progesterone
responder patients in an antagonist cycle.24 c. Severe luteal phase defect
have been nullified by recent studies.18 2. LH surge results in all of the following, except:
• After agonist trigger, intense luteal phase support d. Poor embryo quality
a. Luteinization of granulosa cells
either with progesterone and estradiol or low dose Kisspeptins b. Cumulus expansion
hCG have shown decent pregnancy rates after fresh c. Completion of first meiotic division
transfer, in patients at risk of OHSS.
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• Optimal Dose of Progesterone Support
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• How Long to Give Luteal Phase Support? • Role of Heparin In Luteal Phase Support
research. Mol Hum Reprod. 2009;15:703-11. when a gonadotropin-releasing hormone agonist is used • Is There a Role of Adding Estrogen to Luteal Phase? • Luteal Support in Donor Cycles and Frozen Thaw Cycles
10. Ezcurra, Humaidan. A review of luteinizing hormone and instead of human chorionic gonadotropin for ovulation • Role of Aspirin for Luteal Phase Support
human chorionic gonadotropin when used in assisted triggering: a randomized prospective proof of concept
reproductive technology. Reproductive Biology and study. Fertil Steril. 2011;95:1174.
Endocrinology. 2014;12:95. 21. Shapiro BS, Babayof R, Margalioth EJ, Huleihel M.
11. Youssef MA, Abou-Setta AM, Lam WS. Recombinant versus Gonadotropin-releasing hormone agonist combined with INTRODUCTION
urinary human chorionic gonadotrophin for final oocyte a reduced dose of human chorionic gonadotropin for final The luteal phase is defined as the period from occurrence
maturation triggering in IVF and ICSI cycles. Cochrane oocyte maturation in fresh autologous cycles of in vitro of ovulation until the establishment of a pregnancy or the
Database Syst Rev. 2016;4. fertilization. Fertil Steril. 2008;90(1).
resumption of menses 2 weeks later. In assisted reproduc-
12. The European Recombinant Human Chorionic 22. Haas J, Zilberberg E, Diedrich K. Co-administration of
tion techniques (ART) cycles, luteal phase support (LPS)
Gonadotrophin Study Group. Induction of final follicular GnRH-agonist and hCG for final oocyte maturation (double
maturation and early luteinization in women undergoing trigger) in patients with low number of oocytes retrieved is required in the form of administration of medications
ovulation induction for assisted reproduction treatment– per number of preovulatory follicles-a preliminary report. as there is inadequate luteal support for the implantation
recombinant HCG versus urinary HCG. Hum Reprod. Journal of Ovarian Research. 2014. to happen. Progesterone and estrogen are required to pre-
2000;15(7):1446-51. 23. Decleer W, Osmamagaoglu K, Seynhave B. Comparison of pare the uterus for embryo implantation and to modulate
13. European Recombinant LH Study Group. Human HCG triggering versus hCG in combination with a GnRH the endometrium during the early stages of pregnancy.
recombinant luteinizing hormone is as effective as, but agonist: a prospective randomized controlled trial. Facts The aim of luteal support in ART cycles is to mimic the
safer than, urinary human chorionic gonadotropin in Views Vis Obgyn. 2014;6(4):203-9. natural cycle as much as possible. In natural cycles, cor-
inducing final follicular maturation and ovulation in in vitro 24. Ming-Huei Lin, Wu FS, Lee RK, Li SH, Lin SY, Hwu YM. pus luteum provides LPS and it requires constant stimu-
fertilization procedures: results of a multicenter double- Dual trigger with combination of gonadotropin-releasing lation by luteinization hormone (LH) to secrete human
blind study. J Clin Endocrinol Metab. 2001;86:2607-18. hormone agonist and human chorionic gonadotropin
chorionic gonadotropin (hCG).1 In ART, pituitary suppres-
14. Nakano R, Mizuno T, Kotsuji F, Katayama K, Wshio M, significantly improves the live-birth rate for normal
sion with use of GnRH agonists and antagonists, deprives
Tojo S. ‘Triggering’ of ovulation after infusion of synthetic responders in GnRH-antagonist cycles. Fertil Steril.
luteinizing hormone releasing factor (LRF). Acta Obstetricia 2013;100(5). corpus luteum of this LH drive.2 It is well established that
et Gynecologica Scandinavica. 1973;52(3):269-72. 25. Abbara. Twice-weekly administration of kisspeptin-54 luteal function is compromised in ART cycles leading to
15. Itskovitz J., Boldes R., Levron J., Erlik Y., Kahana L., Brandes for 8 weeks stimulates release of reproductive hormones iatrogenic luteal phase deficiency (LPD). There are many
J.M. Induction of preovulatory luteinizing hormone surge in women with hypothalamic amenorrhea. Clinical reasons for luteal phase abnormalities (Fig. 33.1).1-3
and prevention of ovarian hyperstimulation syndrome by Pharmacology and Therapeutics. 2010;88(6):840-7.
gonadotropin-releasing hormone agonist. Fertility and 26. Jayasena CN, Nijher GMK, Comninos AN, Abbara A, VARIOUS AGENTS FOR LUTEAL
Sterility. 1991:5(2);213-220. Januszewki A, Vaal ML. The effects of kisspeptin-10 on
16. Bodri D, Guillén JJ, Galindo A, MatarÓ D, Pujol A, Coll reproductive hormone release show sexual dimorphism
PHASE SUPPORT Fig. 33.1: Mechanism for luteal phase deficiency in IVF.
O. Triggering with human chorionic gonadotropin or a in humans. Journal of Clinical Endocrinology and • Progesterone Abbreviations: E2, estrogen; P4, progesterone; GnRH, gonadotropin-
gonadotropin-releasing hormone agonist in gonadotropin Metabolism. 2011;96(12):E1963-72. • Estrogen and progesterone (Fig. 33.2) releasing hormone; HPO, hypothalamic-pituitary-ovarian; LPD, luteal
releasing hormone antagonist-treated oocyte donor cycles: 27. Jayasena CN, Abbara A, Comninos AN, Nijher GM. phase deficiency
• hCG injections
findings of a large retrospective cohort study. Fertil Steril. Kisspeptin-54 triggers egg maturation in women undergoing
2009;91(2):365-71. in vitro fertilization. J Clin Invest. 2014;124(8):3667-77. • Gonadotropin-releasing hormone (GnRH) agonists
17. Youssef MAFM, Van der Veen F, Al-Inany HG, Mochtar 28. MacLean DB, Matsui H, Suri A, Neuwirth R, Colombel • Adjuvants like aspirin, sildenafil
MH, Griesinger G, Nagi Mohesen M, Aboulfoutouh I, et M. Sustained exposure to the investigational Kisspeptin Various studies have shown comparable efficacy with
al. Gonadotropin-releasing hormone agonist versus HCG analog, TAK-448, down-regulates testosterone into the
different routes of administration of progesterone regarding
for oocyte triggering in antagonist-assisted reproductive castration range in healthy males and in patients with
implantation rate, clinical pregnancy rate, ongoing preg-
technology (Review): Cochrane Database of Systematic prostate cancer: results from two phase 1 studies. J Clin
Reviews. 2014;10:CD008046. Endocrinol Metab. 2014;99:E1445-53. nancy rate, and live birth rate. Some studies have shown
better pregnancy rates with intramuscular progesterone
compared to vaginal route, but it has inconvenience of pain-
ful injections. The choice of progesterone varies depending Fig. 33.2: Various agents for LPS and their route of administration.
upon side effects, convenience of use, and availability.2,4,5 Abbreviations: LPS, luteal phase support
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290 The Infertility Manual Luteal Phase Support in Assisted Reproduction 291
Table 33.1. Randomized prospective studies comparing P4 with hCG ± P4 with luteal phase supplementation. Subsequent meta-analysis failed to show any benefit the corpora-lutea, at the level of the endometrium,
of adding estrogen to progesterone in both agonist and enhancement of embryo development, or a combina-
Trials Luteal support Conclusion
antagonist cycles.17,18 tion of these mechanisms.27
Claman (1992); Golan (1993) hCG vs. IM P4 Higher live birth rate with hCG (not significant)
• Tesarik et al. showed beneficial effects of GnRH ago-
Araujo (1994); Martinez (2000); hCG vs. IM P4 No differences in ongoing pregnancy rates
Ludwig (2001) ROLE OF ASPIRIN FOR LUTEAL nist triptorelin in IVF patients given injection trip-
torelin on luteal day 6 in both agonist and antagonist
Artini (1995); Martinez (2000); hCG vs. vaginal P4 No differences in ongoing pregnancy rates PHASE SUPPORT cycles. Similarly, other studies have shown improved
Ludwig (2001)
Aspirin is one of the adjuvants used with LPS to increase pregnancy rates with luteal phase administration of
Buvat (1990) hCG vs. oral P4 Higher pregnancy rate with hCG
implantation and pregnancy rates. GnRH agonist.28-30 To the contrary, other studies have
Fujimoto (2002) (hCG + IM P4) vs. IM P4 Higher pregnancy rate with the (hCG + IM P4)
• Low dose aspirin by its positive influence on ovar- shown no improvement in pregnancy rates with addi-
combination
tion of luteal phase GnRH agonist.31
Mochtar (1996); Ludwig (2001) (hCG + vaginal P4) vs. vaginal P4 No differences in pregnancy rates ian and endometrial blood flow has been shown to
improve folliculogenesis and implantation rates. • Most of studies are done with 0.1 mg of inj. Triptorelin
Hubayter. Luteal supplementation in in vitro fertilization. Fertil Steril 2008.
given 3 days after embryo transfer.
• Aspirin when added is used as 75 mg tablet form.
• Studies have shown different results with the use of
The hCG has been shown to be superior or equivalent rate whether micronized progesterone was started on day aspirin, some showing beneficial effect and while the ROLE OF HEPARIN IN LUTEAL
others are not supporting this.19-22
in efficacy to progesterone for LPS. But, owing to increased of hCG trigger, day of oocyte retrieval, or day of embryo PHASE SUPPORT
risk of ovarian hyperstimulation with hCG, progesterone is transfer (Fig. 33.3).11 • A meta-analysis on aspirin use as luteal support did
not support the beneficial role of aspirin on pregnancy Heparin has a proven role in cases of thrombophilia asso-
the preferred agent for LPS in most of in vitro fertilization ciated repeated implantation failure and recurrent preg-
rate and delivery rates in ART cycles.23
(IVF) cases. OPTIMAL DOSE OF PROGESTERONE nancy loss.
Mixed results have been seen when progesterone alone
There is limited data regarding the optimal dose of ROLE OF SILDENAFIL IN LUTEAL • Low molecular weight heparin (LMWH) has been used
was compared to progesterone plus hCG (Table 33.1).5-9
progesterone. empirically in cases of recurrent implantation failure
PHASE SUPPORT
without history of thrombophilias as well.
IDEAL TIME TO START PROGESTERONE • Studies comparing 100 mg and 25 mg of IM progester-
Vaginal sildenafil has also been shown to be useful in the
one showed no difference in clinical pregnancy rates, • Mechanism of action: Heparin has been shown to
• Normally progesterone is started on the day of ovum treatment of women with thin endometrium. enhance implantation through interactions with sev-
ongoing pregnancy, and miscarriage rates.12
pick-up (OPU) and embryo transfer done 3 days later • Comparison of 400 mg and 600 mg of vaginal proges- • Sildenafil citrate, a phosphodiesterase-5 inhibitor, eral adhesion molecules, growth factors, cytokines,
in case of eight-cell embryo and 5 days later in case of terone showed no difference in clinical pregnancy increases the endometrial blood flow by effect on and enzymes such as matrix metalloproteinase in
blastocyst. rates.13 intrinsic vasodilatory effects of nitric oxide. addition to its antithrombotic effect.
• The questions of when to start luteal supplementation • Sildenafil citrate has been used as vaginal sup- • Some studies have shown a positive impact of such
and when to end it are areas that are poorly studied in therapy on implantation32 while others failed to sup-
the literature.
HOW LONG TO GIVE LUTEAL positories in case of thin endometrium to increase
endometrial thickness and vascularity and pregnancy port this.33,34
PHASE SUPPORT? rates.24-26 • Dose recommended is 20–40 mg S/C of LMWH daily.35
Most IVF practitioners arbitrarily start progester-
one supplementation after oocyte retrieval and elect to • In routine IVF cases, LPS is continued till β-hCG is • Dose of vaginal sildenafil used varies from 50 to 100 mg The recent Cochrane review, 20157 on LPS for assisted
continue it, if the patient is pregnant, until 8–10 weeks of checked.14,15 vaginally in divided doses, starting from day of OPU or reproduction concludes:
gestation. Sohn et al.10 in a randomized prospective trial • In donor egg IVF, luteal support is continued till pla- embryo transfer.
cental take over which normally happens around • Progesterone during the luteal phase is associated
reported a significantly lower clinical pregnancy rate when
with higher rates of live birth or ongoing pregnancy
intramuscular (IM) progesterone was started the day 10–11 weeks. GONADOTROPIN-RELEASING than placebo.
before oocyte retrieval compared with the day of retrieval. • In frozen embryo transfer cycles where endometrium
is artificially prepared with hormones, luteal support is
HORMONE AGONIST AS LUTEAL • There is no conclusive evidence that hCG is more effec-
In a randomized prospective study by Mochtar et al., there
was no significant difference in the ongoing pregnancy continued till placental takeover (10–11 weeks). PHASE SUPPORT tive than placebo or no treatment; hCG may increase the
risk of OHSS compared to placebo.
The GnRH agonist use in luteal phase has been shown to
• The addition of GnRHa to progesterone appears to
IS THERE A ROLE OF ADDING improve implantation and pregnancy rates.
improve outcomes. The hCG, with or without proges-
ESTROGEN TO LUTEAL PHASE? • It has been discovered that GnRH receptors are present terone, is associated with higher rates of OHSS than
Effect of addition of estrogen to progesterone for LPS has in endometrium, corpus luteum, and embryo itself. progesterone alone.
shown controversial results. Some studies have shown • At this time, it is unclear if the benefits, if any, of • Neither the addition of estrogen nor the route of pro-
beneficial effect of adding estrogen in long agonist cycles GnRH agonist administration in the luteal phase are gesterone administration appears to be associated
Fig. 33.3: Time of starting of LPS. but not in antagonist cycles.16 because of an indirect gonadotropin stimulation of with an improvement in outcomes.
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292 The Infertility Manual Luteal Phase Support in Assisted Reproduction 293
LUTEAL SUPPORT IN DONOR CYCLES 12. Nosarka S, Kruger T, Siebert I, Grove D. Luteal phase support 26. Urman B, Mercan R, Alatas C, Balaban B, Isiklar A, Nuhoglu GnRH agonist- and antagonist-treated ovarian stimulation
in in vitro fertilization: meta-analysis of randomized trials. A. Low-dose aspirin does not increase implantation rates cycles. Hum Reprod. 2006;21:2572-9.
AND FROZEN THAW CYCLES Gynecol Obstet Invest. 2005;60:67-74. in patients undergoing intracytoplasmic sperm injection: 34. Pirard C, Donnez J, Loumaye E. GnRH agonist as novel
13. Ludwig M, Diedrich K. Evaluation of an optimal luteal a prospective randomized study. Journal of Assisted luteal support: results of a randomized, parallel group,
In donor egg and frozen embryo transfer cycles, LPS con-
phase support protocol in IVF. Acta Obstet Gynecol Scand. Reproduction and Genetics. 2000;17:586-90. feasibility study using intranasal administration of
sists of estrogen and progesterone both. Normally, estro- 2001;80:452-66. 27. Gelbaya T, Kyrgiou M, Stern C, Nardo L. Low dose buserelin. Hum Reprod. 2005;20(7):1798-804.
gen is started in follicular phase and progesterone is added 14. Sohn SH, Penzias AS, Emmi AM, Dubey AK, Layman LC, aspirin for in vitro fertilization: a systematic review and 35. Casanova P, Szlit Feldman E, Rey Valzacchi GJ, Blanco LA,
on the day of donor oocyte pick up in case of donor embryo Reindollar RH, et al. Administration of progesterone before metaanalysis. Hum Reprod Update. 2007;13:357-64. Carrere CA, Torno A, et al. The addition of GnRH agonist
transfer and 3–5 days prior to embryo transfer in frozen- oocyte retrieval negatively affects the implantation rate. 28. Sher G, Fisch JD. Vaginal sildenafil (Viagra): a preliminary for luteal phase support in ovum donation cycles. Fertil
Fertil Steril. 1999;71:11-4. report of a novel method to improve uterine artery blood Steril. 2015;104(3):e346.
thawed embryo transfer cycles depending upon stage at 15. Mochtar MH, Van Wely M, Van der Veen F. Timing luteal flow and endometrial blood flow in patients undergoing 36. Urman B, Ata B, Yakin K, Alatas C, Aksoy S, Mercan R,
which embryo is transferred. Estrogen and progesterone phase support in GnRH agonist down-regulated IVF/ IVF. Human Rep. 2000;15:806-9. et al. Luteal phase empirical low molecular weight heparin
is then continued through luteal phase and till placental embryo transfer cycles. Hum Reprod. 2006;21:905-8. 29. Kim KR, Lee HS, Ryu HE, Park CY, Min SH, Park C, et al. administration in patients with failed ICSI embryo transfer
take over which normally occurs around 10–11 weeks of 16. Check JH, Nowroozi K, Chase J, Nazari A, Callan C. Efficacy of luteal supplementation of vaginal sildenafil cycles: a randomized open-labeled pilot trial. Hum Reprod.
pregnancy. Progesterone use by different routes (vaginal Comparison of luteal-phase support with high- and low- and oral estrogen on pregnancy rate following IVF-ET in 2009;24(7):1640-7.
dose progesterone therapy on pregnancy rates in an in women with a history of thin endometria: A pilot study. 37. Hamdi K, Danaii S, Farzadi L, Abdollahi S, Chalabizadeh
or intramuscular) has been shown to have comparable vitro fertilization program. J In Vitro Fert Embryo Transf.
efficacy. J Womens Med. 2010;3(4):155-8. A, Sabet SA. The role of heparin in embryo implantation
1991;8:173-5 30. Eid ME. Sildenafil improves implantation rate in women in women with recurrent implantation failure in the cycles
17. Chanson A, Germond M, Lagnaux Y, Singh L, Farina M, with a thin endometrium secondary to improvement of of assisted reproductive techniques (without history of
PROBABLE QUESTIONS Raszka A, et al. Comparison of two progesterone dose uterine blood flow; pilot study. Fertil Steril. 2015;104(3):e342. thrombophilia). J Family Reprod Health. 2015;9(2):59-64.
regimens for luteal phase support after embryo transfer: a 31. Hubayter ZR, Muasher SJ. Luteal supplementation in in 38. Akhtar MA, Eljabu H, Hopkisson J, Raine-Fenning N,
1. What are the various mechanisms of luteal phase prospective randomized study. Hum Reprod. 1996;11:170. vitro fertilization: more questions than answers. Fertil Quenby S, Jayaprakasan K. Aspirin and heparin as adjuvants
deficiency in IVF cycles? 18. Stovall DW, Van Voorhis BJ, Sparks AE, Adams LM, Syrop Steril. 2008;89(4):749-58. during IVF do not improve live birth rates in unexplained
CH. Selective early elimination of luteal support in assisted
2. What are the various forms of progesterone available 32. Tesarik J, Hazout J, Hazout A, Mendoza C. Enhancement of implantation failure. Reproductive Bio Medicine Online.
reproduction cycles using a gonadotropin-releasing
for LPS and their routes of administration? embryo developmental potential by a single administration 2013. [online] Available from http://dx.doi.org/10.1016/j.
hormone agonist during ovarian stimulation. Fertil Steril.
of GnRH agonist at the time of implantation. Hum Reprod. rbmo.2013.02.007
3. Elaborate the various adjuvants used during LPS, 1998;70:1056-62.
2004;19:1176-80. 39. Lightman A, Kol S, Itskovitz-Eldor J. A prospective
highlighting the dose and mechanism of action. 19. Nyboe Andersen A, Popovic-Todorovic B, Schmidt KT,
33. Tesarik J, Hazout A, Mendoza-Tesarik R, Mendoza N, randomized study comparing intramuscular with
4. What are the recent Cochrane recommendations on Loft A, Lindhard A, Hojgaard A, et al. Progesterone
Mendoza C. Beneficial effect of luteal-phase GnRH agonist intravaginal natural progesterone in programmed thaw
supplementation during early gestations after IVF or ICSI
the use of LPS in assisted conception? has no effect on the delivery rates: a randomized controlled
administration on embryo implantation after ICSI in both cycle. Hum Rep. 1999;14(10):2596-9.
trial. Hum Reprod. 2002;17:357-61.
REFERENCES 20. Fatemi HM, Popovic-Todorovic B, Papanikolaou E, Donoso P,
Devroey P. An update of luteal phase support in stimulated
5. Ghanem ME, Al-Boghdady LA. Luteal phase support in IVF cycles. Hum Reprod Update. 2007;13:581-90.
ART: an update, Chapter 7. Enhancing Success of Assisted 21. Chul B, Suh CS, Kim SH, Kim YB, Moon SY. Effects of estradiol
Reproduction. In Tech. 2012. pp. 156-72. [online]. Available supplementation during the luteal phase of in vitro fertilization
from http://dx.doi.org/10.5772/51093. cycles: a meta-analysis. Fertil Steril.2010;93:428-36.
6. ASRM Practice Bulletin. Progesterone supplementation 22. Kolibianakis EM, Griesinger G, Venetis CA, Papanikolaou
during the luteal phase and early pregnancy in the EG, Diedrich K, Tarlatzis BC. Estrogen addition to
treatment of infertility: an educational Bulletin. Fertil progesterone for luteal phase support in cycles stimulated
Steril. 2008;90(Suppl 3):S150-3. with GnRH analogues and gonadotrophins for IVF: a
7. Yehia M. Luteal phase support in assisted reproduction. systematic review and meta-analysis. Human Reprod.
Middle East Fertility Society Journal. 2007;12(3):158-60. 2008;23(6):1346-54.
8. Levine H. Luteal support in IVF using the novel vaginal 23. Pakkila M, Rasanen J, Heinonen S. Low dose aspirin does
progesterone gel Crinone 8%: results of an open-label not improve ovarian responsiveness or pregnancy rate in
trial in 1184 women from 16 US centres. Fertil Steril. IVF and ICSI patients: a randomized, placebo controlled
2000;74:836-7. double blind study. Hum Reprod. 2004;20:2211-4.
9. Pritts EA, Atwood AK. Luteal phase support in infertility 24. Hurst B, Bhojwani J, Marshburn P. Low dose aspirin does
treatment: a meta-analysis of the randomized trials. Hum not improve ovarian stimulation, endometrial response, or
Reprod. 2002;17:2287-99. pregnancy rates for in vitro fertilization. J Exp Clin Assist
10. Daya S, Gunby J. Luteal phase support in assisted Reprod. 2005;2:8-12.
reproduction cycles. Cochrane Database Syst Rev. 25. Rubinstein M, Marazzi A, Polak_de_Fried E. Low-dose
2004;3:CD004830. aspirin treatment improves ovarian responsiveness,
11. Van der Linden M, Buckingham K, Farquhar C, Kremer uterine and ovarian blood flow velocity, implantation,
JAM, Metwally M. Luteal phase support for assisted and pregnancy rates in patients undergoing in vitro
reproduction cycles. Cochrane Database of Systematic fertilization: a prospective, randomized, double-blind
Reviews. 2015;7:CD009154. placebo-controlled assay. Fertil Steril. 1999;71:825-9.
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SE C T I O N
6
Assisted Reproduction Techniques
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C HA PTE R
Intrauterine Insemination 34
R Karthigayeni, Divyashree PS
Chapter Outline
• What Is IUI? • Outcomes of Intrauterine Insemination Cycles
• Advantages of IUI • Gonadotropin-releasing Hormone Analogues in Intrauterine
• Rationale Insemination
• Indications of IUI • Luteal Support in Intrauterine Insemination Cycles
• Indications for Insemination with Donor Semen • Intrauterine Insemination endpoints
• Steps of Intrauterine Insemination
INTRODUCTION • Cost effective
• Efficacious modality
Assisted reproduction technique (ART) is a therapy estab- • Low ovarian hyperstimulation syndrome (OHSS) risk
lished for the treatment of infertility in a most of the clini-
and multiple pregnancy
cal situations. It includes a wide range of techniques of
• Decreased psychological burden for the couple
which intrauterine insemination (IUI), in vitro fertilization
• Good compliance and low dropout rates.
(IVF), and intra-cytoplasmic sperm injection (ICSI) are
most popular.
The IUI evolved almost 200 years back. J. Hunter was
RATIONALE
the first person to perform IUI. It was first performed for The rationale is to increase the conception rate in the cou-
a couple with hypospadias in the husband. Performed by ples, by increasing the density of gametes at the time and
injecting his seminal fluid into wife’s vagina with a syringe site of fertilization.
and that resulted in normal pregnancy. Paper regarding IUI was first published in 1962,1 since
then IUI has evolved through innovations in COS proto-
WHAT IS IUI? cols, techniques in sperm preparation, ultrasound moni-
toring, and ovulation induction with human chorionic
Artificial insemination (AI) is a method by which the
gonadotropin (hCG).
sperm is placed into the female reproductive system by a
means other than intercourse.
INDICATIONS OF IUI
In that, IUI is the one most commonly performed and
it involves placing sperms into female uterus. With Husband’s/Partner’s Semen
The IUI is a low-tech solution to infertility issues and it
was the first technique used to assist the couples who have Indication are given in Table 34.1.
difficulty in conception.
Done by using sperm from women’s husband or part- INDICATIONS FOR INSEMINATION
ner [artificial insemination by husband (AIH)] or from WITH DONOR SEMEN
anonymous or known sperm donor [artificial insemina-
tion by donor (AID/DI)]. 1. Donor insemination is considered effective in follow-
Other forms are intratubal insemination (ITI) and ing conditions:18
intracervical insemination (ICI). • Obstructive azoospermia
• Nonobstructive azoospermia
• Severe deficits in semen quality in couples who do
ADVANTAGES OF IUI not wish to undergo ICSI.
• Simple 2. Donor insemination should be considered in condi-
• Safe tions such as:18
• Less invasive • Risk of transmission of genetic disorder to the
• Requires inexpensive infrastructure offspring
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298 The Infertility Manual Intrauterine Insemination 299
Table 34.1. Indications of intrauterine insemination (husband’s semen). •
Risk of transmission of infectious disease to the ovarian stimulation was found to be effective. To avoid
offspring or woman from the man multiple pregnancies, we should aim for two dominant
Indication Rationale Evidence
• Severe rhesus incompatibility. follicles at maximum.23
1. Unexplained infertility COS with IUI might over- In a meta-analysis, it was shown that natural cycle IUI
come any subtle defects in has no added advantage over expectant management in 3. Lesbian couples or single women.18 Mild ovarian stimulation should be considered. In
ovulatory function and that patients with unexplained infertility.2 spite of its negative effect on the endometrium, clomi-
might enhance the pregnancy
In a study,19 it was showed that the cumulative pregnancy
On the other hand, this meta-analysis also showed that phene citrate remains as the first line drug of choice. And
likelihood by increasing the by combining ovarian stimulation and IUI, live birth rates rate (PR) progressively increased after IUI and reached a
egg number available for are significantly improved in couples with unexplained plateau after the fifth cycle. The valuable predictor for AID compared to gonadotropins, CC is less costly and easily
fertilization. infertility,2 but we have to take into account that the o
varian available.23
pregnancy outcomes is prefreezing progressive sperm motil-
stimulation regimen in lot of these studies was rather Looking at cost-effectiveness and the literature, there
aggressive thus resulting in higher multiple pregnancy rate. ity. IVF/ICSI should be recommended when couples have
received more than five AID cycles without pregnancy. seems no role of using GnRH agonist, GnRH antagonist,
In patients with unexplained infertility, controlled ovarian
stimulation and IUI is accepted.3,4 and aromatase inhibitors in IUI.24,25
Ovarian stimulation with IUI is an effective method in STEPS OF INTRAUTERINE
unexplained infertility, compared to timed intercourse (TI).5
INSEMINATION Semen Collection
2. Male factor By IUI, the density of motile Mild-to-moderate male infertility:
•• Azoospermia sperm available to the eggs In a Cochrane review it was shown that there is not much • Highest PR was observed with abstinence interval of
•• Oligospermia are increased, which in turn • Counseling
evidence to suggest whether IUI is effective or not in cou- less than 3 days (14%) and with abstinence of more
•• Asthenozoospermia increase the monthly prob- ples with moderate and mild male factor infertility.6 • Ovarian stimulation
•• Teratozoospermia ability of pregnancy. than 10 days (3%) PR was lower.26
A study involving 17 trials, compared IUI versus timed • Monitoring of ovulation
•• Oligoasthenoteratozoospermia • Collect semen sample by masturbation, directing the
intercourse in mild-to-moderate male infertility.7 • Semen collection
It was shown that IUI performed in both natural cycles and
semen into a clean sample cup. Do not use a lubricant
• Semen preparation
COS cycles improved the probability of conception signifi- which can kill sperms.
cantly. With COS, IUI was found superior to TI cycles.7
• Insemination
• Assisted ejaculation: electroejaculation used in para-
And also IUI overcomes the fertilization failure due to
impaired mucus penetration and poor survival in the
Counseling plegic.
female reproductive tract.8 Counseling plays a key role for the couple undergoing the
Severe male factor infertility:
procedure in terms of completing the treatment with com- Donor Sperm
It has been found that compared to IUI, ICSI is more cost
effective especially when the total motile sperm count is
fort and compliance. Sexually transmitted diseases including HIV should be
<10 million. They are not the candidate for IUI.9 Success rates of the procedure, cost involved, the time prevented from transmission. So in case of donor sperm,
3. Cervical factors IUI can bypass a possible In isolated cervical factor, IUI in natural cycle significantly logistics, etc. should be detailed before the commence- a quarantine period of 6 month is required prior to its use
•• Cervical mucus hostility cervical factor. increased the probability of conception.10 ment of IUI cycles. by cryopreservation.27 But PRs are significantly higher with
•• Poor cervical mucus In a couple with cervical factor infertility, natural cycle IUI Couples should be informed about the beneficial fresh sperm compared to cryopreserved sperm.28
is an effective method.11
effects of a good prognosis. In a trial,20 the selected cou-
4. Immunological IUI overcomes the barrier In male immunological infertility, compared to timed
•• Male antisperm antibodies of antibodies from cervical intercourse with prednisolone, IUI was found to give better
ples without any form of treatment were found to have a Semen Processing
•• Female antisperm antibodies mucus, although it cannot results.12 good prognosis. In them, IUI with controlled ovarian stim-
(cervical, serum) circumvent the damage that ulation (COS) when compared to no treatment did not It is necessary to remove seminal plasma to avoid prosta-
Pregnancy rates improved significantly when cyclical
may be caused by antibodies dose of steroid were used as an adjuvant.13 glandin induced uterine contractions prior to IUI. Insemi-
improve the PR.
in the uterine and tubal fluids.
The treatment options should be given to the couple nation with unprocessed semen is associated with pelvic
5. Endometriosis Minimal endometriosis: infections.29
•• Minimal when both donor insemination and ICSI are easily avail-
Superovulation with IUI is effective.14
•• Mild able. The option for donor insemination should be given The following are the sperm preparation methods:
Minimal and mild endometriosis:
IUI and COS was associated with good outcome in terms
because of the reason that IVF-ICSI might not be afford- • Simple washing
of live birth rates.15 able due to financial issues.21 • Swim up method-migration
6. Ejaculatory failure In ejaculatory dysfunction, In patients with retrograde ejaculation, pulse electric The counseling is particularly important when the Swim up from pellet
•• Anatomical (e.g. hypospadias) there is a failure to deposit stimulation was applied to the epididymis or vas deferens couples want to use donor gametes and it should be given Direct swim up
•• Neurological (e.g. spinal cord semen in the vagina. To to retrieve sperm via a tube cannulated into the vas def-
injury) overcome that, semen sam- erens. After diluting with culture medium, retrieved sperm
for both the recipient and donor couple.22 Swim down
•• Retrograde ejaculation (e.g. ple is collected and artificially were used for IUI. Electric vassal sperm retrieval is found Couples should be assured regarding complete confi- • Density gradient method
multiple sclerosis) inseminated to be a feasible method for collecting sperm from the vas dentiality in case of donor insemination.
•• Psychological (e.g. impotence) deferens in patients with male factor infertility.16 Newer methods in semen preparation:
To prevent transmission
7. Serodiscordant couples [female part- • Magnetic activated cell sorting
ner human immunodeficiency virus
of infection to partner. In case of serodiscordant couples, IUI and IVF are found
to be effective and safe. Evidence-based practice and
Ovarian Stimulation • Glass wool filtration
(HIV) negative and male partner HIV social justice suggest that our field should increase
positive] In couples with mild male subfertility, unexplained sub- • Sperm selection based on membrane charge
access to care for HIV serodiscordant couples.17
fertility and minimal-to-mild endometriosis IUI with mild (Table 34.2).
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300 The Infertility Manual Intrauterine Insemination 301
Table 34.2. Semen preparation techniques. • The recommended lower limit might range from 3 mil- Inseminated Volume
lion motile sperms35 to 5 million36 to 10 million in the
Method Advantages Disadvantages • For both 0.5 mL and 3 mL of inseminated semen, the
insemination sample.34,37
1. Simple washing •• Simple •• Any cell or debris which remained PRs were similar.46
in the inseminate are deleterious to
fertilization.
Insemination Type of Catheter
2. Swim up method •• Simple •• Reactive oxygen species (ROS)
•• Relatively inexpensive •• Less recovery of motile sperm Mode of Insemination • Compared to the less pliable tomcat catheter, by using
•• Requires 1-hour incubation
softer Wallace catheter no difference in PR was found
3. Density gradient •• Thirty-minute of centrifugation is required. •• It takes some time to produce good • The semen sample can be deposited in the uterus, cer-
if done by standard gentle technique47
•• Compared to swim-up technique it takes less time. interphases between layers. vix, or the fallopian tube.
•• Under sterile conditions it is relatively easy to perform. •• Might get contaminated with • IUI is performed using a small catheter without imag-
•• Spermatozoa can be effectively separated from endotoxins. ing guidance and we have to introduce 0.2–0.5 mL Procedure
oligozoospermic semen sample. •• Sperm DNA integrity might get
semen sample into the uterus. • Vagina and cervix is to be cleaned with normal saline.
•• Most of the leukocytes are eliminated from the ejaculate. affected.
• In fallopian tube sperm perfusion (FSP), 4 mL of insem- • Cervical mucus to be aspirated gently with tuberculin
4. Magnetic •• Compared to other sperm preparation techniques, it acts •• Spermatozoa that are viable should
inate volume is used and it fills uterine cavity, fallopian syringe.
activated cell at the molecular level which depends on sperm density be separated from other substances
sorting and motility. such as apoptotic spermatozoa, tube, and some end up inside peritoneal cavity.38 • In unexplained infertility, aspiration of mucus led to
•• This is the only technique which separates apoptotic and leukocytes, and seminal plasma in • In case of frozen semen, IUI was found to be better increased PR.48 PR was found to be 15% in the cervical
nonapoptotic spermatozoa. the ejaculate. For this, magnetic than intracervical insemination (ICI). The chance of mucus aspiration group and 9.9% in the control group.
•• Rapid, noninvasive and convenient activated cell sorting (MACS) should having a live birth after six cycles was twofold higher.39
•• Bead detachment after MACS is not necessary. be used along with other techniques • Avoid holding cervix with tenaculum as it elicits uter-
•• Provides optimal purity and recovery with reliable and like density gradient centrifugation.
• In patients with unexplained infertility two trials has ine contractions.
consistent results. shown that FSP was better than IUI.38,40 • Effectiveness of passive uterine straightening by blad-
•• Can be used to optimize the cryopreservation-thawing • For other indications, FSP was not found to be better der filling was shown in a study.49 The PR was found to
outcome and improve cryosurvival rates following than IUI. be higher in the full bladder group compared to empty
cryopreservation.
bladder group (13.5% vs. 7.4%). The risk of undergo-
5. Glasswool •• Sperm cells having normal condensation of chromatin •• Expensive
filtration can be selected. •• Some debris is usually still present Timing of Insemination ing difficult IUI was also higher in the empty bladder
•• Compared to other methods, from this we obtain higher in the sample after glass wool group than in the full bladder group (10% vs. 37.8%).
• IUI can be done around the ovulation time.
number of spermatozoa with intact acrosome. filtration. The clinical PR was found to be higher in the patients
• To get good success rate, the insemination timing rela-
who had easy IUI compared to who had difficult IUI
tive to ovulation is important. Only few studies were
(12.7% vs. 5.5%).
published regarding optimal time for insemination.41
• US guidance in IUI improves the PRs. It also reduces
• IUI should be done just before ovulation or up to
There were no enough evidence to choose the best sperm A sperm preparation method has to select sperms with the chance of difficult IUI.50
10 hours from ovulation.
preparation techniques, as shown in systematic review.30 reduced DNA damage along with higher motile sperms • IUI can be done in accordance with timing of hCG
A study comparing density gradient centrifugation and also sperms with normal morphology. injection or detection of LH surge.42 Rest after Intrauterine Insemination
(DGC) with simple washing method31 have showed that the Colloidal silica density gradient (CSDG) centrifuga- • A wider time frame can be used. Usually 12–36 hours According to two trials, 10–15 minutes immobilization
PR was 11.6% and 14.3% for wash only and DGC, respec- tion has been found to reduce the viral load from samples after hCG injection, IUI is planned.42 after intrauterine insemination improved the cumulative
tively. In sample with inseminating motile sperm count of carrying an infectious agent like HIV.33
PRs and live birth rates (LBRs).51,52
less than 22 million, PR was found to be 4% in wash only
method and 18% in DGC, respectively. So, in samples with Number of Insemination
good number of motile sperm wash only method can be
Quality of Specimen OUTCOMES OF INTRAUTERINE
• Asystematic review compared two inseminations
used and in semen samples with poor quality DGC should • No consensus regarding the lower limit in semen compared to one. Found no significant difference in INSEMINATION CYCLES
be used for semen processing. parameters are established. So there is no particular the PR per couple.43
In a Cochrane review,32 investigating the clinical out- level at which one would council the couple for ICSI • Most reports recommend a single well timed IUI in
Natural Cycle Intrauterine Insemination
comes after IUI found no clear evidence to suggest one rather than IUI. couple with unexplained subfertility.44 • The cumulative PR was 18% for IUI without COS cycles
sperm washing technique over another technique. It • Lower limits is considered as initial total sperm count • One study reported that double insemination is ben- and 33% for IUI with COS cycles.53 Hence, COH with
did not show any difference between swim-up and DGC or initial total motile sperm count or post wash total eficial in couple with male subfertility.45 IUI was found to give a better PR.
method on PR per couple (30.5% vs. 21.5%). Showed no motile sperm count. • Second IUI might increase the cost and also the psy- • COS doubles PR by IUI in young patients with no prior
difference in PRs (22.2% vs. 38.1%) between swim-up and • If initial total sperm count less than 10 million, the PR chological burden on the couple. So it is better to history of pelvic surgery and with good post wash
centrifugation method. is lower.34 advice double IUI only when proven effective. semen sample.54
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302 The Infertility Manual Intrauterine Insemination 303
• Comparing IUI with timed intercourse in natural • High dose of FSH: In Cochrane review,65 two studies • In the absence of progesterone or if its action is blocked Factors Influencing Success Rate
cycles in case of male factor infertility, IUI was not found that there is no benefit in doubling the gonado- by a progesterone antagonist like mifepristone, the
found to be superior to TI but due to small sample size tropins dosage, although there is an increase in OHSS
of Intrauterine Insemination
endometrium might not be receptive for implantation
firm conclusions were not obtained.55 and multiple PRs. and pregnancy will not occur.72 • The cause of infertility
• The objective of the ovarian stimulation in IUI cycles is • Both partners age
to stimulate the development of multiple follicles and • Infertility duration
Intrauterine Insemination in Cycles Stimu- GONADOTROPIN-RELEASING
the treatment overrides the physiological feedback • Semen parameters
lated with Clomiphene Citrate HORMONE ANALOGUES IN mechanisms which normally ensure that only one or • Number of Cycles
In couples with unexplained infertility, two trials com- INTRAUTERINE INSEMINATION two large follicles reach ovulation.
In couples with unexplained subfertility and mild male
pared IUI and TI cycles using CC.56,57 In both trials, better • In these cycles, because of high level of these hormones
• In stimulated IUI cycles, the premature LH surge inci- subfertility, one cycle of IVF-elective single embryo trans-
PRs were obtained with CC/TI cycle compared to CC/IUI along with inhibin A suppression in luteal phase, LH
dence is 25–30%.66 Because of this timing of IUI may fer was found to be effective when compared to three cycles
cycle. and FSH levels will be on lower side by negative feed-
get affected and it might lead to more cycle cancella- of IUI-COS. In these couples, in order to prevent multiple
The Fast Track and Standard Treatment (FASTT) back mechanism.73
tion and treatment failures. pregnancies as sole reason, elective single embryo transfer
trial58 compared sequence of CC/IUI and IVF with CC, • Hence, LH in low levels might not give luteotropic
• GnRH antagonist prevents premature luteinization is not considered as an effective method.84
gonadotropin-stimulated IUI, and IVF. The PR per cycle support.74 Low levels of LH in GnRH analogue treated
almost completely. But in spite of that there is no
was found to be 7% with CC/IUI in both arms. cycles is associated with poor implantation rates.75
improvement in the PR. INTRAUTERINE INSEMINATION
This trial58 indicated that in couple with unexplained • High level of estradiol will lead to inappropriate prim-
• So poor quality of the growing follicle might not be due
infertility, the PRs per cycle was found to be higher with
to premature luteinization but may be the due to its
ing of the endometrium to hormones.76 ENDPOINTS
CC/IUI treatment, so it can be promoted when compared • In FSH-stimulated cycles, there was advanced matu-
consequence.66 In patients with unexplained infertility, it was found that
to more complex and costly FSH/IUI treatment. ration of the endometrium compared to spontaneous
• The PRs were similar between two groups who under- in the first three cycles of COS and IUI the cycle fecundity
cycles.77
went superovulation/IUI cycles with or without GnRH was higher compared to cycles four to six.85 After three
Intrauterine Insemination in Follicle- agonist. There was no significant difference in the
What Evidence Suggests? cycles of COS and IUI, if the patient fails to conceive, we
ongoing and clinical PR per cycle and live birth.67
stimulated Hormone Stimulated Cycles should advice them regarding IVF or ICSI.86
• Four studies in a Cochrane review,68 has showed that • No clear evidence to suggest that treatment with either
• FSH versus expectant management: FSH/IUI is not by adding GnRH agonist PRs cannot be improved, progesterone or hCG as luteal phase support either in
better than expectant management in couple with although rate of multiple pregnancies were found mildly stimulated cycles or spontaneous cycles is nec-
CONCLUSION
unexplained infertility, but in couples with more than to be more. Three studies found that there were essary to improve the PR.78 • Artificial insemination using husband’s sperm should
3 years of infertility it gives some better results.59,60 no benefits of adding gonadotropins with GnRH • It was found that in hypophysectomized women be considered as a preferable method before starting
Compared with expectant management, one addi- antagonist. undergoing ovarian stimulation using gonadotropins, a more expensive and invasive techniques of assisted
tional pregnancy was obtained for every 11 cycles of • In seven RCTs,68 it was shown that the PR in GnRH it is mandatory to provide luteal phase support in the reproduction, if one tube is found to be patent.
FSH/IUI.59,60 antagonist cycles was only 5.3% greater. form of hCG.79 But women are not totally hypogon- • The IUI success rate can be improved with an insemi-
• FSH versus natural cycle IUI: A meta-analysis61 studied • One-third dose and one-fourth dose of leuprolide adotropic during IUI cycles, even if they are co-treated nating motile count greater than 1 million, a morphol-
in patients with unexplained infertility comparing IUI depot was found to prevent premature LH surge. with potent GnRH antagonist. ogy score more than 4%, a total motile sperm count
with COS and IUI in the natural cycle, have shown that Hence, the minimal dose to prevent premature LH • Regarding the use of progesterone vaginal gel as luteal greater than 5 million, and initial total motile count
live birth was better with IUI in COS cycle. surge was one-fourth dose of leuprolide depot.69 phase support (LPS) in stimulated IUI cycles in couple more than 30%.
• FSH with or without antiestrogens: In a Cochrane • One study has demonstrated that by using GnRH with unexplained infertility and with polycystic ovar- • In cervical factor subfertility, the probability of con-
review,62 seven trials including patients with mild antagonist if a high response gonadotropin-IUI cycles ian syndrome, a study has found that there was no sig- ception is increased by doing IUI in natural cycles.
endometriosis, unexplained infertility, and mild male is converted to IVF cycle, it was found to be a more cost nificant difference in LBR and PR.80 • In couples with unexplained infertility, IUI with ovar-
factor was studied. It was found that compared to anti- effective than regular IVF with minimal morbidity and • When clomiphene and HMG were used for IUI in sub- ian stimulation was found to be better.
estrogens PRs were quiet high with gonadotropins. producing better results.70 fertile couples, it was found that by using luteal phase • The aim is to produce two dominant follicles by COS.
• Classical dose of FSH: There is an increase in the likeli- support with vaginal progesterone the success rate of • In AIH procedures, sperm washing techniques prevent
hood of conception but multiple gestation might occur
LUTEAL SUPPORT IN INTRAUTERINE IUI cycle cannot be improved.81 partner-to-partner transmission but it does not guar-
more frequently and the added benefit of COS is lim- • Another study has also shown that progesterone luteal antee that 100% infections are removed from the post-
ited.63 INSEMINATION CYCLES phase support was effective for IUI cycles with gonad- processed sperm sample.
• Low dose of FSH: In spite of prevention of multiple otropins. But not beneficial in IUI cycles with CC for • Regarding IUI outcome, no sperm preparation tech-
pregnancies by reducing gonadotropins dosage (mild
Why is it Required?
ovulation induction.82 nique was found superior to another technique.
ovarian stimulation), it remains unclear that this can • Progesterone is an essential hormone required for • Also, the outcomes of IUI cycles are not improved by • In couple with male factor subfertility, double IUI was
maintain overall PRs.64 establishment and maintain of pregnancy.71 giving GnRH agonist during the implantation time.83 found to give higher pregnancy rate.
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304 The Infertility Manual Intrauterine Insemination 305
• There should be an interval of 12 and 36 hours between isolated cervical factor: a randomized clinical trial. Fertil follicles on pregnancy rates in intrauterine insemination unexplained infertility: a prospective randomized study.
hCG injection and IUI. Steril. 2007;88:1692-6. with ovarian stimulation: a meta-analysis. Hum Reprod Hum Reprod. 1993;8:890-4.
11. Check JH, Spirito P. Higher pregnancy rates following Update. 2008;14:563-70. 39. Besselink DH, Farquhar C, Kremer JAM, Marjoribanks
• Better results are attained with soft catheters.
treatment of cervical factor with intrauterine insemination 24. Cantineau AE, Cohlen BJ, Heineman MJ. Ovarian J, O’Brien P. Cervical insemination versus intra-uterine
• 10–15 minutes of immobilization is advised after IUI. without superovulation versus intercourse: the importance stimulation protocols (anti-oestrogens, gonadotropins with insemination of donor sperm for subfertility (Review).
• In IUI cycles, prevention of premature LH surge and of a well-timed postcoital test for infertility. Arch Androl. and without GnRH agonists/antagonists) for intrauterine Cochrane Database Syst Rev. 2008:CD00317.
luteal phase support are not required. 1995;35(1):71-7. insemination (IUI) in women with subfertility. Cochrane 40. Cantineau AEP, Heineman MJ, Cohlen BJ. Single versus
• Although IUI treatment is cost effective, the most effec- 12. Lahteenmaki A, Veilahti J, Hovatta O. Intra-uterine Database Syst Rev. 2007;(2):CD005356. double intrauterine insemination (IUI) in stimulated
tive treatment for infertility is IVF. insemination versus cyclic, low-dose prednisolone in 25. Cantineau AE, Cohlen BJ, Heineman MJ. The addition of cycles for subfertile couples. Cochrane Database Syst Rev.
couples with male anti sperm antibodies. Hum Reprod. GnRH antagonists in intrauterine insemination cycles with 2003:CD003854.
1995;10(1):142-7. mild ovarian hyper stimulation does not increase live birth 41. Ragni G, Somigliana E, Vegetti W. Timing of intrauterine
PROBABLE QUESTIONS 13. Robinson JN, Forman RG, Nicholson SC, Maciocia LR, rates-a randomized, double-blinded, placebo-controlled insemination: where are we? Fertil Steril. 2004;82:25-6.
Barlow DH. A comparison of intrauterine insemination in trial. Hum Reprod. 2011;26:1104-11. 42. Cantineau AEP, Heineman MJ, Cohlen BJ. Single versus
1. IUI Indications and advantages.
super ovulated cycles to intercourse in couples where the 26. Jurema MW, Vieira AD, Bankowski B, Petrella C, Zhao Y, double intrauterine insemination in stimulated cycles
2. Semen preparation techniques in IUI. male is receiving steroids for the treatment of autoimmune Wallach E, et al. Effect of ejaculatory abstinence period on for subfertile couples. Cochrane Database Syst Rev.
3. Luteal phase support in IUI. infertility. Fertil Steril. 1995;63(6):1260-6. the pregnancy rate after intrauterine insemination. Fertil 2009;(2).
4. IUI outcome—in natural and COS cycles. 14. Isaksson R, Tiitinen A. Superovulation combined with Steril. 2005;84(3):678-81. 43. Cantineau AEP, Heineman MJ, Cohlen BJ. Single versus
5. Gonadotropin-releasing hormone analogues in IUI. insemination or timed intercourse in the treatment of couples 27. British Andrology Society. British andrology society double intrauterine insemination(IUI) in stimulated
with unexplained infertility and minimal endometriosis. guidelines for the screening of semen donors for donor cycles for sub fertile couples. Cochrane Database Syst Rev.
Acta Obstet Gynecol Scand. 1997;76(6):550-4.
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and in vitro fertilization. Fertil Steril. 2001;75(4):661-8. 22. ESHRE Task Force on Ethics and Law. III. Gamete and Hum Reprod. 1992;7(Suppl. 1):19-24. controlled trial. BMJ. 2009;339:b4080.
10. Steures P, Van der Steeg JW, Hompes PG, Bossuyt PM, embryo donation. Hum Reprod. 2002;17:1407-8. 38. Kahn JA, Sunde A, Koskemies A, Vun Düring V, Sordal T, 53. Guzick D, Carson S, Coutilaris C, Overstreet J, Livak P,
Habbema JD, Eijkemans MJ, et al. Effectiveness of 23. Van Rumste MM, Custers IM, Van der Veen F, Van Wely Christensen F, et al. Fallopian tube sperm perfusion (FSP) Steinkamf M, et al. Efficacy of superovulation & IUI in
intrauterine insemination in sub fertile couples with an M, Evers JL, Mol BW. The influence of the number of versus intrauterine insemination (IUI) in the treatment of treatment of infertility. N Engl J Med. 1999;340:177-83.
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306 The Infertility Manual Intrauterine Insemination 307
54. Hendin B, Falcone T, Hallak J, Nelson D, Vemullapalli S, ganirelix prevents premature LH rises and luteinization in cycles: a Randomized Double Blind, Placebo Controlled subfertility and unfavorable prognosis: a randomized pilot
Goldberg J, et al. Effect of clinical & semen characteristics stimulated intrauterine insemination: results of a double- Study. J Family Reprod Health. 2014;8(4):149-53. trial comparing the effectiveness of in vitro fertilization
on efficacy of ovulatory stimulation in patients undergoing blind, placebo-controlled, multicentre trial. Hum Reprod. 82. Hill MJ, Whitcomb BW, Lewis TD, Wu M, Terry N, DeCherney with elective single embryo transfer versus intrauterine
IUI. J Assist Reprod Genet. 2000;17:189-93. 2006;21:632-9. AH, et al. Progesterone luteal support after ovulation insemination with controlled ovarian stimulation. Fertil
55. Bensdorp AJ, Cohlen BJ, Heineman MJ, Vandekerckhove P. 67. Eskandar MA. Does the addition of a gonadotropin- induction and intrauterine insemination: a systematic Steril. 2011;96(5):1107-11.
Intra uterine insemination for male subfertility. Cochrane releasing hormone agonist improve the pregnancy rate in review and meta-analysis. Fertil Steril. 2013;100(5):1373-80. 85. Aboulghar M, Mansour R, Serour G, Abdrazek A,
Database Syst Rev. 2007:CD000360. intrauterine insemination? A prospective controlled trial. 83. Bellver J, Labarta E, Bosch E, Melo MA, Vidal C, Amin Y, Rhodes C. Controlled ovarian hyper stimulation
56. Karlstrom PO, Bergh T, Lundkvist O. A prospective Gynaecol Endocrinol. 2007:23(10):551-5. Remohi J, et al. GnRH agonist administration at the time and intrauterine insemination for treatment of unexplained
randomized trial of artificial insemination versus 68. The ESHRE Capri Workshop Group. Intrauterine of implantation does not improve pregnancy outcomes in infertility should be limited to a maximum of three trials.
intercourse in cycles stimulated with human menopausal insemination. Hum Reprod Update. 2009;15(3):265-77. intrauterine insemination cycles: a randomized controlled Fertility and Sterility. 2001;75(1):88-91.
gonadotropin or clomiphene citrate. Fertil Steril. 69. Chen HJ, Lin YH, Huang MZ, Seow KM, Huang LW, trial. Fertil Steril. 2010;94(3):1065-71. 86. Custers IM1, Steures P, Hompes P, Flierman P, van Kasteren Y,
1993;59:554-9. Hsieh BC, et al. Dose-finding study of leuplin depot for 84. Custers IM, Konig TE, Broekmans FJ, Hompes PG, van Dop PA, et al. Intrauterine insemination: how many
57. Agarwal S, Mittal S. A randomized prospective trial of prevention of premature luteinizing hormone surge during
Kaaijik E, Oosterhuis J, et al. Couples with unexplained cycles should we perform? Hum Reprod. 2008;23(4):885-8.
intrauterine insemination versus timed intercourse in controlled ovarian stimulation: a pilot study in intrauterine
super ovulated cycles with clomiphene. Indian J Med Res. treatment. Taiwan J Obstet Gynecol. 2016:33(2):235-8.
2004;120:519-22. 70. Balayla J, Granger L, St-Michel P, Villeneuve M, Fontaine
58. Reindollar RH, Regan MM, Neumann PJ, Thornton KL, JY, Desrosiers P, et al. Rescue in vitro fertilization using a
Alper MM, Goldman MB. A randomized controlled trial of GnRH antagonist in hyper-responders from gonadotropin
503 couples assigned to conventional infertility treatment intrauterine insemination (IUI) cycles, J Assist Reprod
or an accelerated track to IVF: preliminary results of the Genet. 2013:30(6):773-8.
fast track and standard treatment (FASTT) trial. Fertil 71. Csapo AI, Pulkkinen M. Indispensability of the human
Steril. 2007;86:S841. corpus luteum in the maintenance of early pregnancy.
59. Guzick DS, Carson SA, Coutifaris C, Overstreet JW, Factor- Luteectomy evidence. Obstet Gynecol Surv. 1978;33:69-81.
Litvak P, Steinkampf MP, et al. Efficacy of superovulation 72. Baird DT. Mode of action of medical methods of abortion.
and intrauterine insemination in the treatment of infertility J Am Med Womens Assoc. 2000;55(Suppl 3);121-6.
Engl J Med. 1999;340:177-83. 73. DiLuigi AJ, Nulsen JC. Effects of gonadotropin-releasing
60. Steures P, van der Steeg JW, Hompes PG, Habbema hormone agonists and antagonists on luteal function. Curr
JD, Eijkemans MJ, Broekmans FJ, et al. Intrauterine Opin Obstet Gynecol. 2007;19:258-65.
insemination with controlled ovarian hyper stimulation 74. Abu-Heija AT, Fleming R, Yates RW, Coutts JR. Pregnancy
versus expectant management for couples with unexplained outcome following exposure to gonadotropin-releasing
subfertility and an intermediate prognosis: a randomized hormone analogue during early pregnancy: comparisons
clinical trial. Lancet. 2006;368:212-21. in patients with normal or elevated luteinizing hormone.
61. Verhulst SM, Cohlen BJ, Hughes E, te Velde E, Heineman Hum Reprod. 1995;10:3317-9.
MJ. Intra-uterine insemination for unexplained subfertility. 75. Tarlatzis BC, Fauser BC, Kolibianakis EM, Diedrich K,
Cochrane Database Syst Rev. 2006:CD001838. Rombauts L, Devroey P. GnRH antagonists in ovarian
62. Cantineau AE, Cohlen BJ, Heineman MJ. Ovarian stimulation for IVF. Hum Reprod Update. 2006;12:333-40.
stimulation protocols (anti-oestrogens, gonadotropins with 76. Macklon NS, Fauser BC. Impact of ovarian hyper stimulation
and without GnRH agonists/antagonists) for intrauterine on the luteal phase. J Reprod Fertil. 2000;55(Suppl l):101-8.
insemination (IUI) in women with sub fertility (Review). 77. Kolibianakis EM, Bourgain C, Platteau P, Albano C,
Cochrane Database Syst Rev. 2007:CD005356. Van Steirteghem AC, Devroey P. Abnormal endometrial
63. Fauser BCJM, Devroey P, Macklon NS. Multiple birth development occurs during the luteal phase of
resulting from ovarian stimulation for subfertility treatment. nonsupplemental donor cycles treated with recombinant
Lancet. 2005;365:1807-16. follicle-stimulating hormone and gonadotropin-releasing
64. Ragni G, Caliari I, Nicolosi AE, Arnoldi M, Somigliana hormone antagonists. Fertil Steril. 2003;80:464-6.
E, Crosignani PG. Preventing higher order multiple 78. Ragni G, Vegetti W, Baroni E, Colombo M, Arnoldi M,
pregnancies during controlled ovarian hyper stimulation Lombroso G, et al. Comparison of luteal phase profile
and intrauterine insemination: 3 years’ experience using in gonadotropin stimulated cycles with or without a
low dose recombinant follicle-stimulating hormone and gonadotropin-releasing hormone antagonist. Hum Reprod.
gonadotropin-releasing hormone antagonist. Fertil Steril. 2001;16:2258-62.
2006;85:619-24. 79. Lunenfeld B. Historical perspectives in gonadotropin
65. Cantineau AE, Cohlen BJ, Heineman MJ. Ovarian therapy. Hum Reprod Update. 2004;10:453-67.
stimulation protocols (anti-oestrogens, gonadotrophins 80. Aytac PC, Bulgan Kilicdag E, Haydardedeoglu B, Simsek E,
with and without GnRH agonists/antagonists) for Cok T, Coban G. Luteal phase support after mild ovulation
intrauterine insemination (IUI) in women with sub fertility induction with intrauterine insemination: an ongoing
(Review). Cochrane Database Syst Rev. 2007:CD005356. debate. Gynecol Endocrinol. 2016;32(7):543-7.
66. Lambalk CB, Leader A, Olivennes F, Fluker MR, Andersen 81. Rashidi BH, Tanha FD, Rahmanpour H, Ghazizadeh M.
AN, Ingerslev J, et al. Treatment with the GnRH antagonist Luteal phase support in the intrauterine insemination
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C HA PT E R
35
Oocyte Retrieval and Embryo Transfer 309
ANESTHESIA Matt et al. in 1991 found no significant detrimental
effect of N2O and isoflurane anesthesia on reproductive
Oocyte Retrieval and Embryo Transfer An ideal anesthesia for oocyte retrieval should provide outcome in ART.13
quick onset, good analgesia with minimal risk, and fast D Jain et al. (2009) in their review article said key of
recovery without compromising reproductive outcome. successful anesthesia is minimal pharmacological expo-
Shivani Singh, Shalu Gupta Options available include general anesthesia, spinal/ sure of anesthesia drugs, aiming to minimizing their pen-
epidural anesthesia, local anesthesia, conscious seda- etration into follicular fluid.14
Chapter Outline tion/analgesia, and combination of these. The mode of
• Brief History • Preparation Before Oocyte Retrieval anesthesia chosen by an ART center depends upon the Vaginal Cleaning
• Comparison of Different Routes • Procedure convenience of that center. Local anesthesia (paracervical
• Anesthesia • Embryo Transfer There is conflicting evidence regarding use of Betadine as
• The Equipment • Types of Embryo Transfers
block) with conscious sedation (a combination of fentanyl,
vaginal disinfectant before oocyte retrieval. Van Os et al. in
• Preparation of Oocyte Retrieval • Factors Affecting Embryo Transfer Results propofol, and midazolam) is most commonly used.5
• Scheduling/Avoiding Weekend Oocyte Retrieval
prospective randomized trial of 334 cycles found signifi-
E Karmon et al. after evaluating 3,525 retrievals concluded
cantly increased clinical pregnancy rates (30.3% vs. 17.2%)
agents like propofol, inhaled anesthetics, fentanyl, and mida-
when only normal saline was used. But there was no signifi-
zolam used at the time of oocyte retrieval does not affect clini-
cant difference in fertilization rate and pelvic infection rate.15
INTRODUCTION The birth of first in vitro fertilization (IVF) baby was through cal pregnancy outcome. However, meperidine was negatively
laparoscopy guided oocyte aspiration. In 1982, Lenz S for Hannoun et al. (2008) stated that Betadine did not
associated with clinical pregnancy per oocyte retrieval.6
The science and technology of assisted reproductive tech- the first time described oocyte aspiration with help of affect the IVF reproductive outcome but was associ-
Goutziomitrou E et al. 2015 in an RCT found no dif-
nology (ART) has progressed enormously in last four dec- transabdominal ultrasound. He had oocyte retrieval rate ated with an increased chances biochemical pregnancy.
ference in fertilization as well as pregnancy rate on com-
ades. In early seventies, laparotomies were performed for of 64% and transient hematuria was the main complica- Hence, advised to cleanse vagina thoroughly before oocyte
paring propofol verses thiopental sodium when used for
oocyte retrieval. It was soon replaced with laparoscopy tion with this technique. Transabdominal guided oocyte retrieval.16 Further studies too have demonstrated vaginal
general anesthesia during TVOR.7 However, Chistiaens
followed by ultrasound guided oocyte aspiration. Trans- retrieval was finally replaced by TVOR.2 povidone iodine disinfection followed by thorough saline
F (1999) found trend toward low fertilization rate when
vaginal-guided oocyte retrieval (TVOR) is easy to learn A special mention to Dr. Subhas Mukherjee, an Indian douching to be an effective in preventing pelvic infection
oocytes were exposed to high dose and longer duration to
and could be performed with minimal complication. scientist who for the first time used vaginal approach— without compromising the IVF outcome.17,18
propofol.8 Another randomized prospective study found
Hence, TVOR is the gold standard technique for oocyte colpotomy—for oocyte collection, freeze the embryos, In our clinic, patients with history of pelvic infection,
the combination of midazolam and ketamine an effective
retrieval.1 and perform frozen embryo transfer resulting in birth of endometrioma, and high risk of infection undergo Beta-
alternative to general anesthesia.9
world’s second IVF Baby in 1978.3 dine vaginal cleaning followed by saline wash.
Matsota P et al. (2015) reviewed potential toxic effects of
BRIEF HISTORY different anesthetic techniques [locoregional, general anes- Prophylactic Antibiotic
Laparotomy was performed to harvest first oocyte in vitro
COMPARISON OF DIFFERENT ROUTES4 thesia (GA), and monitored anesthesia care (MAC)], differ-
but due to high morbidity it was replaced by laparoscopy. Table 35.1 shows comparison of different routes. ent anesthetic agents, and alternative approaches (especially Routine use of prophylactic antibiotic before TVOR is
acupuncture) on the IVF outcome. No conclusions could be debatable with conflicting evidence. Severe pelvic infec-
Table 35.1. Comparison of different routes of oocyte retrieval. drawn regarding different anesthetic techniques. Studies tions are associated with significant morbidity.
Parameters Laparoscopic guidance Transabdominal Transvaginal approach about GA and acupuncture showed conflicting results. MAC A survey done showed wide variation in type and dura-
and locoregional anesthesia were found to be safe alterna- tion of antibiotics used by clinicians at the time of TVOR.
19
Type Invasive Noninvasive Noninvasive
General anesthesia Required Required Optional tive choices with evidence of improved outcome.10 • Forty-three percent of clinician preferred only single
Visualization and Only surface follicles seen Most follicles seen All follicles seen except Cochrane 2013 reviewed 21 randomized controlled dose
accessibility of follicles in high placed ovaries trials and concluded (i) no one particular method or tech- • Nine percent gave antibiotics for 5 days.
Discomfort High Low Low nique is better than another; (ii) simultaneous use of more
Complication rate High Low Low than one method of pain relief and sedation has more Recommendation
Hospital stay Day care Day care Day care effective pain relief than single modality used alone; and • Prophylactic antibiotics, if not routinely used, should
Cost High Low Low (iii) method of anesthesia should depend on the patient be given to patients with high risk of infection—patients
Oocyte retrieval rate Approximately 50% >80% >90% and the clinicians and resources available.11 with history of pelvic inflammatory disease (PID), previ-
Advantage Minor procedures Patient friendly. Patient friendly.
ous surgery, pelvic adhesions, and endometrioma.20
like adhesionolysis, High oocyte recovery rate Minimal complication. Drugs to be Avoided • Donors: The incidence of infection reduced from 0.4 to
coagulation of endometri- even when pelvic adhesions High oocyte recovery rate even
otic spots can be done. were present. when pelvic adhesions were present. Inhalational Agents: Use in Assisted 0%, when prophylactic antibiotic was given.21
Disadvantage CO2 exposure to Ovaries are difficult to assess Difficulty in oocyte aspiration in high
oocytes leading to acidic in obese patients. Needle placed ovaries. Reproductive Technique Controversial Dynamics of Follicular Aspiration
environment. may move away from path. Can injure bladder, ureter, blood
Majority of studies found use of halogenated fluorocar- In 1996, Laboratories of Cook medical technologies con-
Ovaries may not be Multiple structures in vessels, etc.
assessable due to pelvic between ovary and probe. bons with N2O in ART resulted in decreased cleavage rates ducted experiments to study effect of velocity and pressure
adhesions. Hematuria is most common and increased abortion rate.12 of aspiration system on bovine oocyte.22
complication.
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310 The Infertility Manual Oocyte Retrieval and Embryo Transfer 311
Salient Features of the Study vacuum after withdrawal of needle, this oocyte pre- Plastic tubing was replaced by polyethylene tube. Slowly done without removing the needle from follicle. Disad-
sent at tip is exposed to high velocity and turbulence, over years thumb valve changed to foot pedal. Nowadays, vantages of DLN are time consuming and large amount
• The oocyte experiences different pressure level at the thereby high risk of damage due to sudden change of of culture media is used.
in the advanced suction machines negative pressure can
exit of the aspiration device than experienced at the pressure. be adjusted digitally or manually. These machines create
needle tip. Pressure at needle tip is only 5% of that of • Needle and tubing should not have dead space, i.e. Diameter of needle Diameter of needles available vary
low volume vacuum at predetermined negative pressure
the suction machine. Pressure steadily increases from air. This dead space (1.0–1.2 mL) should be filled with from 16 to 20 gauge with length between 33 and 35 cm.
for oocyte recovery at low turbulence. Some machines
needle tip to collecting system and to suction machine. media before beginning of oocyte aspiration. This
may have high vacuum emergency button to clear clogged Outer diameter—16-gauge (1.6 mm), 17 gauge
Thus, the oocyte faces increasing pressure till it reaches is to avoid follicular fluid to come in contact with air
aspiration needles. The negative pressure created ranges (1.4 mm), 18 gauge (1.0 mm), and 20 gauge (0.9 mm). Inner
collecting tube. directly which results in frothing with nonlaminar flow
from 0 to 400 mm Hg. diameter varies as per needle wall thickness.24 In standard,
• Factors determining the pressure experienced at dif- and risk of damaging the oocyte.
needle is 17 gauge with internal diameter of 1 mm.
ferent points in the needle are length and gauge of • Even in laminar flow, high velocity of fluid can dam-
Various studies have shown that 0.8 mm internal
needle, length and gauge of aspiration tube, volume of age oocyte. The velocity of the fluid decreases from Follicular Aspiration Set diameter of aspiration needle gives good oocyte recovery
the collection tube, and size of the vacuum reservoir in the center of the needle toward the wall of needle
the pump.23 (
periphery). Thus the outer layer of COC may be For oocyte aspiration, ready to use follicular aspiration rate without damaging oocyte.25 Cohen et al. (1986) said
• The velocity and flow rate decrease when either the stripped due to “drag effect”. sets are available. They contains needle attached to aspi- reducing the needle’s internal diameter further (<0.8 mm)
internal diameter is decreased or the length of the • The authors emphasized that intact cumulus is impor- ration tubing which ends in cork. The cork snuggly fits may damage the oocyte as well as will make procedure dif-
needle is increased. Hence, to maintain the flow rate, tant factor to make the oocyte resistant to damage. into collecting tube and is connected to suction source to ficult and less efficient.26
velocity needs to be increased in needles with smaller • Spinning movement of needle may damage the oocyte complete the circuit. These sets are mouse embryo assay A prospective randomized study comparing effect
internal diameter or when the length of needle is by its edge in cases where oocyte is “scraped” from the (MEA) and endotoxin tested (LAL) tested and available for of different gauges of needle recommended 19 and
increased. follicular wall especially in collapsed follicle or small single use only. 20 gauged needles due to acceptable ovum pick-up (OPU)
• Laminar flow is less traumatic to cumulus oocyte com- follicles, where the needle size is larger than follicular time, oocyte recovery rate, and lower bleeding loss. They
volume. rejected 21 gauge needle due to low oocyte retrieval rate
plex (COC). The Needle
• It takes few milliseconds for system to equilibrate and and technical difficulties in bending and puncturing the
flow to become laminar. The time taken depends upon THE EQUIPMENT A good needle should be cost effective, causes less pain to follicles.27
vacuum pressure, diameter of needle, and follicular patient, and easy to manipulate. Varieties of needles are Kushnir et al. (2013) study compared 20 G/35 mm
size. Maximum flow of fluid is achieved in steady state
Ultrasound Machine commercially available in the market. The needle can be (thin) with 17 G/35 mm (standard) needle and concluded
of pressure. Hence, vacuum should be applied just Frequency should be minimum of 5–7 MHz to give effec- divided into tip, body, and handle. These needles differ that needle diameter does not affect oocyte recovery rate,
before entering the follicle. tive resolution to visualize uterus and adnexa. in length, size of internal diameter (gauge), sharpness at even patients who are obese or with diminished ovarian
• Follicular pressure was dependent on the shape, size, bevel tip, and type [single lumen needle (SLN) and double reserve. However, the operating time was significantly pro-
and position of the follicle. There is linear relation- Vaginal Probe and its Length lumen needle (DLN)]. The needles are made of stainless longed in thinner needles.28
ship between follicular size and pressure, larger the steel. Wikland et al. (2011) on comparing thinner-tipped
Total length should be of 40–50 cm with gentle curve at
follicle—higher the pressure. On use of blunt needle, needle (50 mm near tip) with standard needle found
handle for easy maneuvring of probe.
the intra follicular pressure may increase as high as Single and Double Lumen Needles significantly reduced overall pain and vaginal bleeding,
60 mm Hg. Hence, small amount of follicular fluid may without prolonging procedure time or influencing the
spill when needle enters follicle. Recommendation
Biopsy Guide In initial years of ART, SLN was used. Recovery rate oocyte yield.29
is to use of sharp needle to avoid spillage of follicular Should fit tightly on vaginal probe and allows easy friction of oocytes was poor with laparoscopic guide oocyte
fluid and lose of oocyte. free gliding of needle through needle hole. retrieval and the follicles were frequently flushed. Flush- Needle sharpness A sharp needle causes less trauma
• When the needle enters the follicle, the open aspira- ing through SLN required removing the cork from col- to ovarian cortex and ovarian tissue and thus decreasing
tion system becomes closed system. Probe Cover lecting tube and retrograde injection of the flushing fluid postoperative pain and bleeding.
• Vacuum to be started just before needle enters follicle through the needle into follicle. Flushing leads to follicle
as to avoid spillage of follicular fluid and lose of oocyte.
A variety of probe covers available. They should be sterile, Echogenic tip For better visualization of needle tip by
getting refilled first with retrograde aspiration fluid pre-
nontoxic, and can be made of latex or latex free material. ultrasound, the needles are etched near the tip either with
• As the follicle collapse, the pressure inside the fol- sent in SLN followed by flushing media. The two main
They are E beam sterilized. laser or embossed. This makes the needle tip more echo-
licle increase almost equivalent to pressure in nee- disadvantage of this were, firstly oocytes if present in the
dle. When vacuum is deactivated inside the follicle, genic and enhances ultrasound resolution during oocyte
the follicle pressure remains high whereas that of Suction Machine dead space of needle and aspiration tubing (1–1.2 mL) aspiration.
were at the risk of damage due to turbulent to and fro
needle decreases to atmospheric pressure. Hence, In early years, manual suction was created using syringe movement and secondly it prolonged the operating time
on withdrawal of needle from follicle after vacuum attached to needle and plastic tubing. Manual pressure
significantly. To make this process of flushing more Handle
is deactivated, there can be back flow of fluid toward was irregular hence had high risk of oocyte damage. This
efficient, needles with double lumen were introduced. Arrow marking on handle indicates bevel point. Handle
the follicle and lose of oocyte which may be pre- was later replaced by special aspiration unit where suction
sent at tip. On the other hand when we deactivate machine could be turned on and off using thumb valve. Advantage of DLN was flushing and aspiration could be shape should be ergonomic, facilitating rotation.
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312 The Infertility Manual Oocyte Retrieval and Embryo Transfer 313
The Aspiration Tubing stimulation on day 2 when cycle starts Friday–Tuesday, Table 35.2. Check list prior to oocyte retrieval. abdomen due to chemical reactions. If punctured acci-
otherwise day 3 and if required we can delay hCG by 1-day First checklist: Before stimulation
dently, we prefer to give broad spectrum antibiotics for
• Translucent tube is made of Teflon to avoid weekends.35 3–5 days.
• Attached to needle on one side and ends in silicon cork Patient’s history Medical risk factors, e.g diabetes
mellitus (DM)/heart disease/bleeding • Minimal tissue should lie between the transducer and
on other side Agonist Cycle follicle.
disorder/screen positive
• Length vary from 50 to 100 cm. • Presence of hydrosalpinx, paraovarian cysts, or
Any allergy/hypersensitivity to any drug
It is easy to plan without compromising the results. Options
Anticipation of Frozen pelvis, anatomical defects encysted fluid in pelvis should be predocumented.
available are:
Collecting Tube difficult OPU Transposed ovaries
• Delay in start of stimulation Procedure
Have a volume of 10 cc and are made of polystyrene. They Previous oocyte retrieval rate
• Delay in hCG for a day.
are kept in test tube warmer 1 hour before the procedure Risk of ovarian hyperstimulation syndrome
1. Check for ultrasound machine and biopsy line.
starts. Previous failures
Antagonist Cycle Investigations
2. Collecting tubes to be prewarmed in alloy blocks
before start of TVOR.
It is trickier to plan in antagonist cycle. Barmat et al. For Wife
Test Tube Warmer reported 22% weekend retrievals in antagonist cycle
3. Set the suction pressure. In our unit, we keep suction
Blood group Hb electrophoresis pressure around 110 to 120 mm Hg.
Simple heating blocks made of autoclavable anodized against 10% in agonist cycle in spite pretreatment with Hemogram Random blood sugar 4. The probe is covered with sterile probe cover followed
aluminum to hold the collecting tube. They maintain the contraceptive pills.36 Options available are: Prolactin Thyroid stimulation hormone by biopsy guide.
temperature of collecting tubes around 37°C. • Pretreatment with combined oral contraceptive pills Rubella IgG Bleeding and clotting time 5. The aspiration set (needle and tube) is flushed with
• Estrogen pretreatment For Both Partners media to remove the dead space to ensure laminar
PREPARATION OF OOCYTE RETRIEVAL • Delay in hCG by 1 day. HIV I and II, hepatitis B antigen, anti-HCV, VDRL flow.
For Husband 6. The probe is inserted in vagina. Follicles and neigh-
For successful ART outcome, oocyte trigger timing dur-
ing ovarian stimulation is one of the crucial steps. If the PREPARATION BEFORE Semen Analysis boring pelvic structures are seen.
Preanesthetic checkup 7. The ovary which is easy to access is aspirated first.
oocyte trigger given early, it can result in higher percent- OOCYTE RETRIEVAL
Consent forms The needle should be removed only after aspirating
age of immature oocytes and if late it can lead to postma-
We at our clinics have three checkpoints to make sure eve- Semen for cryofreezing as backup sample* all the follicles from that side. The needle is reflushed
ture oocytes.
rything goes smoothly. On the day of hCG 2nd checklist and reinserted for other ovary. The vaginal punc-
The choices for trigger available are:
The three checkpoints are: Quick re-look at first checklist tures should be kept to minimal. We prefer only two
• In agonist cycle: human chorionic gonadotropin (hCG) punctures—one for each side. This reduces the risk of
1. Before stimulation Inform the embryology laboratory
• In antagonist cycle: hCG, agonist trigger, and dual trigger complications. Some authors commence with right
Important points to remember before trigger: 2. On day of hCG Name of patient, Husband name, Hospital ID number
ovary followed by left ovary.
• The ovulatory trigger is administered when more than 3. On day of OPU (Table 35.2). Self -IVF or donor OPU
8. Always keep the needle tip in the center of follicle. The
three leading follicles ≥17 mm in mean diameter30 Time of trigger
pressure should be constant and vacuum should be
• No evidence of difference between recombinant hCG PROCEDURE Number of oocytes expected released only after coming out of follicle.
(rhCG), urinary hCG (uhCG) or recombinant human ICSI/IVF
luteinizing hormone (rh LH) in obtaining final follicu-
Rules to Follow during Ovum Pick-up 9. For beginners: Avoid vacuum when needle is in ovar-
Plan for fresh transfer or freeze all ian tissue while focusing for follicular plane. This is to
lar maturation31 • The needle should be kept in ovarian tissue only and avoid blockage of needle with ovarian tissue.
Husband sample or donor
• There is no difference in ART outcome whether 5,000 U always move needle when the needle tip and path is 10. After finishing the procedure, refocus on adnexa and
Screening status
or 10,000 U hCG was used32 clearly visualized. pouch of Douglas for any fluid collection.
On day of OPU: Third checklist
• There is enough evidence to suggest that oocytes can • Differentiate other pelvic structures from follicle: 11. In last, put speculum in vagina to check bleeding from
be aspirated between 35 and 38 hours post-trigger Iliac vessel: May give look of follicle but on r otating Confirm patient identity
puncture sites before shifting the patient out from
without compromising oocyte.33,34 the probe by 90° the round appearance (trans- Check consent form
theatre.
verse section) of iliac vessel will appear elongated Review first check list to see risk factors
SCHEDULING/AVOIDING WEEKEND (
longitudinal section). Doppler ultrasound may See the last follicular monitoring
Temperature Control
also help to differentiate. Confirm date and time of trigger by patient
OOCYTE RETRIEVAL Bowel: Internal area is echogenic and on focusing Spindles and chromosomal of oocytes are temperature
(HCV, hepatitis C virus; HIV, human immunodeficiency virus; ICSI,
At times, oocyte retrieval may need to planned due to the bowel for few seconds peristaltic movement intracytoplasmic sperm injection; IVF, in vitro fertilization; OPU, ovum sensitive. Suboptimal conditions at the time of oocyte
pick-up; VDRL, venereal disease research laboratory). retrieval can affect the fertilization and implantation abil-
certain reasons, e.g. batch IVF, nonavailability of embry- may appear.
*In our unit we offer semen freezing as a back sample to all are
ologist/clinician on certain days or avoiding weekend. • Avoid puncturing endometrioma or dermoid cyst. patients. There have been incidents when husband is not able to
ity of embryo formed. Research has shown that spindles
Michael J Levy et al. in an RCT recommended to start This can act as a nidus of infection or can cause acute produce sample on demand and semen quality has been poor. and chromosomes of human oocytes get disrupted when
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314 The Infertility Manual Oocyte Retrieval and Embryo Transfer 315
oocytes are exposed to room temperature.37 Rewarm- minimal stimulation ART with less number of follicles may only 1.7% cases. It should be performed only when nec- Table 35.3. Complications of Oocytes Retrieval.
ing was found to reverse this effect38 but animal research benefit from flushing.45,46 essary. Patients with pelvic adhesions or altered pelvic
Most common complications
has shown presence of incomplete recovery39 leading to Only two randomized trial studies have compared anatomy with abnormal ovarian location are at higher
Vaginal hemorrhage
reduced pregnancy rates. flushing with aspiration in poor responders. There was risk of transmyometrial oocyte retrieval. The oocyte yield,
Pain
Redding et al. 2006 studied the effects of IVF aspiration nonsignificant increase in the total number of oocytes peritoneal bleeding, and ongoing pregnancy rate were
on the temperature, dissolved oxygen levels, and pH of fol- retrieved with follicle flushing47 with pregnancy rate being comparable with regular TVOR where myometrium is Pelvic infection
licular fluid. They found an average of 7.7°C drop in tem- significantly low in flushing group.48 However, both stud- not injured.52 Peritoneal hemorrhage
perature from follicle to collection tube (not prewarmed). ies are limited by a small sample size of 30 and 50 patients, Less common complications
More than 80% of this fall in temperature occurred in col- respectively. Immature Oocyte Retrieval for Ureteric injuries
lecting tube and was due to water evaporation from folli- Till date, there are no randomized controlled data Vertebral osteomyelitis
cular fluid. The dissolved oxygen amount of follicular fluid evaluating follicle flushing in natural cycle or minimal
In Vitro Maturation
Ovarian torsion
was found to increase due mixing of aspiration fluid with stimulation ART. Immature oocytes can be retrieved from follicles measur-
Injury to bowel/appendix
air in the collecting tube. Also the pH was increased by an It is recommended that more RCTs are needed to ing 2–10 mm. The technique is similar to regular TVOR
Rupture of endometrioma/dermoid cyst
average of 0.04 ± 0.01 pH unit.40 assess the benefit of follicle flushing in poor responders, with few differences. The follicles are small with less fol-
Nowadays the aspiration sets come with Teflon coat- natural cycle ART, and minimal stimulation ART. licular fluid and stiffer walls. Hence, require small gauge Anesthesia Complication
ing which act as insulator, collecting tubes are prewarmed, needles (20–24 gauge needles or Steiner-Tan pseudo dou-
and room temperature is toward body temperature to Follicle Curretting ble lumen needle) with minimal dead space. The pressure
reduce the temperature fluctuation. inside immature follicles are lesser than large follicles, be 8.6%.54 Vaginal bleeding is from either from vaginal lac-
Follicle curretting is gentle and rapid rotation of the needle thus aspiration pressure is kept around 40–60 mm Hg. The eration or punctured vessels.54 These minor vaginal bleeds
in a clockwise and anticlockwise manner inside the folli-
Recommendation procedure time is more and require multiple punctures are managed by pressure, vaginal packing/tamponade,
cle after complete aspiration of the follicular fluid during as the needle gets frequently blocked due to thick ovarian and sometimes may require hemostatic stitch.
• Prewarm collecting tubes. TVOR.49 Theory behind follicle curetting is to dislodge and stroma.
• Do not fill the collecting tube during aspiration above aspirate COC which sometimes gets trapped in collapsed
Precaution Minimize number of vaginal punctures
Seyhan A et al. (2014) on comparing complication rates
the level of heating block. wall of follicle at time of aspiration. Till date only one retro- and pain scores after TVOR procedures for in vitro matu-
• Transport immediately to embryologist. spective study by Dahl et al. has evaluated this technique.
Pelvic Infection
ration (IVM) and IVF cycles found IVM TVOR to require
• Keep the collecting tube in heating block and not hold In 285 cycle studied, there was 22% increase in the number more punctures per ovary with prolonged procedure time Pelvic infection is second most common complication of
in hand during aspiration. of oocytes recovered and mature oocytes (MII) obtained but was well tolerated. Also, complication rates were com- TVOR.55 Incidence of pelvic infection post-TVOR is low but
• Keep the room temperature toward body temperature. without any damage. Thus, the number of embryos frozen parable with that of IVF OPU.53 if untreated it could lead to serious morbidity like acute
in curetting group was higher. However, the operating time abdomen and pelvic abscess.56
Follicle Flushing was increased by 3 min in curetting group. Authors could Complications Incidence of pelvic infection ranges from 0 to 1.5%;
not comment on pregnancy rate because of their freeze all majority of studies have quoted the incidence around
The oocyte recovery rate by laparoscope was only 50%. The technique of TVOR is easy to learn and perform with
policy.50 0.3–0.6%.
Hence, to improve the numbers of oocytes retrieved cli- minimal complication making it gold standard. TVOR is
Rotation movement of needle within follicle is believed
nicians started flushing and aspirating the follicles mul- considered to be a safe minor surgery but every surgi-
Source of infection:54,57
to decrease the chances of the needle lumen from getting
tiple times. It was presumed turbulence caused by flush- cal procedure do have complications. Both minor and • Direct inoculation of ovary or peritoneal cavity by nee-
prematurely blocked by wall of collapsing follicle or large
ing helped in dislodging the COC and aided in oocyte major complications have been reported in literature. To dle through nonsterile vagina (most common)
debris, better visualization of needle tip, and better pen-
recovery. avoid them, we should take all the precautions recom- • Reactivation of latent infection especially in patients
etration into follicle.51
Data from several nonrandomized control studies mended. The patients should be counseled regarding with history of PID or chronically infected ovary
It is also believed that removal of granulosa cells dur-
showed that flushing improved oocyte yield.41,42 How- risks and complications of treatment before starting the • Secondary to bowel injury at time of TVOR.
ing follicle curetting may decrease risk of ovarian hyper-
ever, 2010 Cochrane review showed no benefit of follicle stimulation.
stimulation syndrome (OHSS). High risk patients:
flushing on oocyte recovery rate and ongoing pregnancy Major complication like excessive bleeding and pelvic
In view of lack of RCT studies, it is difficult to recom-
rate.43 infection though rare but are associated with significant • Patients with history of PID
mend follicle curetting.
A systematic review and meta-analysis done recently morbidity and mortality (Table 35.3).54 • Patients with endometriosis and endometrioma
(2012) comparing flushing with aspiration only also found • Patients with history of pelvic surgery/adhesions.
no difference in number of oocyte retrieval rate and preg- Special Situations
nancy rate in normo-responder. The operating time was
Vaginal Hemorrhage Symptoms:
more in flushing group.44
Transmyometrial Oocyte Retrieval Most frequently encountered complication of TVOR. Ste- • Mild: Abdominal pain, fever, leukocytosis, vaginal
They further suggested that selected group of patients Davis et al. (2004) after reviewing 5,115 IVF cases found phen John Bennett et al. (1993) in their prospective study discharge
which include natural cycle ART, poor responders, and that transmyometrial oocyte retrieval was required in of 2,680 cycles of OR found incidence of vaginal bleeding to • Major: Acute abdomen/peritonitis, pelvic abscess.
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Prevention: women with polycystic ovary syndrome were at higher risk hyperstimulated ovaries, radiation therapy, advanced state of • Before inserting the needle for TVOR to use Doppler
of hemorrhage. Other isolated risk factors were activated malignancy, and anatomical anomalies of the urinary tract. for intervening structure, if any.
• To minimize vaginal puncture: Studies have empha-
partial thromboplastin, decrease XI and XII concentra-
sized the importance of keeping the vaginal puncture
tion, factor VIII deficiency, intravenous diclofenac treat- Symptoms Vertebral Osteomyelitis
to one each per ovary.58
ment before TVOR, and anticoagulation therapy.61 • Lower abdominal and pain in flanks
• Prophylactic antibiotic: Controversial, some clinics • Three case reports have been published till date. The
avoid antibiotics, some give routinely and others have • Suprapubic pain route of infection is presumed to be either directly
reserved for high risk cases. There is no consensus on Symptoms • Urinary symptoms like dysuria, urgency, bladder
by OPU needle or hematogenous spread. The organ-
duration and type of antibiotic. Commonly used ones • Diffuse abdominal pain, weakness, nausea, vomiting, tenessmus isms isolated were different—S. faecalis, E. coli, and
are doxycycline, metronidazole, and first generation and severe weakness within 0–28 hour. • Frank hematuria
S. aureus in the three cases.74-76
cephalosporin.59 Use of prophylactic antibiotic, how- • Cardiovascular instability with decreasing hemocrit lev- • Nausea and vomitingFever may be a presenting sign
• Chief complaint: low Back ache with fever. Pain may
ever, does not reduce the incidence of pelvic infection els suggests ongoing intra-abdominal bleeding. Emer- • Clear watery vaginal discharge.
radiate to thighs and knees.
to 0%. gency laparoscopy should be performed in such cases. • Diagnosis: Blood culture and MRI.
Examination
• Vaginal disinfectants: Already discussed. • Management was conservative with antibiotics. In
• Avoid puncture to bowel: Recognize bowel with peri- • Tachycardia
Diagnosis • Abdominal examination: Lower abdominal tender-
resistant cases, surgery may be needed.
staltic movement. • Hence, in patients complaining of backache after
Ultrasound, CT scan. ness with guarding. Signs of peritoneal irritation may
Management There is high variation in time when TVOR, vertebral osteomyelitis should be considered in
or may not be present.
patient presents with symptoms of infection. The diagno- differential diagnosis.
sis becomes more challenging and management difficult, Treatment Diagnosis
especially if symptoms of infection presents at the time of • Conservative management: First line of treatment • USG, MRI, computerized tomography (CT) Scan
Ovarian Torsion
pregnancy. • Surgical management: Laparoscopy • To look for pelvic collections, dilation of the urinary tract The reported incidence of ovarian torsion is 2.3 % in non-
• Topical haemostatic agents may be helpful. Some- (KUB), and extravasations of contrast material to the ret- pregnant women and increases to almost 16 % in pregnant
Diagnosis: Leukocytosis, ultrasound (USG), magnetic res- times, ovariectomy may be needed women. This incidence increased to almost 6 % after ART
roperitoneum. CT scan and MRI should be done in symp-
onance imaging (MRI). • Angiographic uterine artery embolization by an inter- stimulation and reached as high as 16 % with OHSS. Almost
tomatic patient where USG may not show any pathology.
ventional radiologist may be a better option, if availa- • Retrograde cystography and an excretory urography. 90% ovarian torsions occurred 1st or 2nd trimester of preg-
Treatment:
ble. This may avoid abdominal surgery and has greater nancy and remaining 10 % occurred in the 3rd trimester.77
• Pelvic abscess: Medical management with antibiotic. Differential Diagnosis
chances of preserving fertility.
May require abscess drainage (USG/computerized Symptoms78,79
tomography guided) Prevention Adnexal torsion, pelvic infection or abscess, bowel injury,
• Acute abdomen/peritonitis: Surgical management hematoma formation, pelvic vessel injury, and ovarian • Abdominal pain, nausea, and vomiting
• Ask history about prolonged bleeding or episode of cyst rupture. • Acute abdominal pain is most common presenting
(laparotomy or laparoscopy may be needed. Patients
unproved bleeding/clotting symptom, occurring in more than 80 % of cases. Pain
requiring ovariectomy have been reported in literature). Treatment
• Routine evaluation of coagulation system often starting suddenly at night and persist for more
• Always keep the tip of needle in view • Early consultation with urologist than 24 hours
Pain • Minimal vaginal puncture. • Treatment included conservative management with • The diagnosis becomes challenging, if OHSS coex-
Studies have found TVOR a well tolerated procedure, only retrograde ureteral stenting or nephrostomy tube. ist due to nonspecific symptoms. Delay in diagnosis
2–3% patients suffered from severe pain. The probability of Ureteric Injury (Ureteric Fistula/Ureteric Complicated cases may need either open or laparo- increases the risk adnexectomy.
pain increased with number of oocytes retrieved.1 Stricture/Bladder Injury) scopic ureterovesical reimplantation.73
Investigations
The incidence of ureteric injury post-TVOR is very low Prevention
• Total leukocyte count (TLC): Mild leukocytosis. Pre-
Peritoneal Hemorrhage around 0.1%.1 This is surprising low given its anatomical
• To maintain the needle guide in lateral position away caution TLC can be raised in OHSS/pregnancy (mild
position—lateral to cervix.62 Fourteen cases of ureteric injury
Incidence of severe intra-abdominal bleeding is less than 1 from dangerous anterior structure.62 On reviewing all raised TLC may be normal level in pregnancy).
have been reported so far. The insult to excretory system can
in 1,000 procedures. Studies have shown that up to 200 mL 14 cases, we found that right sided ureteric injury is much • Ultrasound: Shows enlarged ovaries with or without
be bladder injury with hematoma and retention of urine,63
of free fluid in peritoneal cavity on evaluation by ultra- more common than left. We hypothesize that this may be signs of ovarian torsion. Doppler flow ultrasound will
ureteric obstruction,64 ureteric fistula,65-72 and uroperito-
sound is normal post-TVOR.60 Intraperitoneal bleeding due to fact that it is uncomfortable to maintain the lateral further help in diagnosis.80 False negative results are as
neum.73 The injury to right sided ureter was more common.
can be caused by injury to ovarian surface, ovarian vein, position of probe while aspirating right ovary because of high 61 % for pregnant women and 45 % for nonpreg-
iliac, or sacral vessels. Risk factors Risk factors are uterine size equivalent to crossing of hands. Hence, we invariably turn the vagi- nant women.81 Hence, diagnosis should be based on
Kazem Nouri et al. in their systemic review of severe 12 weeks pregnancy or more, ovarian cysts 4 cm or larger, nal probe anteriorly making the needle go anterolateral, history, symptoms, and signs and not solely on Dop-
hemoperitoneum by ovarian bleeding reported that lean endometriosis, PID, history of pelvic surgery, enlarged thereby increasing the probably of injuring right ureter. pler flow ultrasound as suggested by some authors.
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318 The Infertility Manual Oocyte Retrieval and Embryo Transfer 319
Differential Diagnosis Management: proper counseling and consents before the procedure, • Propagated by Kato et al. 199385
records to be maintained, and all precautions recom- • Due to invasive nature of the procedure, it invokes
Appendicitis, renal or urethral calculi, renal colic, and 1. Should be documented at time of follicular monitor-
ing. Patient should be properly counseled mended during procedure. higher junctional zone contractions leading to lower
obstructive bowel disease.
2. Adequate help to be available on day of TVOR pregnancy.85,86
Treatment
3. Most of the ovaries become accessible with abdomi-
EMBRYO TRANSFER
Spitzer et al.79 recommended laparoscopic de-rotation of nal pressure and probe manipulation
Embryo Intrafallopian Transfer (ZIFT/GIFT)
Embryo transfer (ET) is the crucial step involved in ART.
adnexal torsion even when ovaries are already ischemic, 4. Pulling of cervix with tenaculum Suboptimal ET Techniques could be a cause of up to 30% This technique requires a laparoscopic approach to the
discolored, and Doppler flow is absent as the first-line treat- 5. Reverse Trendelenburg position with lateral tilt of table of all cycle failures.84 fallopian tube.
ment. They found that in 73 % of cases, de-rotation was suf- 6. If rare cases, transmyometrial aspiration
ficient to preserve ovarian function and fertility. For Arena 7. In some cases, transabdominal oocyte aspiration may TYPES OF EMBRYO TRANSFERS Advantages
et al., if there was complete absence of blood flow in the be needed especially when ovaries are high placed.
The ET can broadly be divided into: • Theoretically, fallopian tubes are the physiological site
ovarian vessels then adnexectomy should be performed.80 Rare complication is rectus sheath hematoma.
for fertilization and early cleavage due to the presence
1. Transvaginal transcervical intrauterine ET
of various growth factors.
Cullen’s Sign (Periumbilical Hematoma) No Oocytes in Follicular Aspirate 2. Transvaginal transmyometrial ET • Lower chances of embryo expulsion due to endometrial
3. Laparoscopic zygote intrafallopian transfer (ZIFT)
Bentov et al. reported two cases with periumbilical hema- If after aspirating one ovary, no oocytes retrieved in folli- contractionsAvoid contamination with vaginal flora.
4. Transvaginal subendometrial embryo transfer
toma post-TVOR. They concluded that appearance of peri- cular fluid. We need to differentiate between false empty
follicular syndrome (FEFS) and genuine empty follicular Disadvantages
umbilical hematoma suggest retroperitoneal hematoma of
syndrome (GEFS) (Flowchart. 35.1).
Transvaginal Transcervical Intrauterine ET
benign nature and will resolve within 2 weeks.82 • Includes surgical as well as anesthesia complications
When agonist is used as trigger it is difficult to distin- • Most widely used • Equivalent pregnancy rate (PR)
Trouble Shooting guish between GEFS and false EFS as we cannot find out • Simplest • Higher ectopic pregnancy rates
whether patient has administered injection properly in • Least invasive • Increases work load of clinic
Flow of aspiration fluid slow or stopped suddenly: dose prescribed or not. • Performed by loading the embryo(s) in the catheter • High cost
• Check suction pump is working and generating together with small volume of media (20 µL) before the
desired negative pressureEnsure all the connection of Conclusion catheter is introduced through endocervical canal, into ZIFT: Meta-analysis by Habana et al., 2001, FS87
aspiration set with collecting tube and suction pump the uterine cavity, where the embryos are expelled gently
Transvaginal oocyte retrieval is simple surgical procedure, Meta-analysis showed ectopic pregnancy rates of 3% in
are secure • Steiner pistol device was introduced for reproducible
easy to learn with minimal complications. We recommend ZIFT as compared to 1.5% in transcervical ET [odds ratio
• Look for cracks in collecting tubes amount of fluid transferred together with embryos into
(OR), 2.05; 95% confidence interval (CI), 0.21, 20.22]
• Aspiration tube kinked or damaged uterine cavity during ET, avoiding pressure changes
Flowchart. 35.1: Stepwise approach if there are no oocytes in the fol-
(stick slip effect). Physician is able to choose pressure,
• Check needle placement—not in follicle/blocked of licular fluid. Hysteroscopic Subendometrial Embryo
follicle wall velocity, and amount of fluid to be transferred with the
click of the trigger. Main advantage—fixed volume and Delivery (SEED)
• Rotate the needle and realign
• Press the emergency button to increase the pressure to pressure during ET and physician can concentrate on • Under hysteroscopic guidance, embryos are trans-
aspirate blood clot/granulosa cell from needle tip the site of embryo release without worrying about the ferred directly into endometrial stroma.
• Disconnect the cork, do retrograde flushing to clear other variables. • First described by Kamrava et al.88 His study showed
the needle. clinical PR of around 33%.
Intravaginal Transmyometrial • Disadvantages: Special training is required for hystero-
Suction machine not working
Embryo Transfer scopic ET; the CO2 used in hysteroscopy may lead to
• Theodoros Kalampokas et al. (2015) used 20-mL lowering of pH of the media resulting in reduced PR,
syringe applied to single lumen follicle aspiration • When transcervical ET becomes difficult or impossi- increased cost, may cause excessive discomfort and
needle to create negative pressure. They were able to ble (cervical agenesis, severe cervical stenosis, acutely
anxiety to patient, and equivalent clinical PR and live
retrieve 8 oocytes of 10 follicles.83 anteverted/retroverted fixed uterus)
birth rate.89
• Disadvantage: Flushing cannot be done. • Uterine cavity is entered through myometrium and
endometrium using a needle similar to the one used FACTORS AFFECTING EMBRYO
for oocyte aspiration under TVS guidance. Needle is
Inaccessible Ovaries provided with an obtorator, to prevent the blockage
TRANSFER RESULTS90
Accessibility to ovaries is sometimes difficult in patients due to tissue impingement during puncture. • Type of catheter used
with endometriosis/pelvic surgeries/adhesions or con- S. hCG, serum human chorionic gonadotropin; UPT, urine pregnancy • It is available commercially as Towako Transmyome- • Ultrasonic guided vs. clinical touch method
genital reproductive abnormality. test. trial ET Catheter System (Cooks Medical) • Experience of clinician
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320 The Infertility Manual Oocyte Retrieval and Embryo Transfer 321
• Catheter loading technique: Air-media versus media endometrial cavity, and anticipate potential problems 2. Straightening of the uterocervical angle (if required): Wallace catheter (Marlow Technologies, Wil-
only at ET. a. By changing patient position: No effect on PR loughby, OH)
• Specific media used, e.g. Embryo Glue • However, a mock transfer remote from the actual ET b. Full bladder during transfer: No effect on PR Frydman 4.5 ultrasoft (Laboratoire CCD, Paris)
• Volume of medium loaded is done under different circumstances and may not be c. By using a tenaculum: No RCTs found • “Hard” embryo catheters:
• Blood/mucous on catheter reflective of actual conditions encountered on the day 3. Check the trial transfer notes prior to embryo transfer TDT (Laboratoire CCD, Paris, France)
• Site of embryo transfer of ET. 4. Per speculum examination to check the cervical and Frydman Classic (Laboratoire CCD)
• Use of a tenaculum/Allis forceps • An RCT by Mansour et al. has clearly shown a signifi- vaginal discharge/mucous Tomcat (Kendell Health Care, Hampshire, MA)
• Removal of all mucus from cervix cant decrease in Difficult Embryo Transfers in women 5. Embryo afterloading Tefcat (Kendell Health Care)
• Hysteroscopy before ET for cavity assessment in whom prior Trial ET (TET) was conducted which • An outer catheter is placed at, or just past, the Rocket ET catheters (Rocket Medical,Watford, UK)
• Leaving catheter in place for at least 1 minute even translated into a significant increase in PR.93 internal cervical os. Once the position is confirmed A review and meta-analysis of prospective trials com-
• Time taken for ET and the passage up to internal os is negotiated, the paring different catheters used for embryo transfer.97
• Rest after transfer Timings of Trial Embryo Transfer inner sheath containing the embryos is passed.
• Trial/mock transfer before treatment This gives the provider the benefit of an immedi- • Soft ET catheters are associated with higher clinical PR
• Prior to stimulated cycle as compared to firm/rigid ET catheters [relative risk
• Uterine relaxants ate mock transfer while minimizing manipulation
• On the day of OPU (RR) 1.34; 95% CI, 1.18–1.54].
of embryos and possibly reducing trauma to the
• On the day of ET
Ease of Embryo Transfer91 endometrium.
Ultrasound Guided or Clinical Touch
• Studies have shown that ET done using the after-
• Easy ET: When ET takes place smoothly, without any
Problematic Cervix loading technique gives significantly higher clini- Method of Embryo Transfer98,99
other instrumentation, the catheter is clean of blood cal PR (52.4%) as compared to preloading method The use of ultrasound to assist embryo transfer was first
and there was no need to change the catheter. • Occasionally, cervical stenosis or acute angulation (34.9%).96
• Intermediate ET: When the primary catheter meets between uterus and cervix limits uterine access. described by Strickler et al. in 1985, who reported that
guided transfer is easier and less associated with catheter
some resistance, leading to the use of cervical forceps • Cervical dilatation performed at the time of OPU Types of Catheter Used distortion.
and/or the outer sheath catheter, after which the trans- resulted in significantly easier ET but significant
More than 70 types of catheters models available based on
fer is smooth with no blood contamination. lower pregnancy rates. A short interval between
• Difficult ET: If at least one of the following problems dilatation and transfer is apparently not sufficient
different material, sizes and length, echogenicity, rigidity, Advantages
and parts.
occur—greater resistance, time consuming, need to for the endometrium to recover from any trauma, • Confirm that the embryos are properly deposited
change to a harder catheter, uterine sounding or cervi- inflammation, or bacterial contamination induced Ideal Catheter • To follow the embryo-associated air bubble
cal dilatation, or blood in any part of the catheter. by the dilation. • Increases the frequency of easy ET
• Soft and flexible to cause minimal endometrial trauma
• In their meta-analysis of Difficult ET and Pregnancy If difficulty is encountered during mock transfer:94 • Decrease cervical and uterine trauma
• Rigid enough to negotiate narrow cervical canals
Rates in 2005, Sallam et al. found a significantly lower • Touching the fundus accidently may increase the uter-
• Hysteroscopic visualization of cervical canal with or • Made of nontoxic material
PR in Difficult Embryo Transfers.92 ine contractions, thus decreasing the PR and increases
without hysteroscopic shaving of any stenotic area • Visible on ultrasound
the chances of ectopic pregnancy.
• Pretreatment with cervical dilatation, hysteroscopy, • Low cost
Difficult ET Management and placement of a 16- to 22-Fr Malecot catheter for an • Should have good memory Cochrane meta-analysis (2007) showed similar live birth
average of 10 days. rates, miscarriage rates, and ectopic PR in both the groups.98
• A commonly used initial approach is to negotiate Material used:
the cervix using the outer sheath of the catheter, with • Polyethylene On the other hand, a meta-analysis by AbouSetta et al. in the
Embryo transfer should be performed 3 weeks to same year showed a significant increase in LBR (OR = 1.78)
its inner needle withdrawn. Once the uterine cav- 3 months after cervical dilatation. • Teflon
• Nylon in ultrasound guided group, although both the groups had
ity is entered, the inner needle is used to deposit the
• Acute ante/retroversion of uterus is an important cause similar miscarriage and ectopic pregnancy rates.99
embryos, taking care to avoid the fundus (After Load- • Polyurethane
of difficult transfer. Uterine position may be changed
ing Technique). • Polyolefin
• The transfer is to make a previous “dummy transfer”
during the ET by filling the bladder or mechanically
• Combination
Echogenic versus Standard Catheter
straightening the cervix using tenaculum. Use of tenac-
or “Trial Transfer” using an uncharged catheter. Cer- Biosafety: • Presence of air bubbles within the inner catheter
ulum may lead to increased uterine contractions and
vical penetrability and any potential trouble related • MEA (1 or 2 cell mouse embryo >80% blastocyst rate (SureView Pro Wallace Catheter), or a small metallic
fundocervical contractions, hence should be avoided,
to catheter introduction must be noted beforehand. A in 96 hours) ring near the tip (EchoTip Softpass Cooks Catheter),
wherever possible.
solution can be decided on and prolonged exposure of • Endotoxin levels (<0.1 EU) makes the visualization of the catheter better during
embryos to unfavorable conditions or their losses aris- the ET, resulting in better placement of the embryos
ing by difficulty during insertion can be avoided
Preparation Prior to Embryo Transfer95 Soft versus Firm and a quicker embryo transfer.
• A trial transfer allows the physician to choose the 1. Abdominal ultrasound to check the bladder condi- • “Soft” embryo transfer catheters: • Studies showed better visibility and a significant shorter
appropriate transfer catheter, measure the depth of the tion and anteversion/retroversion of the uterus Cook catheter (Cook Ob/Gyn, Inc., Bloomington, IN) duration of ET but failed to translate into a better PR.100
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322 The Infertility Manual Oocyte Retrieval and Embryo Transfer 323
Vaginal Ultrasonography-guided by 5–10 μL of medium containing the embryos to be • Abou-Setta et al. failed to find any benefit of Embryo Bed rest102
transferred, and 10 μL of air at the tip of the catheter. glue on implantation rates or pregnancy rates.102
Embryo Transfer No significant difference in pregnancy rates post 30 min-
• In the fluid-only group, the syringe and the entire cath-
utes bed rest versus immediate ambulation group.
The TVS is performed first, carefully visualizing the endo- eter are filled with medium and the embryo-containing Site of Embryo Transfer
metrial cavity in the sagittal plane. USG probe is removed medium (5–10 μL) is aspirated without being brack- Tocolytic agents/general anesthesia
and replaced by a bivalved speculum and outer sheath is • The site of transfer has also been related to poor preg-
eted by air spaces.
nancy outcomes. • Tocolytic agents or prostaglandin synthetase inhibitor
inserted in the cervical canal, taking care not to advance
The use of air brackets around the embryo-containing • Transferring embryos too close to fundus has been did not have a significant effect.
it beyond internal os. Then speculum is removed gently
medium has been debated to be beneficial to the success associated with a higher risk of ectopic pregnancy and • The use of propofol general anesthesia for ET did not
taking care not to move the outer sheath and TVS probe
of the ET: endometrial trauma due to accidental touching of the have a significant effect. However, it could be used in
is reinserted. Outer sheath is advanced gradually, under
USG guidance, just beyond the internal os. The softer inner • By protecting the embryos from the cervical mucus fundus. some patients who experience severe stress and anxi-
stylet is removed and is replaced by soft inner catheter, Prevent accidental discharge before entering the • At the same time, low implantations have been shown ety during ET.
endometrial cavity to have a higher rate of spontaneous abortion and
which is advanced 1.5–2 cm from fundus, at which point Use of antibiotics
the embryos are expelled gently. • Prevent floating of embryos to the end of the catheter cervical ectopic pregnancies.
into the syringe A systematic review and meta-analysis regarding the
103 • The prescription of antibiotics from the day of OPU
• Embryos stick to the air bubbles and rise up to the best site for embryo deposition showed that: up to 6 days (Amoxicillin + Clauvulanic acid) does not
Effect of Blood or Mucus on fundus thereby decreasing the chances of embryo
improve the implantation rate or pregnancy rates.104
• Pregnancy rates are similar when the upper and lower
or in the Catheter expulsion Thus the ideal protocol of embryo transfer can be sum-
halves of the endometrial cavity are compared.
• Blood on the tip means endometrial trauma due to • Better visualization of ET procedure on USG. marized as below:105
• Midcavity transfer (e.g. around 20 mm from fundus)
suboptimal technique or difficult transfer, leading to On the other hand, supporters of the fluid only method is superior to the traditional high transfer (e.g. around • Precycle trial transfer with or without hysteroscopy to
impaired receptivity due to inflammation or bleeding, of catheter loading believe that: 10 mm from fundus). check the uterine cavityTransabdominal ultrasound
initiation of uterine contractions, and/or mechanical guided ET with full bladder
• Nonphysiological with a potential deleterious effect on
plugging of the catheter tip by blood clot.
the embryos and implantation Slow Withdrawal of the Embryo • No routine cervical mucous removal unless exces-
• Mucus plugging of the catheter tip can cause embryo sive secretions. Be extremely gentle in removing the
retention and damage and improper embryo placement.
• Presence of air could increase the likelihood of embryo Transfer Catheter
entrapment mucous from the cervical canal
• Blood or mucus on the catheter tip is associated with a • Some clinicians wait for around a minute before with- • Outer sheath is carried just beyond the internal os
• Increase reactive oxygen species
higher incidence of retained embryos. drawing the ET catheter after the expulsion of the under USG guidance
• Movement of embryos to other areas within the uterus
• Cervical mucus affected the rate of embryo expulsion embryos. This gives time to the embryos to stabilize.
• The occurrence of retained embryos within the catheter • Soft inner catheter, containing 30 µL volume loaded
into the cervix. Also negative pressure of capillary action created by with embryos, either in air media or media type load-
• Cervical mucus may also be a source of infection of the A systematic review and meta-analysis by AbouSetta withdrawing the catheter could draw embryos into the ing according to the preference of the clinician and the
endometrial cavity and the embryos. et al.101 found no difference in the clinical pregnancy rates cervical canal.
and live birth rates. embryologist
• Providers should perform a significant number of • No statistically significant difference in the PR between • Gentle insertion: Use stylet or manipulate cervix with
ultrasound guided trial transfers and intrauterine slow withdrawal of the catheter immediately after speculum or ring forceps only if absolutely necessary
inseminations using a soft catheter to develop the fine The Volume of Fluid Applied with Embryos in embryo deposition and a 60-second delay before cath- to negotiate internal os
motor skill which is mandatory for embryo transfer Embryo Transfer eter withdrawal.102 • Use ultrasonography to avoid catheter tip disrupting
technique.
• It has been advised that while transfer volumes more endometrium; avoid touching fundus
than 60 µL may result in expulsion of the embryos Mechanical Pressure on the Portiovaginalis • Inject embryos slowly in the midcavity as confirmed by
Removal of Cervical Mucus Prior to through the cervix into the vagina. of the Cervix102 ultrasound and withdraw catheter slowly
Embryo Transfer • It was also suggested that transferred volumes less • Inspection of catheter by embryologist for blood,
• Applying gentle mechanical pressure on the portio- mucus, or retained embryos
than 10 µL as well as the presence of air bubbles may
Derks et al. failed to find any benefit of removing the cervi- vaginalis of the cervix for few minutes post-ET, by
have a negative influence on the implantation rate.
cal mucus on clinical PR and live birth rate.95 loosening the screws of vaginal speculum significantly
improved the clinical pregnancy and implantation
PROBABLE QUESTIONS
The Embryo Transfer Medium
Air-Media versus Media Only rates by reducing embryo expulsion from the uterus 1. Types of embryo transfer catheters.
• It has been postulated that the presence of hyaluronan caused by uterine contractions. 2. Trouble shooting in oocyte retrieval.
Catheter Loading in the culture media prior to ET may increase the PR. • Some authors, however, have expressed concern that 3. What are the complications of transvaginal oocyte
• In the air-fluid group, embryos are loaded as follows: • Embryo Glue medium is an ET medium containing this approach increases uterine contractility and may retrieval?
10 μL of air in the proximal part of the catheter, followed high amount of hyaluronan reduce the chances of successful ET. 4. Empty follicle syndrome. Write a short note.
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324 The Infertility Manual Oocyte Retrieval and Embryo Transfer 325
6. Describe the various factors affecting outcome of 14. Jain D, Kohli A, Gupta L, Bhadoria P, Anand R. Anaesthesia 28. Kushnir VA, Kim A, Gleicher N, Barad DH. A pilot trial of pH of follicular fluid. Assist Reprod Genet. 2006;23(1):37-40.
embryo transfer with evidence. for in vitro fertilisation Indian J Anaesth. 2009;53(4): large versus small diameter needles for oocyte retrieval. Published online 2006 Mar 15.
408-13. Reprod Biol Endocrinol. 2013;11:22. 41. Waterstone JJ, Parsons JH. A prospective study to
15. Van Os HC, Roozenburg BJ, Janssen-Caspers HA, 29. Wikland M, Blad S, Bungum L, Hillensjö T, Karlström PO, investigate the value of flushing follicles during
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C HA PT E R
36
Prognosticating and Optimizing In Vitro Fertilization Outcome 329
Prognosticating and Optimizing Duration of Subfertility Table 36.1. Live birth rates according to the duration
of infertility.
In Vitro Fertilization Outcome • As the duration of subfertility increases, there will be
associated reduced chance of natural conception10-13
Duration of Number of Live birth rate (%) per
infertility (years) cycles treatment cycle
(adjusted hazard rate 0.83; 95% CI 0.78–0.88).14
G Ashwini Sidhmalswamy 0 2,258 13.3 (12.0–14.8)
• In IVF, pregnancy rates were slightly lower in couples
with increased duration of subfertility (OR 0.99, 95% 1–3 8,407 15.3 (14.6–16.1)
Chapter Outline
CI: 0.98–1.00),3 even after adjustment for age.15-17 4–6 13,483 14.0 (13.4–14.4)
• Steps to Optimize in vitro Fertilization Outcomes
• Although the meta-analysis did not find a significant 7–9 7,017 12.9 (12.2–13.7)
association between type of subfertility (primary or
10–12 3,701 12.4 (11.4–13.6)
INTRODUCTION 1. Step I: Predictors while selecting and preparing the secondary subfertility) and chance of pregnancy with
>12 2,092 8.6 (7.4–10.1)
patient IVF (unadjusted OR 1.04 95% CI: 0.65–1.43),3 two
“If you quit on the process, you are quitting on the 2. Step II: Optimizing ovarian stimulation protocol recent large studies did find an association.
result.” 3. Step III: Oocyte retrieval and sperm selection • Increased duration of subfertility is associated with
—Idouv Koyenikan 4. Step IV: ET procedure and laboratory procedure • On the other hand, another study suggested that
decreased chance of spontaneous pregnancy. The
“How you tried to achieve something matters more 5. Step V: Leuteal support. women with pathology in tube had higher possibility
Human Fertilization and Embryology Authority
than what you achieved.” of pregnancy post-IVF compared with couples with
(HEFA) register demonstrated the reduced live birth
—Sharad Vivek Sagar Step I: Predictors While Selecting and rate with rising duration of infertility after age adjust-
unexplained subfertility, though not significant.21
• Although two studies (N = 2,628 cycles) reported that
It was in 1978 that the world’s first IVF baby was born Preparing the Patient ment (Table 36.1). couples with male subfertility have lower pregnancy
through natural cycle in vitro fertilization (IVF). This
Nine predictive factors in IVF were evaluated in a recent chances than those with unexplained subfertility,22,23
is considered as milestone in the treatment of infertil-
ity. There were several advances after this in the field
systematic review and meta-analysis:3 Indication for In Vitro Fertilization a very large cohort study (N = 144,018 cycles) showed
1. Female age that couples with only male subfertility had increased
of IVF which has improved the success rate of IVF • Through the years, several studies have reported on
2. Duration of subfertility pregnancy chances compared to couples with unex-
tremendously. the relation between the reason for IVF and pregnancy
3. Type of subfertility plained subfertility.15
The IVF does not guarantee success as it is thought with IVF without consistent results.
4. Indication for IVF • The reason for infertility has a limited effect on the
by many; almost 38–49% of couples will remain childless
5. Basal follicle-stimulating hormone (bFSH) • Women with pathology in tube alone had lower possi- probability of live birth following conventional IVF.
after starting IVF. This is the case even if they undergo six
6. Fertilization method bility of pregnancy compared to women having unex- Templeton et al. after adjusting for age and dura-
IVF cycles.1 Therefore, subfertile couples should be well
7. Number of oocytes plained subfertility or other indications.15,18-20 tion of subfertility showed that there is no significant
informed about the chances of success with IVF before
8. Number of embryos transferred effect on live birth rate per IVF cycle.17 (Tables 36.2
starting their first or before taking on a new IVF cycle.
9. Embryo quality to 36.4).
Based on a couple’s specific probability, one should decide
whether the chances of success with IVF justify the bur-
den, risks, and costs of the treatment.
Female Age Lifestyle Factors
As IVF is currently used as an empirical treatment and • Female age is one of the most important predictor for
success of IVF cycle. The association of lower preg- The Table 36.5 shows the impact of lifestyle on infertility.
not as a causal intervention for a specific disorder, there is
a strong need that a good and a poor prognosis couple are nancy in IVF, increased with increasing female age Obesity
to be distinguished.2 (Odds ratio (OR) 0.95, 95% CI: 0.94–0.96).3
• The decrease in fertility sets in with aging. There is a • Obesity is associated with higher doses of medication
This chapter provides the most up to date evidence on
marked decline of fertility after 35 years for both spon- required for ovarian stimulation in obese women.
ART cycles from systematic reviews of randomized con-
taneous and IVF-induced pregnancies.3-5 • There is decreased chance of clinical pregnancy in
trolled trials. Using the best available evidence to optimize
• The explanation for the declined fertility with increas- obese woman.36
outcomes is the best practice in order to improve live birth
ing female age is most likely due to the diminished • In a systematic review, it was found that overweight
rates and reduce cycle cancellation.
ovarian reserve. There is decrease both in quantity and women (BMI >25 kg/m2) going through IVF have a
quality of oocytes with increasing age of a female.6
STEPS TO OPTIMIZE IN VITRO 10% lesser live birth rate than a normal weight woman
• With reduced ovarian reserve, there is diminished (BMI <25 kg/m2) (OR 0.90; 95% CI, 0.82–1.0).23
FERTILIZATION OUTCOMES response to gonadotropin therapy and this is a hin- • Lifestyle modification is the first-line treatment for
drance to a successful IVF cycle (Fig. 36.1).7,8 obese women. Adjunctive medical therapy comes later.
This chapter particularly looks at prognostic and predic- Fig. 36.1: The effect of female age on the rate of pregnancy
tive factors in IVF, and provides key principles to optimize The decline in fertility with aging is due to ovarian fac- evaluated from studies in 10 different populations that did not use • Bariatric surgery is adjuvant to lifestyle modification
contraceptives.9 and medical therapy for weight loss.
IVF outcomes. These points will be addressed in five steps. tors rather than endometrial.
330 The Infertility Manual Prognosticating and Optimizing In Vitro Fertilization Outcome 331
Table 36.2. Live birth rate by cause of infertility. History of Previous Pregnancy Table 36.5. Impact of lifestyle on infertility.
Secondary infertility is a good prognostic factor in infer- Lifestyle factors that affect infertility. Impact on fertility Study
Number of Live birth rate (%) per
Cause of infertility cycles treatment cycle tile couples. The pregnancy from previous IVF treatment Obesity (BMI >35) Time taken to conceive increased by twofold Hassan and Killick, 200430
increases the chances of pregnancy (Table 36.6). Underweight (BMI <19) Time taken to conceive increased by fourfold Hassan and Killick, 200430
Tubal disease 19,096 13.6 (13.0–14.0)
Smoking RR of subfertility increased fourfold Clark et al.,199831
Endometriosis 4,117 14.2 (13.2–15.3) Repeated In Vitro Fertilization Attempts Alcohol RR of subfertility increased 60% Eggert et al., 200432
Unexplained 12,340 13.4 (12.9–14.1) The chance of conception per cycle being low, there is a Caffeine (>250 mg/day) Fecundability reduced by 45% Wilcox et al.,199833
Cervical and uterine 4,232 14.2 (13.2–15.3)
need to understand whether the chance decreases after Illicit drugs RR of subfertility increased 70% Mueller et al.,199034
repeated attempts at IVF (Table 36.7). Toxins, solvents RR of subfertility increased 40% Hruska et al.,200035
BMI, body mass index; RR, relative risk
Note: 1. Table reprinted from the article impact of lifestyle on infertility, published in 2008, Fertil Steril. 2008;90(Suppl):S1-6.
2. Practice Committee. Optimizing natural fertility. Fertil Steril. 2013.
Table 36.3. Interventions done for the cause of infertility.
Cause Study/evidence Conclusion
Ovarian Reserve Test Antiphospholipid Antibody
Tubal pathology Johnson 2010 Cochrane Treatment with surgery is to be considered for women diagnosed with
review24 hydrosalpinges before IVF stimulation. • Garcia et al. in 1983 were the first to report the phe- A meta-analysis concluded there is no significant asso-
Occlusion of tube through laparoscopy is an alternative treatment to sal- nomenon of poor responders. About 10% of the ciation between antiphospholipid abnormalities and live
pingectomy in improving pregnancy chance in women with hydrosalpinges birth (95% CI 0.66–1.75) in IVF patients.
going in for IVF. patients that undergo controlled ovarian stimulation
are found to be poor responders. Hence no measurements of antibodies is required in
Endometriosis Benschop 2010 No difference in pregnancy rates between surgery (cystectomy or cyst patients undergoing IVF.24
Cochrane review25 aspiration) prior to ART. No difference as well between ablation and cystec-
• Hence it is vital to screen every patient for their ovarian
tomy before IVF in women with endometrioma. reserve prior to starting ovarian stimulation.
• Several tests have been proposed but a basal day 3 FSH
Step II: Optimizing Ovarian
Myoma Klatsky PC 2008 sys- IVF success rates are less in women having intramural myomas, more so if
tematic review26 the size exceeds 5 cm in diameter. along with age is considered among better predictors Stimulation Protocol
Hysteroscopic myomectomy is done for intracavitatory myomas and sub- of poor response. The IVF success depends on adequate follicle response
mucous myomas with 50% of their volume inside the uterine cavity. • Screening with anti-Müllerian hormone (AMH) levels by using controlled ovarian stimulation through gonad-
have shown to be promising tool. otropins. There are various protocols in use. The Table
• A history of previous poor response cycle is also best 36.8 gives complete overview with recent evidence.
predictor of poor response.
Table 36.4. Line of management depending on the indication for assisted reproductive technique (recent r eviews). • An increase in basal FSH value is related with reduced Ovarian Stimulation Protocols
Number of rand- pregnancy rates post-IVF (OR 0.94; 95% CI: 0.88–1.00).21
omized controlled Ovarian stimulation protocol in poor responders It is
Higher number of oocytes was related with increased
Review article trials (RCTs) Total population Interventions Control groups Comments and limitations shown in Table 36.9
pregnancy rates (OR 1.04, 95% CI: 1.02–1.07).3
Pandian 201227 Six RCTs 733 couples In vitro Expectant manage- The review stated that
management of with unex- fertilization ment, intrauter- IVF treatment is much
unexplained plained infertility ine insemination better than IUI with ovar- Table 36.7. Live birth rate by no. of previous
infertility (IUI), intrauterine ian stimulation. But, due Table 36.6. Effect of previous pregnancies. unsuccessful IVF attempts.
insemination + ovarian to the lack of evidence No. of previous No. of Live birth rate (%) (95% No. of previous Live birth rate (%) per
stimulation the effectiveness of IVF pregnancy cycles CI) per treatment cycle treatment cycle No. of cycles treatment cycle
compared with other
None 19,997 12.5 (12.0–12.9) 0 18,239 14.0 (13.5–14.5)
above treatment is not
proven >1 not by IVF 1 8,123 13.0 (12.2–13.7)
Yossry 200628 No available Not applicable IVF vs. tubal Nonavailability of rand- 2 3,706 11.4 (10.4–12.5)
No previous live birth 8,388 13.7 (13.3–14.1)
RCTs reanastomosis omized controlled trial 3 1,786 11.5 (10.1–13.4)
(poststerliza- evidence to back their >1 previous live birth 5,336 15.3 (14.3–16.3)
4 864 8.9 (7.2–11.2)
tion of tubes) review questions >1 by IVF
5 389 9.3 (6.7–12.9)
Siristatidis No available N/A In vitro matura- IVF Intracytoplasmic Nonavailability of RCT No IVF live birth 1,786 16.6 (14.9–18.4) 6–9 483 10.2 (7.7–13.7)
200929 RCTs tion in women sperm injection evidence to back their
with PCOS review questions >1 IVF birth 1,451 23.2 (21.1–25.6) >10 111 6.2 (2.6–12.6)
PCOS, polycystic ovary syndrome.
332 The Infertility Manual Prognosticating and Optimizing In Vitro Fertilization Outcome 333
Oocyte Maturation and Trigger Protocol Sperm Selection Techniques Embryo Selection and Embryo Transfer CONCLUSION
It is described in Table 36.10. It is described in Table 36.12. These are discussed in Table 36.14. There should be some prognosticating and prediction
models for IVF outcome in clinical practice. This need
Step III. Oocyte Retrieval and Sperm Selection Step IV: Embryo Transfer and Embryo Transfer Techniques can be explained by the inability of diagnostic tests to
Oocyte Retrieval Laboratory Phase These are shown is Table 36.15. detect factors that indicate subfertility with near 100%
certainty in patients. Providing the highest possible
Both single lumen and double lumen needles have been Laboratory Phase Step V: Luteal Phase Support pregnancy rates requires continual evaluation of patient
used successfully for collection of oocytes. Table 36.11
It is shown in Table 36.13. These are discussed in Table 36.16. selection, stimulation protocol, IVF laboratories embryo
gives inference of a systematic review in this regard.
These are shown in Table 36.17. transfer technique, and luteal support. Every center
Table 36.8. Ovarian stimulation protocols.
No. of Interven- Comparison inter- Comments and Table 36.10. Oocyte maturation and trigger protocol.
Review article RCTs Total population tion vention/control Outcomes limitations Review article No. of RCTs Total population Outcome measures Conclusion
Al-Inany HG 2016 73 12,212 women GnRH antagonist protocol vs There was no significant difference Youssef 7 1,136 Recombinant versus urinary There was no conclusive evidence of a
Cochrane review37 going through ART GnRH agonist protocol between the groups in live birth rates MA 201646 human chorionic gonadotro- difference between rhCG and uhCG There
Siristatidis CS 2015 20 1,643 Long protocol vs. short There was no significant difference in Cochrane pin for final o
ocyte matura- was no significant difference between
Cochrane Review38 protocol. live birth and ongoing pregnancy rates Review tion triggering in IVF and rhCG and uhCG in the incidence of mod-
Gibreel 14 2,536 (12 trials) Clomiphene citrate +/- addi- This review found no significant ICSI cycles erate to severe OHSS either
201239 infertile women tional treatments difference between the groups in terms Youssef 201447 17 1,847 women GnRH agonist vs. hCG There was evidence suggestive of
going through Alternative treatments of live births, but use of clomiphene led going through reduced live birth rate, lower pregnancy
ART for c ontrolled ovarian to a decrease in the incidence of OHSS ART rate, and higher chances of miscarriage
hyperstimulation in women who was given GnRH agonist
Pouwer 6 3,753 women with Long acting FSH vs. daily FSH This review stated no significant compared to women given hCG but the
201540 subfertility difference between the groups in live incidence of OHSS was reduced in the
birth rates or OHSS GnRH agonist group
Mochtar 33 5,624 infertile Recombinant luteinising No significant difference between OHSS, ovarian hyperstimulation syndrome; rhCG, recombinant human chorionic gonadotropin; uhCG, urinary human chorionic gonadotrophin.
200741 women hormone plus Recombinant the groups
follicle stimulating hormone
Van Wely 42 9606 women Recombinant follicle No significant difference between Table 36.11. Oocyte retrieval.
201142 going through stimulating hormone vs the groups Review article No. of RCTs Total population studied Intervention/outcome measures Conclusion
ART Urinary gonadotrophins
Wongtrangan S 4 208 women going To study whether follicular aspiration There was no
Martins 5 351 women going Low dose HCG in the late No significant difference between 2010 Cochrane through ART and flushing increases the number significant differ-
201343 through COH for follicular phase vs FSH the groups Review48 of oocytes yield and live births over ence between the
ART. throughout COH aspiration alone groups
Smulders 23 2603 infertile Combined OCP, Progesterone, Found no significant difference with
201044 women Oestrogen ,Placebo or no regard to the number of live births when
treatment used as pre treat- using a pre-treatment Table 36.12. Sperm selection techniques.
ment prior to IVF
No. of Total population
GnRH, gonadotropin-releasing hormone; OHSS, ovarian hyperstimulation syndrome
Review article RCTs studied Interventions Conclusions and comments
Teixeira DM 1 168 ICSI versus ultra-high magnification (IMSI) There was no significant differ-
Table 36.9. Ovarian stimulation protocol in poor responders. 2013 Cochrane sperm selection for assisted reproduction ence between IMSI and ICSI.
review49 Results from RCTs do not support
No. of Total population Comparison Intervention/
Review article RCTs studied Intervention control Conclusion the clinical use of IMSI
Pandian 201045 10 625 women GnRH antagonist (GnRHa) Long protocol, spontaneous No significant McDowell S, 1 482 To study the effect of advanced sperm No significant difference between
diagnosed protocol, stop protocol, natural cycle IVF, m odified difference between Kroon B 2014 selection techniques on IVF outcomes. the groups was found
as poor GnRH antagonist, low dose long protocol ,GnRHa the groups was Cochrane Advanced sperm selection techniques in-
responders’ to GnRHa flare-up protocol, flare-up protocol, found review50 clude: selection according to surface charge;
COH GnRHa flare-up proto- Mini dose long agonist sperm birefringence; sperm apoptosis;
col, multiple dose GnRH protocol sperm morphology under ultra-high magnifi-
antagonist flare-up protocol cation; and ability to bind to hyaluronic acid
334 The Infertility Manual Prognosticating and Optimizing In Vitro Fertilization Outcome 335
Table 36.13. Laboratory phase. Table 36.16. Luteal phase support.
Review No. of Total no. of Review
article RCTs population Intervention Controls Outcome/conclusion article Total population studied Intervention Conclusion
Van Rumste 1 415 subjects Intracytoplasmic In vitro fertilization There was insufficient evidence to reach van der 94 RCTs 26,198 women Luteal phase support post IVF: Progesterone used as luteal phase support
200351 with normal sperm injection a conclusion Linden with any cause of subfer- progesterone, hCG vs. placebo increases live birth rate when compared to placebo
factor subfertility 201563 tility undergoing ART or no treatment. hCG, proges- There is no evidence that hCG is better than
Bontekoe 11 2,422 Embryo culture Embryo culture with Evidence suggested improved effect of terone + estrogen, progester- placebo or no treatment. hCG may have higher
201252 with low oxygen atmospheric oxygen culturing in low oxygen concentration; one + GnRH agonist risk of OHSS compared to placebo
concentrations concentrations improvement was found in live birth rate Progesterone with GnRHa appears to improve
Huang 8 733 Brief coincuba- Standard overnight Coincubation improves the ongoing outcomes
201353 tion of gametes insemination proto- pregnancy and clinical pregnancy rates Boomsma 14 RCTs 1,879 infertile Preimplantation glucocorticoids No conclusive evidence
in women going col for women going 201264 couples vs. no glucocorticoids placebo
through IVF through IVF
Akhtar 3 RCTs 386 infertile wom- Heparin vs. placebo no treat- No conclusive evidence
Carney 31 5,728 Assisted No assisted The live birth rate was not improved by 201365 en going through ART ment
201254 hatching hatching AH, and multiple pregnancy rates were
ART, assisted reproductive techniques; GnRH, gonadotropin releasing hormone; GnRHa, gonadotrophin-releasing hormone agonist; hCG,
significantly increased in the AH groups human chorionic gonadotropin; IVF, in vitro fertilization; OHSS, ovarian hyperstimulation syndrome.
Armstrong 3 994 Time lapse Conventional No significant evidence to prove it
201555 systems embryo incubation
Twisk 9 1,589 women IVF/ICSI with IVF/ICSI with no Preimplantation genetic screening Table 36.17. Assisted reproductive technique and adjuvant strategies.
200656 going through preimplanta- preimplantation significantly reduced live birth rates in Adjuvant strategies Study/evidence Conclusion
IVF or ICSI tion genetic genetic screening women of advanced female age and
screening those with r epeated IVF failure Endometrial injury done a month Nastri 201566 It was associated with higher live birth rate and ongoing
prior to ART cycle pregnancy rate
Hysteroscopy Bosteels 2013 67
The hysteroscopic correction of endometrial pathologies (e.g.
Potdar 201268 polyps and leiomyomas) improved the possibility of implantation
Table 36.14. Embryo selection and embryo transfer.
The endometrial injury associated with hysteroscopic procedure is
Review article Study details Outcome measures Comments and conclusion thought to improve the possibility of implantation
Glujovsky 23 RCTs Blastocyst stage (Day 5 to 6) There significant difference in live birth rate Growth hormone Duffy 201069 Growth hormone used in poor responders may have increased
201257 3,241 women going embryo transfers (ETs) vs. per couple favoring blastocyst stage ET chance of live birth rates
through ART cleavage stage (Day 2 to 3) ETs.
Metformin treatment in women with Tso 2014 70
The use of metformin increased clinical pregnancy rates and
Pandian 14 RCTs 2,165 couples Single-cycle SET versus The live birth rate and multiple pregnancy PCOS undergoing ART decreased the risk of OHSS in PCOS women
201358 going through ART single-cycle DET rate was significantly lower in the SET group
Acupuncture in ART Cheong YC There is no evidence as to acupuncture increases live birth or
DET, double embryo transfer; SET, single embryo transfer 201371 pregnancy rates
Ovarian cyst aspiration prior to ART McDonnell 201472 No evidence of improvement in clinical pregnancy rate
ART, assisted reproductive technique; OHSS, ovarian hyperstimulation syndrome; PCOS, polycystic ovary syndrome.
Table 36.15. Embryo transfer techniques.
Review article Total population Outcome measured Conclusion
Bontekoe 17 RCTs 3,898 Embryo transfer media with adherence No significant difference was should conduct regular audits to improvise the success 6. Which ovarian stimulation protocol is best to prevent
201459 women going compounds (hyaluronic acid or fibrin sealant) vs. found between the groups rate of IVF. OHSS in IVF cycle?
through ART embryo transfer media without any compound in it. 7. Does assisted hatching improve IVF outcome?
Derks 10 RCTs 1,693 Dummy transfer to straighten uterocervical angle, No significant implications to
200960 women (9 RCTs) embryo afterloading, cervical and endometrial improve the IVF outcome were
PROBABLE QUESTIONS 8. Is IMSI better than ICSI—what does evidence say?
9. Which has better outcome in IVF cycle—USGguided
going through IVF preparation, vs. no intervention made 1. What are the prognosticating factors one should be embryo transfer or clinical touch transfer?
Brown 17 RCTs 6,524 Ultrasound guided embryo transfer (UGET) vs. UGET was associated with a aware of before starting an IVF cycle? 10. Does progesterone supplement as luteal phase
201061 subfertile women clinical touch transfer higher possibility of live birth 2. What does evidence say about dealing with hydrosal-
undergoing ART rate compared to clinical touch
support improve IVF outcome?
pinx before IVF cycle?
Abou-Setta 4 RCTs 1,392 Post-embryo transfer interventions like bladder No significant conclusion was 3. Why is it vital to screen every woman with ovarian
201462 infertile women emptying, bed rest, fibrin sealant, mechanical derived
pressure on cervix vs. no intervention
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Evers JL, te Velde ER. Two new prediction rules for reversal) for subfertility after tubal sterilisation. Cochrane
44. Smulders B, van Oirschot SM, Farquhar C, Rombauts L, 2012;(7):CD002118.
spontaneous pregnancy leading to live birth among Database of Systematic Reviews. 2006;(3):CD004144. Kremer JAM. Oral contraceptive pill, progestogen or estrogen
29. Siristatidis CS, Maheshwari A, Bhattacharya S. In vitro 58. Pandian Z, Marjoribanks J, Ozturk O, Serour G,
subfertile couples, based on the synthesis of three previous pre-treatment for ovarian stimulation protocols for women Bhattacharya S. Number of embryos for transfer following
models. Hum Reprod. 2004;19:2019-26. maturation in subfertile women with polycystic ovarian undergoing assisted reproductive techniques. Cochrane in vitro fertilisation or intra-cytoplasmic sperm injection.
15. Nelson SM, Lawlor DA. Predicting live birth, preterm syndrome undergoing assisted reproduction. Cochrane Database Syst Rev. 2010;(1):CD006109. Cochrane Database Syst Rev. 2013;(7):CD003416.
delivery, and low birth weight in infants born from in Database of Systematic Reviews. 2009;(1). 45. Pandian Z, McTavish AR, Aucott L, Hamilton MPR, 59. Bontekoe S, Heineman MJ, Johnson N, Blake D. Adherence
vitro fertilisation: a prospective study of 144,018 treatment 30. Hassan MA, Killick SR. Negative lifestyle is associated Bhattacharya S. Interventions for ‘poor responders’ to controlled compounds in embryo transfer media for assisted
cycles. PLoS Med. 2011;8:e1000386. with a significant reduction in fecundity. Fertil Steril. ovarian hyper stimulation (COH) in in-vitro fertilisation (IVF). reproductive technologies. Cochrane Database Syst Rev.
16. Templeton A, Morris JK, Parslow W. Factors that affect outcome 2004;81:384-92. Cochrane Database Syst Rev. 2010;(1):CD004379. 2014;(2):CD007421.
of in-vitro fertilisation treatment. Lancet. 1996;348:1402-6. 31. Clark AM, Thornley B, Tomlinson L, Galletley C, 46. Youssef MA, Abou-Setta AM, Lam WS. Recombinant versus 60. Derks RS, Farquhar C, Mol BW, Buckingham K,
17. van Loendersloot LL, van Wely M, Repping S, Bossuyt Norman RJ. Weight loss in obese infertile women results urinary human chorionic gonadotrophin for final oocyte Heineman MJ. Techniques for preparation prior to embryo
PMM, van der Veen F. Individualized decision-making in in improvement in reproductive outcome for all forms of maturation triggering in IVF and ICSI cycles. Cochrane transfer. Cochrane Database Syst Rev. 2009;(4):CD007682.
IVF: calculating the chances of pregnancy. Hum Reprod. fertility treatment. Hum Reprod. 1998;13:1502-5. Database Syst Rev. 2016;4:CD003719. 61. Brown J, Buckingham K, Abou-Setta AM, Buckett W.
2013;28(11):2972-80 [Epub ahead of print]. 32. Eggert J, Theobald H, Engfeldt P. Effects of alcohol 47. Youssef MA, Van der Veen F, Al-Inany HG, Mochtar MH, Ultrasound versus ‘clinical touch’ for catheter guidance
18. Bancsi LF, Huijs AM, den Ouden CT, Broekmans consumption of female fertility during an 18-year period. Griesinger G, Nagi Mohesen M, et al. Gonadotropin- during embryo transfer in women. Cochrane Database Syst
FJ, Looman CW, Blankenstein MA, et al. Basal Fertil Steril. 2004;81:379-83. releasing hormone agonist versus HCG for oocyte triggering Rev. 2010;(1):CD006107.
C HA PTE R
338 The Infertility Manual
62. Abou-Setta AM, Peters LR, D’Angelo A, Sallam HN,
Hart RJ, Al-Inany HG. Post-embryo transfer interventions
for assisted reproduction technology cycles. Cochrane
Database Syst Rev. 2014;(8):CD006567.
63. van der Linden M, Buckingham K, Farquhar C, Kremer JA,
Metwally M. Luteal phase support for assisted reproduction
associated with suspected major uterine cavity abnormalities.
Cochrane Database Syst Rev. 2013;(1):CD009461.
68. Potdar N, Gelbaya T, Nardo LG. Endometrial injury
to overcome recurrent embryo implantation failure: a
systematic review and meta-analysis. Reprod Biomed
Third-party Reproduction
37
Online. 2012;25(6):561-71. Nivedita Shetty
cycles. Cochrane Database Syst Rev. 2015;(7):CD009154. 69. Duffy JMN, Ahmad G, Mohiyiddeen L, Nardo LG,
64. Boomsma CM, Keay SD, Macklon NS. Peri-implantation Watson A. Growth hormone for in vitro fertilization.
glucocorticoid administration for assisted reproductive Chapter Outline
Cochrane Database Syst Rev. 2010;(1):CD000099.
technology cycles. Cochrane Database Syst Rev. • Sourcing the Donors and Surrogates • Surrogacy
70. Tso LO, Costello MF, Albuquerque LET, Andriolo RB, • Sperm Donation • The Surrogacy (Regulation) Bill 2016, as Introduced in the Lok
2012;(6):CD005996.
Macedo CR. Metformin treatment before and during IVF or • Egg Donation Sabha
65. Akhtar M, Sur S, Raine-Fenning N, Jayaprakasan K,
ICSI in women with polycystic ovary syndrome. Cochrane • ART Guidelines for Donor Gametes—2014 Draft • The Future of Third-party Reproduction
Thornton JG, Quenby S. Heparin for assisted reproduction.
Cochrane Database Syst Rev. 2013;(8):CD009452. Database Syst Rev. 2014;(11):CD006105.
66. Nastri CO, Lensen SF, Gibreel A, Raine-Fenning N, 71. Cheong YC, Dix S, Hung Yu Ng E, Ledger WL,
Ferriani RA, Bhattacharya S, et al. Endometrial injury in Farquhar C. Acupuncture and assisted reproductive tech- INTRODUCTION Specifically, there are two variants of oocyte donors:
women undergoing assisted reproductive techniques. nology. Cochrane Database Syst Rev. 2013;(7):CD006920.
72. McDonnell R, Marjoribanks J, Hart RJ. Ovarian cyst aspiration • Third party reproduction involves a third person, other 1. Patient donors:
Cochrane Database Syst Rev. 2015;(3):CD009517.
67. Bosteels J, Kasius J, Weyers S, Broekmans FJ, Mol BWJ, prior to in vitro fertilization treatment for subfertility. than the couple, in order to help them reproduce. Who enter an agreement with the infertility clinic
D’Hooghe TM. Hysteroscopy for treating subfertility Cochrane Database Syst Rev. 2014;(12):CD005999. However, this process is like a Pandora’s box and can to donate a proportion of their oocytes to others, in
raise various ethical questions. order to receive subsidized infertility treatment
• The chapter deals with the indications for borrowing 2. Non-patient donors:
sperms, eggs and the uterus. It also deals with screen- a. Volunteer donors
ing both the couple and the donor and also about pre- b. Known donors
paring them for the procedure. c. Commercial donors
• A section also deals with third party reproduction pro-
cedures, which are still experimental in nature, but
SPERM DONATION
hold promise for the future.
1. Indications
DEFINITION 2. Evaluation of the sperm donor
3. Evaluating the recipient couple
Third party reproduction refers to a process, where a third
person, other than the couple, provides sperm or eggs or
embryos or where another woman provides her uterus, in
Indications for Donor Insemination1
order to help them reproduce. • The male partner has azoospermia or severe oligoas-
However, the third party’s involvement is limited to the thenospermia and not willing for intracytoplasmic
reproductive process and does not extend into the raising sperm injection (ICSI).
of the child. • The male partner has ejaculatory dysfunction (if not
It involves: willing for ICSI after testicular sperm aspiration [TESA]).
• Sperm donation • Prior failure to fertilize after intracytoplasmic sperm
• Egg donation injection.
• Embryo donation • The male partner has a significant genetic defect or the
• Surrogacy couple previously has produced an offspring affected by a
condition for which carrier status cannot be determined.
SOURCING THE DONORS AND • The male partner has a sexually transmissible infection
SURROGATES that cannot be eradicated as in HIV (relative indication
Types of donors in general: as with ICSI the risk of transmission is very low).
• The female partner is Rh-negative and severely
• Volunteer donors (donation without financial reward—
altruistic) Rh-isoimmunized, and the male partner is Rh-positive.
• Commercial donors (donation with monetary com- • Females without male partners, though in India the
pensation) current draft (2014) of the ART bill requires the couple
• Known donors (donation to known recipients) to be married for ART treatment.
340 The Infertility Manual Third-party Reproduction 341
Evaluation of the Donor anti-Müllerian hormone (AMH), and antral follicle Table 37.2. Oocyte donation. specific information like identity and address not to be
count (AFC) provided.
• Normal semen analyses Recipient Donor
• Obtain informed consent from recipient and partner • The age of the sperm donor should be between 21 and
• Medical history to evaluate any inheritable disease 1. Evaluation •• Screening
• Transvaginal scan: Screening for uterine pathology 45 years of age.3
• Sexual history of multiple partners 2. Synchronizing with donor •• Synchronizing with
like fibroids indenting the uterine cavity, adenomyo- • The age of the oocyte donor should be between 23 and
• Screening for HIV, hepatitis B antigen, hepatitis C, and menstrual cycle recipient menstrual cycle
sis, tubal pathology like hydrosalpinges 35 years of age.3
venereal disease research laboratory (VDRL). After 3. Hormone replacement •• Ovarian stimulation of
• Semen analyses for the male partner • Semen sample should be quarantined for at least
donation, anonymous donor specimens must be quar- with estrogens to achieve donor
• Infection screening for both partners: Sexual history, a endometrial thickness of 6 months before being used.
antined for a minimum of 180 days. The donor must HIV, hepatitis B antigen, hepatitis C virus, VDRL • The sperm of a single donor should not be used more
at least 7 mm
be retested after the required quarantine interval, and • Well being of the couple especially the recipient than 25 times.
4. Semen collection from the •• Oocyte retrieval
specimens may be released only if the results of repeat (including cardiac checks if over 40 years of age) com- partner • The sperm donor, if married, requires the consent of
testing are negative. plete blood count (CBC), blood sugar (RBS), thyroid- 5. Progesterone supplement his spouse.
• Genetic evaluation: Detailed personal and family stimulating hormone (TSH), blood group, serum cre- for the recipient from the • Mixing of semen from two individuals is not permitted.
history for genetic disorders especially in parents, sib- day of oocyte retrieval
atinine • Oocytes from one donor can be shared between two
lings, and offsprings. 6. Embryo transfer for the •• Follow-up to rule out
• Psychological assessment of both partners recipients only provided that at least seven oocytes are
recipient OHSS
Some centers perform karyotyping and genetic screen- • Mock cycle to assess endometrial thickness and if available for each recipient.
7. Early pregnancy
ing according to ethnic background. In the north and needed evaluation of the uterine cavity by hysteros-
management—HRT till
copy or mock embryo transfer.
certain western regions of India, thalassemia screening of 9 weeks maintaining the SURROGACY
donors is a must. American Society for Reproductive Med- pregnancy
Surrogacy refers to a contract in which woman carries a
icine (ASRM) guidelines recommend routine screening for Screening the Oocyte Donor Abbreviations: HRT, hormone replacement therapy; OHSS, ovarian
cystic fibrosis carrier status in donors. hyperstimulation syndrome pregnancy for another couple.
Table 37.1 describes the screening of oocyte donor.
Evaluating the Recipient Couple High Success Rate with Donor Programs3,4 Definitions and Terminologies5
Preparing the Donor and the Recipient The donor’s age is one of the most significant factors con-
• Male partner: HIV, hepatitis B antigen, hepatitis C, and • Genetic couple or commissioning couple: The couple
VDRL after confirming the necessity of donor insemi- Table 37.2 describes how to prepare the donor and the tributing to the high success rate. The recipient’s age does who provide both sets of gametes or at least one set of
nation recipient. not necessarily contribute to the prognosis. gametes.
• Female partner: HIV, hepatitis B antigen, hepatitis C, The most reliable predictive factor for pregnancy in • Surrogate: The woman receiving the embryos created
and VDRL oocyte donation cycles are the quality of the embryos and by the gametes of the genetic couple.
Tubal patency evaluation Table 37.1. Screening of the oocyte donor. the recipient’s midcycle endometrial thickness. • Traditional surrogacy: In this situation, the gestational
Follicular monitoring for confirming ovulation and 1. Ovarian reserve Age, FSH, AMH, AFC The main reason for high pregnancy rates is the lack carrier not only carries the pregnancy but also pro-
timing the donor insemination. assessment of ovarian hyperstimulation and very high estrogen levels vides the oocytes. This is not approved by the current
2. Transvaginal AFC, accessibility of ovaries, affecting the endometrial receptivity.3 ART guidelines in India.
scan pathology of ovaries like
EGG DONATION endometriomas, polycystic ovaries
• Gestational surrogacy: In this situation, the surrogate
ART GUIDELINES FOR DONOR only carries the pregnancy and does not provide the
Indications 2 3. Infection Sexual history, HIV, HBsAg, HCV,
GAMETES—2014 DRAFT3 gametes.
screening VDRL, High vaginal swab
• Women with nonfunctioning ovaries • Commercial surrogacy: When the surrogacy is paid for
4. Donor CBC, RBS, blood group especially • Known donors for either oocyte or sperm are not per-
Premature ovarian failure in addition to her medical expenses. This is legal in
well-being if recipient is R
h-negative mitted.
Iatrogenic ovarian failure due to ovarian surgery or India.
5. Genetic •• Family (parents, siblings, • The ART clinics shall obtain donor gametes from ART
radiation • Altruistic surrogacy: When the surrogate is paid only
screening offspring) history of any
banks only. for her medical expenses.
Menopause inheritable diseases
•• Genetic testing for carrier status • Screening of gamete donors and surrogates; the col-
• Women with functioning ovaries
lection, screening, and storage of semen; and provi-
Risk of inheritable disease in child 6. Psychological •• Assessment of readiness for egg
sion of oocyte donor and surrogates shall be done by
Indications
Poor responders or poor quality oocytes assessment donation
•• Spouse approval with informed an ART bank. 1. Without uterus:
consent • Nonspecific information in respect of donor of gam- a. Congenital absence of uterus as in Mayer-
Evaluating the Oocyte Recipient and Partner Abbreviations: FSH, follicle stimulating hormone; AMH, anti- etes including height, weight, ethnicity, skin color, Rokitansky-Küster-Hauser (MRKH) syndrome
Müllerian hormone; AFC, antral follicle count; HIV, human
• Confirm the need for oocyte donation—detailed immunodeficiency virus; HBsAg, hepatitis B surface antigen; HCV,
educational qualifications, and medical history of the b. Hysterectomy for cancer or postpartum hemor-
history, follicle stimulating hormone (FSH),
hepatitis C virus; VDRL, venereal disease research laboratory donor, including HIV/AIDS should be provided but rhage (PPH) or rupture uterus
342 The Infertility Manual Third-party Reproduction 343
Table 37.3. Screening the genetic couple. Table 37.4. Screening the surrogate. • Surrogacy for foreigners in India shall not be allowed Central Database of Donors
but surrogacy shall be permissible to overseas citizen
1. Semen analysis 1. Age—current guideline—between 23 and 35 years Maintaining a database of donors along with data of preg-
of India (OCIs), people of Indian origin (PIOs), non-
2. Assess ovarian reserve 2. Married and have at least a child of her own and also nancy outcome is a necessity.
have consent of the spouse
resident Indians (NRIs), and foreigner married to an
3. HIV, HBsAg, HCV for the couple Indian citizen. • In the event of any adverse outcome in pregnancy,
3. Should not have any severe medical disorder which it gives an opportunity for tracing the donor, even if,
4. Blood group for couple especially if recipient is • A commissioning couple shall not have the service of
might make pregnancy difficult without any personal details being exposed to the
Rh-negative more than one surrogate at any given time.
4. Should not have alcohol, smoke, or indulge in recipient.
5. Psychological screening for the couple • A couple shall not have simultaneous transfer of
substance abuse
embryos in the woman and in a surrogate. • Besides, the ART guidelines have restricted semen
5. Screen for infectious diseases—HIV, HBsAg, HCV, donation to only 25 times and only 10 pregnancies per
VDRL • A surrogate shall not act as an oocyte donor for the
couple, as the case may be, commissioning surrogacy. donor. Oocyte donation can be done only once in life-
2. With uterus: 6. CBC, TSH, serum creatinine, blood sugar time and in a married lady with at least one child of at
a. Repeated miscarriages 7. Psychological assessment of the surrogate and part- least 3 years.
b. Repeated implantation failure ner/screening for social issues THE SURROGACY (REGULATION) • These restrictions are required to prevent inadvert-
c. Untreatable Asherman syndrome 8. Transvaginal ultrasonography to rule out uterine
3. Medical conditions which make pregnancy anomalies
BILL 2016, AS INTRODUCED IN ent consanguineous conceptions. But, implementing
these guidelines can be made possible, if a database
life-threatening. 9. Assessment of uterine cavity—HSG/hysteroscopy THE LOK SABHA of donors is maintained along with data of pregnancy
10. Trial HRT cycle to assess endometrial thickness The salient features of the bill are: outcome.
Screening the Genetic Couple Abbreviation: HSG, hysterosalpingography
1. To allow only altruistic ethical surrogacy to intending
and Surrogate infertile couple. Egg Banking
Tables 37.3 and 37.4 define screening the genetic couple 2. The infertile couple should be between the age of 23 • The first human birth from frozen sperm was reported
and surrogate. • All expenses during pregnancy and delivery to be and 50 years and 26 and 55 years for female and male, in 1963.6
Table 37.5 describes how to prepare the surrogate and borne by the commissioning couple. respectively. • First human birth from a frozen embryo, reported in
the commissioning woman. • The surrogate may also receive monetary compensa- 3. The intending couples should be legally married for 1984.7
tion from the commissioning couple, for agreeing to at least 5 years. • In 1986, the first human birth from a frozen oocyte was
act as surrogate. 4. The intending couple should be Indian citizens.
Preparing the Genetic Mother and • Surrogate mother should be a married Indian woman
reported.8
5. The intending couples have not had any surviving
the Surrogate between 23 and 35 years of age and shall have at least child biologically or through adoption or through sur-
However, it is only now that, oocyte freezing is no
longer considered experimental.9
one live child of her own with minimum age of 3 years rogacy earlier except when they have a child and who
ART Guidelines for Surrogacy—2014 Draft • No woman shall act as a surrogate for more than one
The improved efficiency in oocyte freezing has made
is mentally or physically challenged or suffer from
the concept of egg banking, as in sperm banking, a
• The commissioning couple and the surrogate shall successful live birth in her life and with not less than life-threatening disorder with no permanent cure.
possibility.
enter into a surrogacy agreement. 2 years interval between two deliveries. 6. The intending couples shall not abandon the child,
born out of a surrogacy procedure under any
condition.
Disadvantages to Fresh Oocyte Donation
Table 37.5. Preparing the surrogate and the commissioning woman. 7. The child born through surrogacy will have the same • Long waiting lists due to difficulty in sourcing the
Commissioning woman Surrogate rights as are available for the biological child. donor
•• Evaluation •• Screening 8. The surrogate mother should be a close relative of the • Limited choice of donor
•• Synchronizing with surrogate menstrual cycle with oral •• Synchronizing the menstrual cycle with oral intending couple and should be between the age of 25 • Complexity in synchronization between donor and
contraceptive pills contraceptive pills and 35 years. recipient
•• Controlled ovarian stimulation of the woman •• Hormone replacement with estrogens to achieve a 9. She can act as surrogate mother only once. • Long periods of estradiol replacement due to uncer-
endometrial thickness of at least 7 mm tainty regarding the date of the donation
•• Semen collection from the commissioning male partner •• Progesterone supplement for the surrogate from the day • No quarantine period (HIV and others infectious
•• Oocyte retrieval from the female partner of oocyte retrieval
THE FUTURE OF THIRD-PARTY agents) as in sperm donation
•• Follow-up to rule out OHSS •• Embryo transfer for the surrogate REPRODUCTION • Supernumerary embryos cryostorage which increases
•• Early pregnancy management—HRT till 9 weeks 1. Central database of donors the workload of the egg bank.
maintaining the pregnancy 2. Egg banking—quarantining This improved efficiency in egg freezing combined
•• Handover of the baby to the commissioning couple 3. Carrier genetic testing—compatibility with donors with the disadvantages of fresh oocyte donation have led
along with birth certificate in the name of the couple to the emergence of a new phenomenon: commercial
4. Three parent baby
344 The Infertility Manual Third-party Reproduction 345
“egg banks”(CEBs),10 although, still at an infantile stage in dementia, and blindness; many of these conditions
India. are fatal.
• The reported pregnancy rate per oocyte was 7.5% using • Cytoplasmic transfer was pioneered in the late 1990s
vitrification. by clinical embryologist Dr. Jacques Cohen and his
• The recommendation is to obtain a minimum of six team at the St Barnabus Institute in New Jersey.
oocytes (range of four to seven) per recipient consid- • The US Food and Drug Administration (US FDA) has
ering oocyte survival, fertilization, and cleavage. banned it in the USA after some babies were born as
• Safety of long duration of storage: two of the fetuses developed genetic disorders, and
In one study, no differences in survival, fertiliza- the technique was halted by the US FDA. The problem
tion, cleavage, embryo quality, implantation, and may have arisen from the fetuses having mitochondria
live-birth rates were observed in oocytes cryopre- from two sources as they injected mitochondrial DNA
served with slow-freeze and thawed after up to from a donor into another woman’s egg, along with
48 months compared to earlier thaws.11-13 sperm from her partner.
In another retrospective study, embryos derived • However, The Human Fertilisation and Embryology
from cryopreserved oocytes demonstrated Authority (HFEA, UK) has concluded that given the
impaired blastulation but equivalent rates of tremendous individual and social benefits involved, it
euploidy, implantation, and live birth compared would be ethical to proceed with these techniques in Fig. 37.1: Method 1—Maternal spindle transfer.
with blastocysts derived from fresh oocytes, clinical trials (Figs. 37.1 and 37.2).
supporting the safety and efficacy of oocyte A baby boy was born on 6th April 2016 using the mater-
cryopreservation.14 nal spindle transfer technique. The boy’s mother carries
genes for Leigh syndrome, a fatal disorder that affects the
Compatibility Testing for Donors developing nervous system. Around a quarter of her mito-
chondria have the disease-causing mutation. While she is
• The global prevalence of all single gene diseases at
healthy, Leigh syndrome was responsible for the deaths of
birth is approximately 10/1,000 (WHO data).
her first two children. The couple sought out the help of
• Currently, in humans so far approximately 4,000 auto- John Zhang and his team at the New Hope Fertility Center
somal recessive diseases are known. in New York City. The process of three parent baby was
• Incidence of autosomal recessive diseases: 2.5 in 1,000
carried out in Mexico, due to the restrictions in the United
newborns. The risk for a healthy person to be a genetic
States.
carrier of a recessive disease is 1:10 to 1:200 (carrier
frequency of normal population.
• Genetic testing for carrier status is being done cur- CONCLUSION
rently using massive next generation sequencing.
The entire process of third party reproduction is a like a
Screening for carrier status can be offered for about
Pandora’s box where ethical questions are concerned.
600 monogenic diseases. Hence, every patient should be treated on an individual- Fig. 37.2: Method 2—Pronuclear transfer.
• Carrier genetic match (CGT), can be offered, in order ized basis, also, keeping the law of the land in view.
to match the donor and the recipient in order to avoid
any autosomal recessive disorders manifesting in the
offspring. PROBABLE QUESTIONS REFERENCES 4. Noyes N, Hampton BS, Berkeley A, Licciardi F, Grifo J,
1. 2008 Guidelines for gamete and embryo donation: a Practice Krey L. Factors useful in predicting the success of oocyte
1. “Third-party reproduction is like a Pandora’s box
Committee report Practice Committee of the American donation: a 3 year retrospective analysis. Fertil Steril.
Three-parent Babies—a Form where ethical issues are concerned”. Comment on the Society for Reproductive Medicine, and the Practice 2001;76(1):92-7.
above statement. Committee of the Society for Assisted Reproductive 5. Surrogacy. Infertility, Chapter 73. Principles and Practice
of Third-party Reproduction 2. Debate about the pros and cons of the surrogacy reg- Technology. Fertil Steril. 2008;90:S30-44. of Assisted Reproductive Technology, Volume 1. pp. 807-18.
• About one in 5,000 births and likely an even higher ulation bill. 2. The Assisted Reproductive Technology (Regulation) Bill, 6. Sherman JK. Improved methods of preservation of human
2014. Government of India Ministry of Health and Family spermatozoa by freezing and freeze drying. Fertil Steril.
proportion of fetuses that never make it to birth have a 3. Write a short note on “egg banking”.
Welfare, Department of Health Research. 1963;14:49-64.
mitochondrial DNA mutation. 4. Sourcing and screening of donors. 3. Oocyte and embryo donation. Infertility, 7. Trounson A, Mohr L. Human pregnancy following
• Mutations in mitochondrial DNA can cause rare 5. Future of third-party reproduction—looking through Chapter 71. Principles and Practice of Assisted Reproductive cryopreservation thawing and transfer of an eight cell
but progressive disorders, including heart failure, the crystal ball. Technology, Volume 1. pp. 791-7. embryo. Nature. 1983;305:707-9.
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346 The Infertility Manual
8. Chen C. Pregnancy after human oocyte cryopreservation.
Lancet. 1986;884-6.
9. Fahy GM, Wowk B. Mature oocyte cryopreservation: a
guideline. Fertil Steril. 2013;99:1.
10. Quaas AM, Melamed A, Chung K, Bendikson KA, Paulson
RJ. Egg banking in the United States: current status of
12. Potdar N, Gelbaya TA, Nardo LG. Oocyte vitrification in
the 21st century and post-warming fertility outcomes:
a systematic review and meta-analysis. Reprod Biomed
Online. 2014;29:159-76.
13. Parmegiani L, Garello C, Granella F, Guidetti D,
Bernardi S, Cognigni GE, et al. Long-term cryostorage does
Adoption: Review and
Current Status
38
Suvarna A Rathor
commercially available cryopreserved oocytes. Fertil Steril. not adversely affect the outcome of oocyte thawing cycles.
2013;99:827-31. Reprod Biomed Online. 2009;19:374-9.
Chapter Outline
11. Cobo A, Meseguer M, Remohi J, Pellicer A. Use of cryo- 14. Goldman KN, Kramer Y, Hodes–Wertz B, Noyes N,
banked oocytes in an ovum donation programme: a McCaffrey C, Grifo AJ. Long-term cryopreservation of • Reasons for Adoption • Procedure
• Adoption Triad • Cost
prospective, randomized, controlled, clinical trial. Hum human oocytes does not increase embryonic aneuploidy.
• Forms of Adoption • Disruption of Adoption
Reprod. 2010;25:2239-46. Fertil Steril. 2015;103:3. • Legislative History of Adoption • Psychological Effects
• Guidelines Governing Adoption of Children 2015 • Current Amendments
• Preference for Placement of Indian Children in Adoption
INTRODUCTION Following are the authorities or agencies for adoption
within the country:
The chapter explains the legal meaning of adoption,
various agencies involved in the process and different • Court of Competent Jurisdiction who can pass order
types of adoption. It provides insight into the guidelines for Adoption
• Central Adoption Resource Authority (CARA)
governing the process of adoption. It also describes the
• State Adoption Resource Agency (SARA) or Adoption
psychological issues faced by adoptee as well as the
Coordinating Agency (ACA)
adoptive parents. Current amendments are also high-
• Specialized Adoption Agency (SAA)
lighted in brief.
Adoption can be a wonderful gift for childless couples, Following are the authorities or agencies for adoption
single people as well as homeless children. Through adop- between different countries:
tion biologically unrelated persons can establish a parent- • Court of Competent Jurisdiction who can pass order
child relationship amongst them. Adoption is a means for adoption
through which a person undertakes the parenting of a • Central Adoption Resource Authority (CARA)
child, from that child’s biological or legal parents, and also • Central Authority in the receiving Country (CA)
permanently transfers all rights and liabilities, along with • Indian Diplomatic Missions Abroad
filiation, from the biological parents. • Foreign Diplomatic Missions in India
• Authorized Foreign Adoption Agency (AFAA)
• State Adoption Resource Agency (SARA) or Adoption
REASONS FOR ADOPTION Coordinating Agency (ACA)
• Infertility • Recognized Indian Placement Agency (RIPA)
• Infertile couple not willing for infertility treatment • Adoption Recommendation Committee (ARC)
• When pregnancy imposes serious health issues for
mother’s health
• Single persons who want to experience parenthood
• Couple having inheritable disease
• To have children of particular sex
• To avoid contributing to perceived overpopulation
• Adults who themselves were adopted as children
ADOPTION TRIAD
The triad is formed by the birth parents, the adoptive par-
ents, and the child who are connected by organizations
like adoption agencies as shown in Figure 38.1. Fig. 38.1: Adoption triad.
348 The Infertility Manual Adoption: Review and Current Status 349
FORMS OF ADOPTION Table 38.1. Age criteria for adoption.
• Independent Adoption: In this no agency is involved, Maximum Maximum
Age of the child composite age of age of single
rather the adoptive parents and the prospective plac-
(years) PAPs (years) PAP (years)
ing parents have located each other by themselves.
Up to 4 90 45
The adoption is mediated through an attorney or other
Above 4, up to 8 100 50
intermediaries as defined by the state law. It is not
advisable for international adoptions. Above 8, up to 18 110 55
• Agency adoption: The process is through an author-
ized agency which adheres to licensing and procedural
standards. It includes different types of adoption like: GUIDELINES GOVERNING ADOPTION
In open adoption identity information can be trans- OF CHILDREN 20152
mitted between the biological parents and adoptive
Adoption laws are different from country to country. We
parents. It also allows interaction between biologi- need to know various principles, policies, laws, regulations,
cal relatives and the adopted person. social issues, and procedures affiliated with adoption.
In closed adoption identities of the biological rela-
tives, adoptee, and adoptive parents are not dis- 1. Fundamental principles governing adoption:
closed. But nonidentifying information such as • While deciding the adoption child’s best interest is
religion and medical history are available. of paramount importance
In semi-open adoption is mediated by an agency. • In-country or within country adoption of the child
This may include contact between adoptive and is preferred
biological parents through the agency. • Process of adoption is guided by a set of procedures
• No one shall obtain any profit through adoption
2. Person competent of being adopted are as below:
LEGISLATIVE HISTORY OF ADOPTION • Orphan
Fig. 38.2: Procedure of adoption.
• The Guardians and Wards Act, 1890: Complete adop- • Abandoned or surrendered child
tion is not recognized in certain religions like Chris- • A child declared free for adoption by the Child Table 38.2. Cost of adoption procedure.
tianity, Muslims, Jews, and Parsis. People of these Welfare Committee (CWC) Cost for Indian Cost for foreign/NRI PAP Cost for foreign PAP
• If the child is 7 years or older his consent has to be Procedure PAP residing in foreign country residing in India US$
religions who wish to adopt a child can only take
the child in “guardianship” under the provisions of The taken before placing him for adoption Home Study Report Rs 6,000/- As per country norms 300
Guardians and Wards Act.1 3. Person competent to adopt: Child Study and Medical Examination Report, others Rs 40,000/- 5,000 US$ 4,700
• The Hindu Adoptions and Maintenance Act (HAMA), • Any individual whether single or married Counseling and follow-up postadoption Rs 2000/- per visit As per country norms
1956: This act is applicable only to Hindus.1 With the • Couple already having kids of any sex
help of this act, Hindu children can be adopted by • A couple having no child
Hindu individuals. 4. Eligibility Criteria for Prospective Adoptive Parents
completed but this is inapplicable when the two COST
are siblings
• Juvenile Justice (Care and Protection of Children) Act, (PAPs): The approximate cost of adoption is shown in Table 38.2.
• A single male cannot adopt a girl child
2000: The prime motive of the act is to provide a per- • A couple married for 2 or more years
• A single female can adopt a child of any gender
manent substitute family for an abandoned child.1 • Consent of both husband and wife is required DISRUPTION OF ADOPTION
• Juvenile Justice (Care and Protection of Children) • Live-in relationship couples are ineligible
Amendment Act, 2006:1 Under this act, 26 revisions • Age criteria for adoption are as per Table 38.1. PREFERENCE FOR PLACEMENT OF It is the termination of adoption, may it be before or after
the legal finalization. It is a process initiated by the PAP via
were made. This act formulates the legal proceedings • The age difference between the child and adoptive INDIAN CHILDREN IN ADOPTION a court petition. It is unique to adoptive parents, and is not
and forms a basic structure for care, protection, treat- parents should be at least 25 years applicable to a biological kin.
• Indians in India
ment, and rehabilitation of children with legal conflict • Couples having five or more children cannot be
• Nonresident Indians
and those in need of care. considered for adoption
• People of Indian origin
PSYCHOLOGICAL EFFECTS
• Guidelines issued by Central Adoption Resource Author- • The PAP should be financially sound so as to take
• Foreigners Adoptee
ity (CARA): It is an independent organization. It comes good care of the adopted child
under the Ministry of Women and Child Development, • The PAP should be healthy physically and mentally Generally adopted individuals lead a normal life. Some-
Government of India. It is authorized to supervise and • Adoption of a second child is permissible only PROCEDURE times, some adopted persons might have certain dis-
regulate the adoptions within and outside the country. after the legal adoption of the first child has been The procedure for adoption is illustrated in Figure 38.2. turbing experiences which have an impact on their lives.
350 The Infertility Manual Adoption: Review and Current Status 351
A multitude of issues may arise when children become Fast-track Adoption in Special 3. Borders LD, Penny JM, Portnoy F. Adult adoptees and their 6. Miller BC1, Fan X, Christensen M, Grotevant HD, van
aware of their adoption like: friends: Current functioning and psychosocial well-being. Dulmen M. Comparisons of adopted and nonadopted
Circumstances Family Relations. 2000;49:407-18. adolescents in a large, nationally representative sample.
• Feelings of loss, grief, rejection, and abandonment 4. Sharma AR, McGue MK, Benson PL. The emotional
• In case prospective parents have been registered with Child Dev. 2000;71(5):1458-73.
• Identity development and behavioral adjustment of United States adopted
a wrong agency and waiting for more than 3 years 7. Juffer F, Marinus H. van IJzendoorn. Behavior problems
• Self esteem: Some studies have shown that adopted adolescents: Part I. An overview. Children and Youth
Services Review. 1996;18(1/2):83-100. and mental health referrals of international adoptees a
persons have lower self-esteem and self-confidence • If parents are likely to leave for overseas on a long-
5. Baden AL, O’Leary Wiley M. Counseling adopted persons meta-analysis. JAMA. 2005;293(20):2501-15.
compared to the nonadopted persons3,4 term assignment and cannot come back in the near 8. Eldrige S. 20 Things Adoptive Parents Need to Succeed.
in adulthood: Integrating research and practice. The
• Behavioral and mental health: Studies have shown future Counseling Psychologist. 2007;35:868-901. New York, NY: Random house; 2009.
that adopted individuals suffer more from mental • If the biological child of the prospective parent has
health issues.5 Adoptees are at higher risk of depres- died (due to sickness or accident, but not neglect)
sion, substance abuse, anxiety, and psychological dis- • Facts or circumstances that indicate hardship or irrep-
orders like post-traumatic stress disorder or attention arable damage to parent or child.
deficit hyperactivity disorder. A large nationally rep-
resentative sample study done in the USA also shows Thus, adoption is a rewarding challenge which involves
that adopted adolescents are prone to substance a commitment for lifetime. It gives a person an opportunity
abuse, lying, and poor physical and psychological to experience the pleasure of parenting and gives a child
well-being.6 Another meta-analyses showed that com- privilege of parental love and care. Adoption is a journey
pared to the nonadopted controls, adopted individuals of time, healing, faith, challenges, learning, attachment,
require more referral to mental health services.7 and grace. This noble act of adoption has to be regulated
to prevent swindling of prospective parents and help chil-
Adoptive Parents dren meet their unfound families. Though it seems to be a
Like the adoptee, adoptive parents also encounter an array complex and time consuming process but may be worth
of difficulties like: for those who are in need.
• Difficulty in developing attachment toward the child
• Loss of confidence and suspicion about the parenting PROBABLE QUESTIONS
capabilities
• Postadoption depression syndrome (PADS): This syn- 1. Define adoption. Motives for adoption.
drome may occur within few weeks of finalization of 2. Write a short note on Central Adoption Resource
the adoption process. In this syndrome, the parents Authority (CARA).
who are yearning for months and years anticipating 3. What are the eligibility criteria for prospective adop-
parenthood suddenly lose all the interest and excite- tive parents?
ment of adoption and start experiencing depression or 4. Brief about the adoption procedure in India.
sadness.8 5. Define adoption and different types of adoption.
The adoption process might take the persons involved 6. What is an adoption triad? Brief about the challenges
through a spectrum of emotions, which ranges from exhil- faced by the adoptee and the adoptive parents.
aration to anguish. Specialized adoption agencies, support 7. Brief about the guidelines governing adoption and
groups, and therapists skilled in adoption can help indi- the recent amendments.
viduals to cope with these feelings. 8. Write a note on fast-track adoption.
CURRENT AMENDMENTS
REFERENCES
• All the adoption agencies must have registration with
CARA. 1. Roy D. Legal Service India-Adoption under Juvenile Justice
Act: a clarion call to secularism. [online] Available from
• All adoptions should happen through an online regis-
http://www.legalserviceindia.com/article/l327-Adoption-
tration process. under-Juvenile-Justice-Act.html.
• Allowing prospective parents’ age norm to be waived 2. Central Adoption Resource Authority, Ministry of Women
off if PAP are adopting a child more than 5 years along & Child Development Government of India. [online]
with his/her sibling, or a special child, or a sick child. Available from http://www.cara.nic.in.
SE C T I O N
7
Laboratory Management
C HA PTE R
Quality Control
in the ART Laboratory
39
Hemalatha Ravikumar
Chapter Outline
• Quality Control • Micromanipulator and Stereo Zoom Microscope
• Total Quality Management • Air Quality in IVF Laboratories
• Quality Assurance • Instruments for Oocyte Retrieval
• References of Quality Guidelines • Quality Control of Culture Medium and Disposables
• Components of Quality Control Program • Sterile Culture Conditions
• Laboratory Director Responsibilities • Correct Handling and Identification of Patients and Their Gametes
• Laboratory Supervisors and Embryos
• Clinical Embryologists • Daily Check-list
• Quality Management • Weekly Check-list
• Quality Control of Laboratory Equipment • Pressure Modules (Air Purifier)
• CO2 Incubators • Quality Control Using Evaluation of Results
• Laminar Flow Workstation
“Quality control (QC) and quality assurance (QA) in the in Incubators
vitro fertilization (IVF) laboratory play an important role Refrigerators
in the success of any IVF program. The role of QC proce- Freezers
dures in the IVF laboratory is to fine tune existing protocols
in order to more effectively help infertile patients in their
quest to have a healthy baby.” TOTAL QUALITY MANAGEMENT
The role of QC procedures in the IVF laboratory is • A management approach of an organization centered
to fine tune existing protocols. Effectiveness, to have a on quality, based on the participation of all its mem-
healthy baby in infertile patients. bers, and aiming at long-term success through cus-
tomer satisfaction and benefits to all members of the
QUALITY CONTROL organization and society. The services provide in the
• All elements required for QC in the laboratory, which laboratory should cover every aspect including, outside
includes all activities and operational techniques has the laboratory also to continuously improve the total
to meet QC. Elements include protocols, documenta- quality management (TQM). Not restricted to IVF labo-
tion, record keeping, personnel working in laboratory, ratory but includes every function in the assisted con-
instruments, equipment, supplies, and techniques of ception unit. This ensures QA and QC of the whole unit.
the laboratory. Measurements of elements are taken to
compare with established norms, to maintain optimal QUALITY ASSURANCE
standardization of results.
• The three most important physical conditions in the • The objective of QC is not only to maintain the stand-
IVF laboratory that can be controlled are: ard (QC), but to also improve the outcome. This also
Temperature of the IVF culture medium involves monitoring and evaluating the whole process
pH of the IVF culture medium to ensure the highest standard of treatment. The total
Osmolarity of the IVF culture medium sum of all activities required in order to establish con-
• Documentation of above mentioned physical condi- fidence that the product or service meets the deter-
tions must be monitored regularly to maintain the pro- mined quality requirements.
gram of QC and QA.
• Monitoring and documentation of temperatures REFERENCES OF QUALITY GUIDELINES
inside the below mentioned equipments is essential as
they are an integral part of routine day to day QC in the • Association of Clinical Embryologists 1996. Accredita-
IVF laboratory: tion Standards & Guidelines for IVF laboratories.
356 The Infertility Manual Quality Control in the ART Laboratory 357
• ISO 15189/2002—Medical laboratories—particular 7. Management of laboratory staff training and contin- QUALITY MANAGEMENT Data should include:
requirements for quality and competence. ual scientific and biomedical education.
• American Society for Reproductive Medicine (ASRM) 8. For QC and QA purposes, periodic or regular review According to the European Directives and Recommen- • Morphological characteristics of gametes and embryos.
2008—Guidelines for human embryology and androl- of key performance indicators (KPIs) of all laboratory dations (European Commission, 2006a, c, 2012; Coun- • Detailed information of the procedures, including tim-
ogy laboratories. procedures should be updated and implemented. cil of Europe, 2013), QMS is compulsory, laboratory ing, and staff involved.
• European Society of Human Reproduction and Embry- 9. Reporting of clinical data and adverse events accord- personal has to work in line with recommendation done by • Data collected should contain, all the informa-
ology (ESHRE) 2012—Guidelines for good practice in ing to European and/or national regulations. European Commission, related to the management and tion, which is mandated by international or national
IVF laboratories. 10. Approval of research projects by competent organization, also related to materials, equipment and registry.
authorities. facilities in the premises and also recommendations done • Should contain all the information communicated by
COMPONENTS OF QUALITY CONTROL related to personnel working in laboratory documenta- the consultants with patient or patient with consultant
PROGRAM LABORATORY SUPERVISORS tion and records maintained by staff in the laboratory, and discussed, etc. related to counseling done, information
includes quality review done. related to laboratory procedures.
Some laboratories may require additional managerial
These are key factor which includes QC in ART • Taking into account the high degree of attention
positions. These require specific qualifications like: • All personnel in the laboratory must be competent and
laboratories. needed during laboratory work, distractions should be
• At least a BSc in biomedical sciences qualified, which includes their responsibilities well minimized.
• Laboratory personnel, laboratory equipment’s, labora-
• Three years of documented human embryology defined. • Proactive risk assessments should be made and pre-
tory procedures, laboratory records maintained, labo-
experience • Having validated, written instructions for each process
ratory periodic review of daily QC records, laboratory ventive actions taken to minimize nonconformities.
• Preferably attainment of the ESHRE clinical embryolo- (SOP), including management of adverse events.
data storage, laboratory plastic disposables and labora-
gist certification or similar certification. • Records are maintained of all individual persons of (Referred from ESHREE revised guidelines for good
tory culture media (laboratory materials and supplies),
and laboratory environment. Definitive identification Laboratory supervisor responsibilities also include specific activities related in laboratory. Records are practice in IVF laboratory 2015.)
of patients and their biological requirements is a must. ensuring: maintained in such a way that cells and tissues of
• Laboratory personnel is one who is appropriately • Efficient organization of daily work of their areas of
patient are traceable and also of materials and equip- QUALITY CONTROL OF LABORATORY
qualified, experienced, responsible, has required quali- responsibility ment used. EQUIPMENT
fications, and expertise in the field of embryology and • Effective communication with laboratory staff and • Appropriate assays are used to test the Medias,
biological or medical sciences according to national reagents, and disposables for their quality and • The laboratory equipment must be adequate for labo-
clinical colleagues
rules (ESHREE Guidelines). Continues to attend con- • Continuous improvement where possible confirmed. ratory work and easy to clean and to disinfect.
tinuing medical education (CME) program for both, • Structured training of staff members and students. • Ensuring proper and periodic equipment mainte- • Critical items of equipment, including incubators and
new or senior embryologists. To upgrade the knowl- nance, service, and calibration. frozen gamete, zygote, and embryo storage facilities,
edge and skills by attending national and international
CLINICAL EMBRYOLOGISTS • Verifying conformance to specifications. should be appropriately alarmed and monitored. An
conferences and workshops. Has to undergo periodic automatic emergency generator backup in the event
• Taking corrective action to keep procedures under
assessment of competency. • In daily clinical practice, the first line of participation is of power failure should be in place. A minimum num-
represented by clinical embryologists. An undergrad- conformity.
• Reviewing performance to ensure continuous and sys- ber of two incubators is recommended. Gas cylinders
LABORATORY DIRECTOR uate degree of biomedical sciences like BSc is a must
should be placed outside or in a separate room with an
for this kind of program. Clinical experienced embry- tematic QMS improvement.
RESPONSIBILITIES ologists should train a new staff. Clinical experienced • Providing risk assessment analysis for all laboratory automatic backup system. Incubators should be fre-
embryologists should supervise, while a new staff is quently cleaned and sterilized.
ESHREE—2015 Guidelines: activities.
trained, to follow the protocol or structural training • A clinical embryologist is held accountable for the • All QC values should be recorded in the morning prior
1. To reach the highest standards in clinical IVF, by pro- program. to opening the incubators and prior to beginning any
management of QC in the laboratory is recommended.
viding with most advanced materials and implement • Clinical embryologists with 3 years’ experience procedure. The QC data are thus reflective of stable
of latest procedures available. • Written, authorized, signed, and up-to-date SOPs
should endeavor to apply for the ESHRE clinical levels maintained during the night preceding any pro-
2. According to National and/or European regulations, should exist for all processes in order to optimize out-
embryologist certification, whereas those with higher cedures performed.
facilities in laboratory equipment’s must be appropri- comes.
degrees and 6 years’ experience should endeavor to
ate, latest, and safe. apply for the ESHRE senior clinical embryologist cer- • The patient must be provided with, unique identity for
3. Implementation of a quality management system tification. the maintenance of confidentiality of patient, for their CO2 INCUBATORS
(QMS). tissues, and reproductive cells in the QMS. • Incubator in the IVF laboratory play an important role
Clinical embryologist responsibilities include:
4. Implementation of a laboratory risk management and • Corrections should be traceable, if made electronic in providing a stable and appropriate culture environ-
prevention policy. • Execution of standard operating procedures (SOPs) mode or written. ment required for optimizing embryo development
5. Sufficient laboratory staff members with the appro- • Participation in daily practice, communication, and • Database must contain, relevant data concern- and clinical outcome. Incubator also maintains a very
priate skills. organization ing recorded in laboratory work and also during KPI constant environment and a clean environment for
6. A comprehensive orientation and introduction of • Contribution to laboratory clinical decisions extraction and statistical analysis. embryo culture.
programs for all new staff members. • Training of staff members and students
358 The Infertility Manual Quality Control in the ART Laboratory 359
• The most commonly used environment conditions for environment. The stereo microscope has to be built
human IVF incubators are: into the laminar flow cabinet (LAF) cabinet, so
5% CO2 in air that, the entire table top can be heated to maintain
37°C temperature temperature at 37°C. This station should be used for
100% humidity handling oocytes and embryos. Test tube warmers
• Variation in pH and temperature affects the embryo are used for maintaining the temperature of follicular
quality, in turn affecting pregnancy rate. Therefore aspirates contained in test tubes at 37°C which can be
proper maintenance of CO2 incubator is very impor- placed in laminar hood to screen follicular fluid and
tant. The measurement of CO2 percentage and temper- dish warmers which maintain 37°C to preequilibrate
ature of incubator is to ensure stability of pH and tem- 65 × 30 mm dish for follicular screening.
perature. Separate readings of temperature and gases, • Vertical laminar airflow is preferable to avoid cross
calibrated thermometer inside the incubators as stand- infection. In embryology laboratory, laminar air flow
ards (Figs. 39.1A to C). Monitor CO2 supply cylinders hood must be kept, for maintenance of aseptic manipu-
regularly; ensure that auto changers are functional. For A
lation of gametes and embryos. Vertical laminar airflow
humidified incubators, check water levels daily. should be switched 1 hour before ovum pick-up (OPU). Fig. 39.2: Micromanipulator with inverted microscope.
Each incubator should have a 24 hours surveillance Vertical laminar airflow must be run throughout the
system, with alarm accessible to a staff when at home day and switched off when the day’s work is over.
or work. • The filter should be changed every 6 months.
development, reduction in viability, implantation and
• Regular cleaning is to be done by surface disinfectant
• A secondary back up power source should be installed pregnancy rates. Toxic materials are particulate mat-
with Oosafe or 70% alcohol after the day’s work.
and a contingency or alternate plan should be made ter, bacteria, dust, and chemicals; VOCs are believed
• If there is any soiling/spillage happens during work,
available to all laboratory staff with backup arrange- to exert a range of effects on above mentioned condi-
it should be cleaned by 70% alcohol. Minimum of
ments clearly outlined in writings or printout fixed at tions. These effects may or may not be evident mor-
30 minutes gap should be given, to resume the work
important places in laboratory. phologically. Fine particulate matter has effects like:
after exposure to the alcohol.
• A log book is maintained for proper documentation. Decreased implantation rates
• Laminar hood should not be placed near incubator, as
• Incubator must be regularly cleaned and decontami- Pregnancy rates
the air flow can cause dramatic changes in the osmo-
nation done. Increased miscarriage rates
larity of the media inside the incubators.
• Water in incubator pan (to maintain humidity) must • Particle counts are performed at various points in the
• Daily cleaning of surface.
be regularly changed, before new batch starts. laboratory and other critical areas by Third party Certi-
B • Regular servicing, cleaning of HEPA filter is a must.
• A sperm survival test is done before every batch to fication Company. Individual and mean counts for dif-
• Ensure the HEPA filter is working properly by testing
ensure proper functioning of incubator. ferent sizes (0.3, 0.5, 5 microns) are measured. Reports
the density of the particulate matter in the air.
• Annual maintenance contract with the supplier for are compared with previous records and standards are
servicing must be regularly maintained. set for any trends of increased air borne particulates.
• Internal air filter may be added to decrease the volatile
MICROMANIPULATOR AND STEREO • Results or report is done as mentioned below:
organic compound (VOC) level inside the incubators. ZOOM MICROSCOPE Not within limits
• Incubators should not be opened frequently and • Daily measurement of temperature of the stage warmer Potential contamination source identification is
widely. Consecutive opening and closing for two to done
is required.
three times within 5 minutes causes a significant fall • Clean rooms equipped with carbon-impregnated fil-
• Lack of clarity noted and rectified.
in CO2 concentration and temperature which will be ters for air and incubators results in better good qual-
• Procedures for alignment of the optical path should be
detrimental or bad for both the oocytes and embryos. ity embryo formation, cleavage, and pregnancy rates.
readily available.
• QC of image quality is to be recorded daily and also to Clean rooms equipped with carbon-impregnated fil-
LAMINAR FLOW WORKSTATION be documented daily. ters for air and incubators results in control of air pollu-
• Regular cleaning with Oosafe (surface disinfectant) tion in ART laboratory, operating, and transfer rooms.
• Laminar flow hoods together with high-efficiency
C after each procedure must be followed (Fig. 39.2). Construction of clean rooms equipped with carbon-
particulate absolute (HEPA) filtration. HEPA filtration
Figs. 39.1A to C: (A) Digital display of CO2 level in incubator. (B) Moni- impregnated filters for air and incubators results in
captures and removes airborne contaminants.
toring of CO2 tank pressure. (C) External CO2 analyzer. lowering spontaneous abortion rates. Measures to be
HEPA filtration also provides a particulate free work AIR QUALITY IN IVF LABORATORIES taken to improve air quality in the laboratory are: high
• Toxic materials present in air of IVF laboratory have positive pressure air module or air curtain at the door.
effect on fertilization failure rate, delayed embryonic Activated carbon pre-filters near the incubators.
360 The Infertility Manual Quality Control in the ART Laboratory 361
• HEPA filter which purifies air of harmful volatile com- STERILE CULTURE CONDITIONS Once the sample is collected and it arrives in the
pound, bacteria, and molds. HEPA filter is 99.97% laboratory along with the file, the name on the jar is
effective in the removal of particles with more than • To ensure that your embryos are happy in the IVF lab, cross checked with one on the file. We analyze the
0.3 m diameter. Laminar air flow equipped with HEPA we need to ensure optimal culture conditions. These sample and transfer the sample to sterile centrifuge
filter is used for manipulation of gametes. include: tube, which is labeled with his name. The cap is
• Suitable extraction system in operation theater (OT) also labeled.
IVF Laboratory Cleanliness
for removal of anesthetic gases used for OCR. Positive The sperms will be processed while they are in this
pressure air modules, such as Coda tower, within the • Daily Cleaning of IVF laboratory with proper disinfect- tube and will remain in this tube till we use them
laboratory to eliminate dust particles, microbes sus- ant is mandatory. for IVF procedure.
pended in the laboratory air. • The disinfectant should be nontoxic and odorless. The culture dishes required for the IVF Procedure
Benzyl-alkyldimethyl chloride (Oosafe) or 70% alcohol are prepared well in advance. All the dishes are
is used as disinfectant to do so. labeled with name. The shelf of the incubator in
INSTRUMENTS FOR OOCYTE RETRIEVAL
• Alcohol-based solutions can be embryo toxic and which the dishes are kept is also labeled.
• Ultrasound machine with transvaginal probe and should not be used. • Verification of patients’ identity should be performed
biopsy attachment. at critical steps:
• Aspiration pump: An aspiration pump with foot con- CO2 Incubator Cleanliness Before egg collection, at semen recovery and
trol is used to puncture follicles and aspirate follicular embryo transfer procedures
• Regular cleaning and disinfection of CO2 incubator
fluid containing oocyte corona cumulus complex by The nurse and OT staff cross check patient file
should be performed once in every 3–6 months.
single or double lumen needles. The pump should pro- before taking, patient inside the OT for procedure
• Aseptic precautions while handling gametes and
vide a smooth and low volume suction to avoid injury The doctor and embryologist are to be informed by
embryos:
to the oocyte. concerned nurse, while the patient is being taken
One should wash his hands properly before enter-
• Dry bath incubator is required for warming tubes and inside the OT
ing and before handling the dishes containing
media. During the transfer of embryo, the nurse stands as
embryos.
While preparing dishes and handling culture witness, while the catheter is loaded with patient
QUALITY CONTROL OF CULTURE medium, gloves should be worn. embryos
Patient name is confirmed loudly or audibly with
MEDIUM AND DISPOSABLES the clinician before handing over the catheter Fig. 39.3: Verification of patients’ identity.
• (ESHREE guidelines—2015): When commercially CORRECT HANDLING AND loaded with patient embryos.
produced media are used, integrity of the packages IDENTIFICATION OF PATIENTS AND • Critical procedures like insemination of oocytes, dur-
and appropriate delivery conditions should be con- ing replacement of embryos, while freezing of embryo,
THEIR GAMETES AND EMBRYOS
trolled. thawing of embryo, are to be cross checked by two per- equipped with stages and heating blocks pre-warmed
• Documentation of QC testing specifies in the certificate Written procedures should be present describing the vari- sons in concerned laboratory. at 37°C. Tissue culture grade disposable items should
of analysis (COA) should be supplied by the manufac- ous phases of IVF techniques. Rules concerning the cor- • Liquid nitrogen (LN2) are regularly filled up in cryo- be used in the laboratory procedures for culturing eggs
turer for any commercially produced media distribu- rect handling and identification of gametes and embryo cans, the samples of sperm and embryo are cross and embryos.
samples should be established by a system of checks. checked thrice and prior labeling is done, before these
tors. • Aseptic technique should be used at all times.
Where needed, double-checks are to be done.
• Documentation of shelf life and batch number is samples are stored in LN2 filled cryocans. • Appropriate measures should be taken to be main-
maintained. • All material obtained from the patients, i.e. tubes with • Sperm and embryo samples are double-checked by tained at 37°C during handling/observation or while
• Culture media—tested using: follicular fluid containing eggs and containers con- the embryologists to ensure that correct samples are using stage warmers or other systems while checking
An embryo development assay, e.g. Mouse embryo taining sperm, should bear the names of the treated used. Samples are then checked by the senior embry- oocytes and embryos.
assay, sperm survival assay, ensure nontoxicity of couple. ologist, IVF physician, and nurse-in-charge, prior to • Tissue culture grade disposables should be used for
plastic materials, use of an oil overlay, and carbon When the husband collects a semen sample for embryo transfer or use during intracytoplasmic sperm handling gametes.
filters for volatile organics. an IVF procedure, semen freezing or semen analy- injection (ICSI) (Fig. 39.3). • Pipetting devices (Pasteur, drawn pipettes, tips, etc.)
• Stock keeping with reorder level is maintained to avoid sis, the person in-charge will ask the patient his used for procedures should be disposed of immedi-
shortage. full name to avoid any spelling mistakes and the
Handling of Oocytes and Spermatozoa ately after use.
• Proper maintenance of refrigerator, i.e. cleaning and same name will be written on semen collection jar. • The laboratory procedures regarding the handling of • One patient must be treated always; simultaneous
regular temperature measurement for storage of cul- The jar is labeled with husband’s name with unique gametes for assisted should be easy, simple and effec- treatment should never be done with other patient in
ture medium. patient ID number and his wife’s name. tive and must be performed in a laminar flow hood the same working place.
362 The Infertility Manual Quality Control in the ART Laboratory 363
Protective Measures Quality Control of Lasers QUALITY CONTROL USING EVALUATION 4. What are the sources of laboratory errors?
5. What are the advantages and disadvantages of quality
The purpose of the protective measures is also to ensure • QC with every use. Frequent checking, for confirma- OF RESULTS
control?
aseptic conditions for gamete and embryos: tion of alignment of beam. Frequent checking, for con- On regular basis, results are to be evaluated. The factors to
• Use of nontoxic (nonpowdered) gloves and masks firmation of lasers power (set by adjustment of beam be evaluated regularly are listed below:
• Use of vertical laminar-flow benches intensity and duration of light pulse). Documentation
BIBLIOGRAPHY
• Fertilization rates 1. Air quality in the assisted reproduction laboratory: a mini-
• Use of mechanical pipetting devices of above mentioned is important.
• Embryo quality review. Journal of Assisted Reproduction and Genetics.
• Use of disposable material; after usage, it must be dis-
• Pregnancy rates 2015;32(7):1019-24.
carded immediately in the proper waste containers. Monitoring Liquid Nitrogen Levels • Multiple pregnancy rates 2. Boone WR, Higdon III HL, Jhonson JE. Quality management
Potential infectious materials must be disposed of in a issues in the assisted reproductive laboratory. J Reprod
• Measuring stick should be used to measure the level. • Implantation rates
manner that protects laboratory workers and mainte- Stem Cell Biotechnol. 2010;1(1):30-107.
Stick should be lowered till the bottom of the tank and • Survival rate for frozen embryos 3. IVF Success & Temperature Control in IVF lab. The IVF lab.
nance, service, and housekeeping staff from exposure
to infectious materials in the course of their work. also allowed to cool for a few seconds. Stick is then Posted by IVF World, Shivani on July 16, 2016.
• Needles and other sharps should be handled with taken out and shaken. Frost or condensation takes Record Keeping 4. Kastrop P. Quality management in the ART laboratory.
places where the measuring stick had contact with Reprod Biomed Online. 2003;7:691-4.
extreme caution and discarded in special containers. • QC recording and maintenance is a must. 5. Mayer JF, Jones EL, Dowling-Lacey D, Nechiri F, Muasher
Special containers are used to discard used broken LN2. The height of LN2 is recorded by seeing the con-
• Available for review in the future. SJ, Gibbons WE, et al. Total quality improvement in the IVF
glassware and Pasteur pipettes. densation value, which is at the highest point. laboratory: choosing indicators of quality. Reprod Biomed
• Helps in analysis later, when problems arise or
Online. 2003;7(6):695-9.
improvements are desired.
Quality Control of Procedures Electronic Monitoring Functions • Helps in determining corrective actions (QA) in future.
6. Rao KA. Assisted Reproduction Technology.
7. Referred from ESHREE revised guidelines for good practice
• Every laboratory should have a manual detailing of all • Early alarm and alarm of sound and light for LN2 level in IVF lab, 2015.
the procedures done in the laboratory. • Temperature display, digital temperature adjustment, Paper Records: (Disadvantages) 8. Revised Guidelines of Human Embryology and Andrology
• The procedures should include sperm assessment calibration. Threshold-crossing alarm signal should be Laboratories. The Practice Committee of the American
and preparation for ICSI or IVF insemination, oocyte • Conventional method Society for Reproductive Medicine and the Practice
set accurately.
• Analysis not possible Committee of the Society for Assisted Reproductive
retrieval, oocyte culture, IVF insemination, ICSI, ferti- • Level of LN2 in the tank should be measured daily. The Technology. Birmingham, Alabama.
lization check, embryo culture, preparation of embryo level should not be allowed to drop below 5 inches. 9. Rush Center for Advanced Reproductive Care, Chicago.
culture dishes, embryo selection, embryo transfer, and • Lower levels of LN2 could allow some of the product Electronic Records: (Advantages) Illinois, Marathon Products San Leandro.
cryopreservation of gametes and embryos. stored to thaw. Levels below 2 inches are critical. 10. Sharma L, Tarchala S, Nakagawa J, Perry J, Rawlins R. Next
• Records in spreadsheets/database generation quality control in the IVF laboratory. Using data
• Laboratory protocols have to be transposed into SOP.
• Analysis possible immediately loggers to monitor real time incubator temperature.
All techniques details, done in the ART laboratory has
DAILY CHECK-LIST • Ensure data backup 11. Sharma L, Tarchala S, Nakagawa J, Perry J, Rawlins R. Next
to be written clearly and also all the activities done in Generation Quality Control in the IVF Laboratory Using
the ART laboratory. • CO2 Incubator (temperature, CO2, humidity) Data Loggers to Monitor Real Time Incubator Temperature.
• All SOPs are to be dated and signed by embryologist. • CO2 cylinder before and after procedure over10 PROBABLE QUESTIONS Rush Center for Advanced 9.9. Reproductive Care, Chicago.
Information related to SOPs is to be updated on regu- • Petridish warmer should be maintained at 37°C 1. What is quality control? Illinois.
lar basis to all staff. • Dry bath, heating block, and heated stages should be 12. The Practice Committee of the American Society for
2. What is the difference between quality control and
Reproductive Medicine & the Practice Committee of the
• Compliance of the SOP is a must. No excuse to be maintained at 37°C quality assurance? Society for Assisted Reproductive Tecnology. Birmingham,
entertained for noncompliance of SOPs and has to be • Microscopes weather light is working and to check 3. What are the quality control tools called as? Alabama.
punished severely. objectives
• Refrigerator. LN2 level in cryocans and storage tank
Quality Control of pH • Media and consumables stock and to check shelf-life.
• Measured by digital pH meter. pH meter requires daily
calibration QC to be documented. Easier to control the WEEKLY CHECK-LIST
pH by controlling the % CO2. • pH meter check10
• Incubator humidity, water change if any contamination
Quality Control of Humidity
• High humidity maintained inside incubator, 90–100%.
Humidity maintained by pan of water. Measured by
PRESSURE MODULES (AIR PURIFIER)
digital hygrometer. Quality control to be documented • Regular servicing
on weekly basis. • Regular cleaning of HEPA filter (once in 6 months)
C HA PT E R
40
Follicular Fluid Screening 365
Follicular Fluid Screening
Hemantha Kumar
Chapter Outline
• Why Follicular Fluid Screening? • Instruments
• Preparation for Follicular Screening • Oocyte Nuclear Maturity Evaluation
• How to Do Follicular Fluid Screening? • Oocyte Dysmorphisms
INTRODUCTION and BMP decreased with reproductive aging. So proteome
analysis of FF might be helpful in evaluating oocyte qual-
By understanding the process of folliculogenesis, we
ity and identifying predictive markers for oocyte develop-
believe that initial hormonal growth is not hormonal
mental potential prior to fertilization and the success in
dependent. Each day an average of 50 follicles start to A B
assisted reproductive technology (ART).2
grow in the human ovaries. But not all follicles reach the Figs. 40.1A and B: (A) The presence of cumulus in the FF can be seen in the naked eye at 11’O clock position. (B) Oocyte cumulus complexes
maturation stage and survive till ovulation (to be precise (OCCs) can be visible as transparent gelly mass.
in normal female every month only few follicles reach
WHY FOLLICULAR FLUID SCREENING?
the maturation stage and only one follicle ovulate and • Follicular fluid screening aids in identification of
produce egg). Most of them, undergo atresia. Actually to oocyte and its further assessment. • Blower should be on in the LAF throughout the FF • Once FF reaches in the IVF laboratory, FF poured into
complete the growth phase, it requires 90 days and three • Follicular screening also gives first sign of information screening. a Petri dishes 65 mm prewarmed at 37°C.
ovarian cycles. about the quality of oocyte. Check the heating blocks, LAF workstation are main- • Pour the FF not more than half of the tubes per dish.
We know that as follicles grow, the production of fol- • Studying the oocyte cumulus complex (OCC) in FF tained the temperature at 37°C and media to be used • Then carefully inspected the FF for the presence of
licular fluid (FF) increases its accumulation in the intracel- tells about its maturation: for flushing and it must be equilibrated before 8 hours. cumulus.
lular space eventually connecting to form a cavity called 1. If it has a dense compact cumulus oocyte might be Prewarm the Petri dishes (65 mm) and 14 mL ster- • The cumulus can be identified with the naked eye (in
antrum. immature. ile test tubes for the FF collection. Moreover to the Figs. 40.1A and B) but the presence of an oocyte
2. Expanded cumulus and slightly compact corona minimize the temperature changes, the collection can only be assessed under stereo zoom microscope
Follicular antrum has FF and surrounds the ovum in
can be intermediate stage. tubes should kept in a warmer before 30 minutes of (Fig. 40.2).
an ovarian follicle. It has hyaluronic acid and portion ovar-
3. Mature stage: Well expanded cumulus and corona the procedure. • Once identified the OCC, flush the glass pipette
ian follicle filled with liquor folliculi. Presence of the folli-
radiata distribute evenly around oocyte. Check the pressure and make sure aspiration nee- (1,000 µ) to pick the OCC, otherwise it will stick to the
cular antrum is the first sign that a follicle has reached the dle connected properly to the suction pump. glass pipette.
4. Postmature stage: Irregular and zona not com-
next stage of maturation to form a secondary follicle. Align the stereo microscope and check the light • Then collect OCC, washed two to three times in the
pletely visible, dark granular ooplasm
Follicular fluids protein composition gives an infor- source for the better visualization. buffered medium to remove red blood cells (RBCs)
5. Atretic stage: Cumulus will be in small amounts,
mation for the presence of various types of protein. Gon- • Lower magnification (1*10X) can be used for screening and other cellular components (Figs. 40.3A and B).
irregular corona, zona will be visible and ooplasm
adotropins and steroids in the FF as the markers for oocyte the FF. • Then dish containing all the OCCs is placed in the CO2
in irregular shape.3,4
maturation are proven by some of the studies. The other • Oocyte identification in the cumulus can be verified in incubator for next 2–3 hours for incubation (cytoplas-
factors, such as vascular endothelial growth factor (VEGF), zooming the object with (1X, 2X, 3X, 4X, 6.5X). mic maturation).
inhibin A and B, anti-Müllerian hormone, lactoferrin,
PREPARATION FOR FOLLICULAR Collected OCC immediately transfer to the CO2 incu-
insulin-like growth factor-II (IGF-II), hyaluronan, nitric SCREENING HOW TO DO FOLLICULAR bator. Because of temperature and pH fluctuation, it affects
oxide, leptin, 25-OH vitamin D, glucose, bone morpho- the oocyte competence.4
• To prevent risk of contamination or infection, class II FLUID SCREENING?
genetic proteins (BMPs), and interleukin 8, correlate with vertical laminar air flow is recommended for in vitro
• The FF is collected in sterile round bottom test tube
oocyte maturation and folliculogenesis.1 fertilization (IVF) laboratory. Follicular Fluid Contaminated with Blood
(14 mL) held at 37°C.
The FF composition alteration is associated with • Follicular fluid cannot be examined immediately.
• Those FF tubes aseptically passed to the IVF laboratory • During multiple ovarian punctures at oocyte retrieval,
diminished reproductive capacity. Some of the FF mark- • FF should collected in the sterile test tube (14 mL) with
by hand relay in the heating blocks. chances of blood contaminating the FF is high.
ers such as interleukin-8, GDF-9 and transforming growth strictly maintained 37.0°C until to poured in the pre-
factor beta 1 (TGF-β1), inhibin, anti-Müllerian hormone, warmed Petri dishes (65 mm).
366 The Infertility Manual Follicular Fluid Screening 367
INSTRUMENTS
Vacuum Pump
The oocyte retrieval is carried out using an aspiration
unit composed of vacuum pump, which connected to a
23-gauge needle connecting to the 14 mL test tube. Maxi-
mum pressure should be 120–140 mm Hg. Because if the
oocytes are exposed to an increased pressure that may
affect their integrity and may damage the oocyte.
Laminar Air Flow
Laminar air flows creates working area free from con-
taminants. Because laboratories require sterile work-
ing environments in order to handle the oocyte/sperm/
Fig. 40.2: Presence of oocyte in the OCC at 6 O’clock position (black embryos. Fig. 40.4: Schematic diagram of a class I biological safety cabinet. Fig. 40.5: Schematic diagram of a class IIB1 biological safety cabinet.
dot) with the magnification (×1–10). Abbreviations: RA, room air; FO, front open; PCA, principal component Abbreviations: HEPA, high efficiency particulate air; RA, room air
Why Laminar Air Flow Cabinets? analysis
• These bloody samples will often clot despite of the Laminar flow cabinet provides a clean air environment
presence of heparin in the flushing media. and particle free working area by air filtration system and • Most compound microscopes use objectives designed for
• Follicular fluid blood clots are difficult to assess for the exhausting it across unidirectional air stream. The air par- specimens mounted on slides and enclosed under cover
ticles 0.3 µm in diameter are removed through the high slip, objects are commonly flattened or cut into thin sec-
presence of OCC because of the bloody fluid.
efficiency particulate air (HEPA) filter. tion, so they can be viewed with transmitted light, which
• When the FF is contaminated with blood, thinner layer
should be prepared by pouring less amount of fluid passes through the object before it enters the objective.
into Petri dish.
Why Class II Biosafety Cabinate? • Stereo microscope view most objects using incident
• Any blood clot present in the FF is gently teased apart • A class I bio safety cabinet (BSC) is an open-fronted light. That is, light is reflected from the object before
using hypodermic needles in order to release the OCC ventilated cabinet with a unidirectional inward airflow entering the lens. And Most stereo microscope have
from the blood clot, then OCC recovered from the away from the operator. Exhaust air is hard-ducted dual objectives designed for viewing without cover
blood clot should be washed thoroughly using flush- through HEPA filters (Figs. 40.4 and 40.5). The HEPA slips. They’re designed to view objects at relatively low
ing media then transferred to center well dish (65 mm) filter traps 99.97% of particles of 0.3 µm in diameter magnification typical 10X-40X.10
containing fertilization media. and 99.99% of particles of greater or smaller size. • Stereo microscope magnification is a combination of
the eyepiece magnification (most commonly 10X) and
the objective lens magnification (anywhere between
Fig. 40.6: Microscope eyepiece magnification of 10X. 0.1X and 6.5X). If you are using a stereo microscope
with 10X, eyepieces and the zoom knob is set to 6.5X
• The class I BSC provides protection for the worker (Figs. 40.6 and 40.7).
and the environment but does not protect the product
against contamination. Oocyte Cumulus Complex Grading
• A class II BSC additionally provides protection of the
product against contamination by recirculating part of What are Cumulus Complex?
the HEPA-filtered air in a laminar vertical flow.
• Cumulus complex (CC), which is in contact with the
oocyte, develops projections and they cross the zona
How Does a Stereo Zoom Microscope Work? pellucida and form gap junctions with oolemma. This
organized structure is called OCC.13,14
About the Stereo Zoom Microscope • CCs are formed from undifferentiated granulosa cells
• It uses illumination to light up specimen. That means it (GCs). GCs are the primary cell type in the ovary and
A B
utilizes light that is naturally reflected from the object they provide physical support and the microenviron-
Figs. 40.3A and B: Image after oocyte retrieval. (A) OCC in the center well dish contain fertilization media, visualized under stereo microscope
which is ideal when dealing with thick or opaque s amples. ment required for the developing oocyte.
(x1–10). (B) The oocyte is surrounded by an expanded cumulus complex identified at (x6–10) magnifications.
368 The Infertility Manual Follicular Fluid Screening 369
Table 40.1. Cumulus-oocyte complex grading.
Groups OCC morphology Image
Mature •• Expanded cumulus
•• Radiant corona
•• Distinct zona pellucida clear ooplasm
•• Expanded well-aggregated membrane granulosa
cells
•• Clear, visible oocyte
Immature •• Dense compact cumulus
•• Compact layer of corona cells
•• Nonexpanded cumulus
•• Oocyte barely visible
A B
Figs. 40.7A and B: The adjustment for the zoom objective lens, number is printed on the knob.
Postmature •• Expanded cumulus with clumps
• The cumulus and corona cells and the entire cumulus What is Cytoplasmic Maturation? •• Radiant corona radiata
mass provide clues on oocyte maturity. FSH depend- •• Irregular or slightly granular, dark ooplasm
Cytoplasmic maturation entails proper relocation of orga- •• Partly loss corona cells
ent expansion of the cumulus and corona cell complex
nelles, synthesis of proteins, and post-translation modifi-
run parallel to nuclear maturation in spontaneous nat-
cations of mRNAs accumulated during oogenesis required
ural cycles. When exogenous gonadotropins are given,
for successful completion of meiosis.12
cumulus expansion may be disturbed and is not cor-
The migration of cortical granules toward the oolemma
related as regultory with nuclear maturation.
and their positioning along the periphery of the cortex are Atretic •• Rarely with associated cumulus mass
•• Clumped and very irregular corona radiata
essential steps of this cytoplasmic maturation, crucial to
•• Membrane granulosa cells with very small clumps
Cumulus Corona Oocyte Complex Grading establish efficient defense against polyspermy. of cells
The cumulus-oocyte complex of cumulus was graded as •• Dark and misshapen ooplasm
compacted, expanded, or hyperexpanded, and the oocytes Denudation
were classified as mature or immature according to per- To denude the oocyte from surrounding cumulus and corona
centage of dark versus light. cells prior to intracytoplasmic sperm injection (ICSI).
The oocyte maturational may be scored by several
scoring system to assess the oocyte nuclear maturity and Using Hyaluronidase and
the cumulus mass, corona cells, and membrane granulosa
Mechanical Methods
cell use to grade the OCC (Table 40.1).11
1. After 2–3 hours of OCCs incubation, add 100–200 µL
of hyase (80 IU/mL) prewarmed at 37°C to the center
Incubation Time
well dish (65 mm).
• Once ovum pick up is completed, OCC have to keep it 2. In the lid of the dish, make the 50 µL of droplets from
in the fertilization media at CO2 incubator for next to the buffered medium (Figs. 40.8A and B).
1–2 hrs for complete the cytoplasmic maturation. 3. The enzyme used here is hyaluronidase in 80 IU/mL
• Preincubation time between oocyte collection and which is extracted from human sperm. Commercially
denudation may not increase the percentage of mature available hyase comes with dilution and also without
oocytes but improves the fertilization and implanta- dilution. Gone through the kit insert of the product
tion rates.7 before the procedure.12,17
A B
Figs. 40.8A and B: (A) Center well dish containing 100–200 µL of hyase. (B) Droplets which made from buffer medium 50 µL.
370 The Infertility Manual Follicular Fluid Screening 371
to identify, the best quality oocyte. This is because the Oocyte Nuclear Maturity Evaluation
health of embryo, fetuses, neonates, and adults is unques-
It is shown in Figures 40.11 to 40.13.
tionably rooted in a healthy oocyte.
Denudation is a prerequisite for ICSI, as only denuded
oocytes can be successfully manipulated by the hold-
OOCYTE DYSMORPHISMS
ing pipette. This ancillary effect of ICSI allows us to focus Description regarding an “ideal” oocyte has been given.
on oocyte nuclear maturity and also its morphological However, all the ideal characteristics may not be found
abnormalities. in all the oocytes retrieved during controlled ovarian
hyperstimulation (COH); this is true even in donor IVF
Currently, oocyte nuclear maturity is assessed by the
cycles.
presence of an extruded first polar body (IPB) in the perivi-
Following denudation, specific anomalies or dysmor-
telline space (PVS) and by the absence of germinal vesicle.
phisms of the oocytes may be detected by examination
• Germinal vesicle: The chromatin in the meiotically with light microscopy. These anomalies or dysmorphisms
arrested oocytes is encapsulated by a nuclear structure have been observed in a high proportion (63%) of meta-
A B
known as the germinal vesicle. phase II (MII) oocytes obtained after retrieval.
Figs. 40.9A and B: (A) Oocyte partially denuded in hyase. (B) Oocyte after complete denudation.
• IPB: As a part of reductional division, oocytes undergo Oocyte abnormalities can be broadly categorized as
disproportionate cytokinesis and extrusion of half of shown in Figure 40.14.
their genetic material within in the IPB.
During controlled ovarian hyperstimulation Granular Cytoplasm
cycles, approximately 85% of the oocytes are MII, 10% Two types of granulations homogenous or heterogeneous,
are germinal vesicle (GV), and 5% are MI. and granulations can be observed inside the cytoplasm
• MII oocyte: Otherwise called mature oocyte or egg (Figs 40.15 and 40.16).
should have following characteristics: Severity of granularity is based on its depth and diam-
Round, clear zona pellucida eter (>50% severe)3
Small PVS containing a single unfragmented IPB
Cytoplasm that is pale and moderately granular Vacuoles
and with no inclusions Vacuoles are membrane bound cytoplasmic inclusions
GV oocyte: Germinal vesicle is present in the ooplasm filled with fluid virtually identical with perivitelline fluid.
MI oocyte: No visible GV in the ooplasm and no IPB Vacuoles vary in size as well as in number. They can be
in the PVS present in the oocyte on the collection day (day 0) or may
A B be artificially created by ICSI (day 1) or may be seen in
Figs. 40.10A and B: (A) Buffered droplets prepared for oocyte washing to reduce the hyase concentration. (B) 135 µm (yellow), 175 µm (red) and
stripper holder for oocyte/embryo handling.
4. Maximum five OCC at a time are exposed in hyase, Enzyme Concentration
and glass pasture pipette (1,000 µ) should be used.
To minimize the toxic effect on the oocyte, enzyme con-
Exposure time in hyase not exceed 30 seconds
centration and exposure time should be less (30–60 sec-
(Figs. 40.9A and B).
onds). If enzyme concentration is more than 80 IU/mL, it
5. Partially denuded oocytes are transferred to 50 µL may induce parthenogenesis, and its significantly reduce
of buffered droplet which would be prepared earlier the degeneration rate and the 1-PN activation in the oocyte
held at 37°C to reduce the effect of hyaluronidase after ICSI.
(Figs. 40.10A and B).
6. Partially denuded oocytes are further denuded using
OOCYTE NUCLEAR MATURITY
denudation pipette (130/135/140 µm diameter);
remove excess cells by doing in and out repeatedly, EVALUATION
during this process care should taken, because it may First, we need to know how to differentiate the mature Fig. 40.11: MII oocyte. 1. Round, clear zona pellucida; 2. Small perivi-
damage the zona and may cause dysmorphism in the and immature oocytes and also to identify different oocyte telline space (PVS); 3. Single unfragmented first polar body (IPB); 4. Fig. 40.12: MI oocyte. (No germinal vesicle in the ooplasm. No visible
Pale, moderately granular ooplasm. IPB.)
oocyte and rupture the zona pellucida. abnormalities. In assisted reproductive techniques using
372 The Infertility Manual Follicular Fluid Screening 373
A A
Fig. 40.16: Oocyte with centrally placed granularity.
B C B
Figs. 40.13A to C: (A) Germinal vesicle (GV) oocyte (X40). Oocyte recovered at the GV stage, note the presence of germinal vesicle (arrow).
(B) GV oocytes (X20). All oocytes are at GV stage and despite overnight incubation did not proceed to GV breakdown. (C) Oocytes in different
stages of maturation. (1) MII oocyte with the presence of IPB; (2) MI oocyte without IPB and GV; (3) GV oocyte with the presence of germinal vesicle.
Fig. 40.18: Oocyte with smooth endoplasmic reticulum (SER) cluster
(arrow).
embryos associated with developmental arrest. The later
the vacuole arouse, the poorer the oocyte perform on blas-
tocyst formation (Figs. 40.17A to C).4
Smooth Endoplasmic Reticulum Cluster
Under inverted microscopy, smooth endoplasmic reticu-
lum (SER) clusters can be morphologically distinguished
from fluid-filled vacuoles. SER clusters are pronuclear
C
sized, flat clear disc-like structures. Presence of SER clus-
ter has been associated with poor fertilization and embryo Figs. 40.17A to C: (A) Single large vacuole in MII oocyte. (B and C) Mul-
tiple vacuoles.
development (Fig. 40.18).18,23
Fig. 40.14: Classification of oocyte abnormality. Fig. 40.15: Oocyte having homogenous granular cytoplasm.
374 The Infertility Manual Follicular Fluid Screening 375
Refractile Bodies According to Ebner et al., five categories of IPB has
been described:
The refractile body, so called because of its nature under
bright-field microscopy. The evolution of this structure 1. Grade 1: Round or ovoid, intact (smooth surface)
and its relationship to oocyte maturity and viability are not 2. Grade 2: Round or ovoid, intact (rough surface)
yet fully understood (Figs. 40.19A and B). 3. Grade 3: Fragmented
4. Grade 4: Broken in two
Polar Body 5. Grade 5: Huge first polar body
First polar body is believed to indicate the postovulatory
age of the oocyte. Different authors have found a signifi-
cant relationship between IPB morphology and blasto-
Perivitelline Space
cyst formation, implantation, and ongoing pregnancy Abnormalities of PVS could be enlargement at one or more
(Figs. 40.20A to F). areas or PVS granularity/fragments (Figs. 40.21A to C).
C D
A B E F
Figs. 40.19A and B: Refractile body (arrow). Figs. 40.20C to F: (C) First polar body detached from ooplasm (arrow). (D) Fragmented polar body. (E) Polar body broken into two. (F) Large first
polar body.
A B
A B
Figs. 40.20A and B: (A) Polar body with round surface. (B) Polar body with rough surface.
Figs. 40.21A and B: (A and B) Increased perivitelline space with septa.
376 The Infertility Manual Follicular Fluid Screening 377
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AC. Effects of different hyaluronidase concentrations and 143-9.
mechanical procedures for cumulus cell removal on the 25. Ebner T, Moser M, Sommergruber C, Yaman C, Pfleger U,
outcome of intracytoplasmic sperm injection. Human Tews G. First polar body morphology and blastocyst formation
Reprod. 1997;12(10):2246-50. rate in ICSI patients. Hum Reprod. 2002;17:2415-8.
13. Talukder MNS, Iqbal A, Khandoker MAMY, Alam MZ. 26. Ebner T, Moser M, Yaman C, Feichtinger O, Hartl J, Tews G.
Collection grading and evaluation of cumulus-oocyte- Elective transfer of embryos selected on the basis of first
complexes for in vitro maturation in sheep. Bangl Vet. polar body morphology is associated with increased rates
2011;28(1):31-8. of implantation and pregnancy. Fertil Steril. 1999;72:
14. Predictive value of oocyte morphology in human 599-603.
IVF: a systematic review of the literature HYPERLINK 27. Oehninger S. Biochemical and functional characterization
“https://academic.oup.com/humupd/article/17/1/34/ of the human zona pellucida. RBM Online. 2003;7(6):641.
javascript;” Laura Rienzi HYPERLINK “https://academic. 28. De Sutter P, Dozortsev D, Qian C. Oocyte morphology does
oup.com/humupd/article/17/1/34/javascript;” Gábor not correlate with fertilization rate and embryo quality
Vajta HYPERLINK “https://academic.oup.com/humupd/ after intracytoplasmic sperm injection. Human Reprod.
C D article/17/1/34/javascript;” Filippo Ubaldi Human 1996;11:595-7.
Reproduction Update. 2011;17(1): 34-45, https://doi. 29. Balaban B, Urman B, Sertac A, Alatas C, Aksoy S, Mercan R.
Figs. 40.22A to D: (A) Thick zona pellucida (ZP) surrounding the oocyte. (B) Two oocytes were recovered within a single ZP, the smaller oocyte
org/10.1093/humupd/dmq029: Published: 16 July 2010. Oocyte morphology does not affect fertilization rate, embryo
has a GV and larger oocyte is M II. (C) Oocyte with absent zona (one of the reason could be high suction pressure due to which ZP can break).
(D) Oocyte with double zona.
C HA PTE R
378 The Infertility Manual
quality and implantation rate after intracytoplasmic sperm
injection. Human Reprod. 1998;13:3431-3.
30. Ebner T, Moser M, Sommergruber M, Gaiswinkler U,
Shebl O, Jesacher K, et al. Occurrence and developmental
consequences of vacuoles throughout preimplantation
development. Fertil Steril. 2005;83;1635-40.
34.
morphology and nuclear maturity of oocytes collected in
stimulated cycles for intracytoplasmic sperm injection.
Fertil Steril. 1999;71:937-40.
Ebner T, Moser M, Shebl O, Sommergruber M, Yaman C,
Tews G, et al. Blood clots in the cumulus-oocyte complex
predict poor oocyte quality and post-fertilization
IVF, ICSI, IMSI, IVM
41
Ratnakar Majalekar, Vinay Kumar, Kamini A Rao
31. Otsuki J, Okada A, Morimoto K, Nagai Y, Kubo H. The development Reprod Biomed Online. 2008;16:801-7.
relationship between pregnancy outcome and smooth 35. Ng ST, Chang TH, Wu TC. Prediction of the rates of
endoplasmic reticulum clusters in MII human oocytes. fertilization, cleavage, and pregnancy success by cumulus- Chapter Outline
Hum Reprod. 2004;19:1591-7. coronal morphology in an in vitro fertilization program. • History • Insemination by In Vitro Fertilization
• Indication of Intracytoplasmic Sperm Injection • Children Born after Intracytoplasmic Sperm Injection
32. Rosália Sá, Mariana Cunha, Joaquina Silva, Ana Luís, More. Fertil Steril. 1999;72:412-7.
• Advantages of In Vitro Fertilization • Intracytoplasmic Morphologically Selected Sperm Injection
Ultrastructure of tubular smooth endoplasmic reticulum 36. Lin YC, Chang SY, Lan KC, Hsuan-Wei Huan. Human • Sperm Preparation • In Vitro Maturation in Assisted Reproductive Technique
aggregates in human metaphase II oocytes and clinical oocyte maturity in vivo determines the outcome of • Assessment of Oocytes before In Vitro Fertilization/Intracytoplas-
implications. Fertil Steril. 2011;96:143-9. blastocyst development in vitro. J Assist Reprod Genet. mic Sperm Injection
33. Rattanachaiyanont M, Leader A, Leveille MC. Lack of 2003;20:506-12.
correlation between oocyte-corona-cumulus complex 37. Available from www.vitrolife.com. Accessed October, 2017.
Edward and Steptoe created history in medical field by Fertilization process in IVF involves following events:
producing first in vitro fertilization (IVF) baby by fertilizing
1. Acrosome reaction and zona binding
an oocyte in vitro and transfer of resulting embryo.
Initially IVF and embryo transfer was mainly done for 2. Sperm penetration of zona pellucida and corona
tubal factor infertility. radiata
Later on other causes of infertility like male factor 3. Fusion of cell membrane of oocytes and sperm
infertility, unexplained infertility, reduced ovarian reserve, 4. Oocyte activation
endometriosis, and cervical factor infertility were treated 5. Male and female pronuclei formation
with IVF with good success rate. 6. Migration of pronuclei at center of oocyte.
Fertilization procedures in assisted reproductive tech-
In ICSI, first and second events are bypassed.
nique (ART) have evolved over a period of time from IVF
to intracytoplasmic sperm injection (ICSI) with recent
advances like intracytoplasmic morphologically selected INDICATION OF INTRACYTOPLASMIC
sperm injection (IMSI).
SPERM INJECTION
Success rate for male factor infertility has dramatically
improved because of micromanipulation techniques for • Severe male factor infertility (oligozoospermia, asthe-
handling of gametes. ICSI was milestone achievement in nospermia, teratospermia).5
the field of infertility. • Surgically retrieved spermatozoa.6
With newer technique like IMSI, sperms with better • Previous fertilization failure with IVF.7
morphology can be selected for fertilization procedure • Frozen thawed oocytes fertilization: denudation of
which will improve ART outcome. oocytes and freezing may alter zona pellucida (ZP).8
• Oocytes abnormality like thick zona.
HISTORY • Unexplained infertility.
• 25th July 1978: Due to efforts of Steptoe and Edward, • Preimplantation genetic testing: to confirm monosper-
Louise Brown—world’s first IVF conception baby was mic fertilization and avoid risk of extra sperm attached
delivered in Oldham, Greater Manchester, UK.1 to zona pellucida in IVF.
• 1988: Ng, et al. reported birth of baby by injecting sper- • IVM oocytes fertilization: Culturing oocytes in matura-
matozoa into subzonal space.2 tion media may alter zona pellucida.
• 1992: Andre Van Steirteghem and Paul Devroey from
• HIV discordant couples to minimize the exposure of
Brussels developed ICSI of single sperm in human
the oocyte to a large number of spermatozoa.9
oocyte for fertilization.
• 1991–92: First birth after in vitro maturation (IVM) of For all ages and with the different types of sperms used,
immature oocytes by Cha.3 fertilization after ICSI is approximately 80% with clinical
• 2003: Bartoov et al. described IMSI.4 pregnancy rate up to 45%.10
380 The Infertility Manual IVF, ICSI, IMSI, IVM 381
ADVANTAGES OF IN VITRO INSEMINATION BY IN VITRO • Oocyte morphology is assessed by zona pellucida, Preparation for Intracytoplasmic
perivitelline space, polar body, and cytoplasmic char-
FERTILIZATION FERTILIZATION Sperm Injection
acteristics.
• Natural selection of sperm Success of an IVF procedure depends on the design of IVF • In a sterile Petri dish, pipette 10 µL of polyvinylpyrro-
• Cost is reduced compared to ICSI laboratories, good air handling units, and filters. Insemination by Intracytoplasmic lidone (PVP) solution in the centre of the dish in lon-
• Micromanipulation and technical expertise not required. Sperm Injection gitudinal plane.
4-Well Dish • PVP helps in sperm handling and prevents sperm cells
• Magnification of 200–400 × is normally required to vis-
SPERM PREPARATION • Take 0.5 mL of culture media covered with oil in each
from sticking to the injecting pipette during ICSI but
ualize procedures. prolonged exposure to PVP can also cause damage to
• Sperm preparation technique for selecting morpho- well and culture three to four oocytes in each well. • Before starting ICSI, all conditions such as temperature, sperm membranes.
logically normal and motile sperm is important com- • Take washed semen sample with measured volume pH, equipment setup, and alignment should be checked. • Pipette three to five droplets of 5 µL of warmed oocyte,
ponent of IVF/ICSI. so that sperm concentration in each well should be • Micro-tools: two types: (1) holding pipette and washing droplets in a circular way around the PVP
• Standard swim up method can be used for preparation around 100,000–150,000 progressive motile sperms. (2) injection pipette droplet, as close as possible but being careful not to
of sperm sample with good count and good rapid pro- • Check under microscope the concentration of motile Both these micro-tools are bent to an angle of mix droplets. Cover with mineral oil.
gressive motility. sperms to the oocytes and add more motile sperms, if 30–35° at the terminal end which helps in horizon- • This dish is then incubated for ½ to 1 hour in CO2 incu-
• In case of low count, poor rapid progressive motility, required. tal positioning in the culture dish. bator.
or high concentration of debris, density gradient cen- • After insemination keep the 4-well dish immediately Holding pipette: • Two to three hours after oocyte denudation, inject 3–5 µL
trifugation is the method of choice. in the CO2 incubator to avoid any pH change. ■■ Used to hold the oocyte at the blunt end. of prepared sperm sample to the PVP droplet in previ-
• Hypo-osmotic swelling test can be used to differentiate ■■ Outer diameter 0.08–0.15 mm and inner diam- ously prepared ICSI dish.
between nonmotile sperms and dead sperms. Viable Denudation eter 0.018–0.025 mm. • After 10–15 minutes, motile sperms are normally seen
sperms will show swelling of their tails with coiling while • Denudation of mature oocytes is a prerequisite before Injection pipette: on the edge of PVP droplet.
dead spermatozoa will not show any swelling of tail. ICSI. ■■ Used to inject the sperm inside the oocyte
• Oocyte cumulus complex (OCC) interferes with micro- ■■ Outer diameter 0.006–0.007 mm and inner Intracytoplasmic Sperm Injection
ASSESSMENT OF OOCYTES injection. Denudation helps to overcome this problem. diameter 0.005–0.006 mm. Procedure
■■ To avoid oocyte damage, microscopes are
BEFORE IN VITRO FERTILIZATION/ • Oocyte maturity assessment is better after denudation.
• Injection pipette is brought at the edge of PVP droplet.
• Incubation of 3–4 hours is required at 37°C with 5–6% placed with a heating stage to maintain the
INTRACYTOPLASMIC SPERM INJECTION of CO2, for retrieved oocytes before denudation. temperature at 37°C. • Morphologically, normal appearing sperm is selected
■■ Two identical micromanipulators are mounted and immobilized by hitting and crushing its tail against
• Germinal vesicle (GV): Oocytes yet to resume meiosis • Denudation dish: Prepared ½ hr before, by pipetting
on the microscope with holding pipette on the the bottom of the dish. It helps in sperm head decon-
with large nucleus visible. 50–75 µL of hyaluronidase with culture media and cov-
left side and injecting pipette on the right side. densation.
• Metaphase I (M I): Meiosis resumes with GV breakdown, ered with equilibrated oil.
■■ Equipment is placed on a vibration free table • Then sperm is aspirated in injecting pipette with tail
nucleus disappears, and first polar body yet to appear: • Denudation is carried out with denuding pipette of
top in a dust-free laboratory (Fig. 41.2). first and positioned 10–15 µm from the tip.
IVM can be tried for M I oocytes before fertilization. diameter 170–200 µm by pipetting two to three oocytes
• Oocyte is aspirated from the culture droplet with the
M I oocytes can be incubated for 2–3 hours and if in hyaluronidase droplet for 40–50 seconds followed by
use of holding pipette.
they extrude polar body then can be used for ICSI.11 changing pipette to smaller diameter of 130–150 µm.
• Oocyte is positioned with polar body at 12 O’clock or at
• Metaphase II (M II): Mature oocytes with extrusion of • Wash oocytes in droplets of culture media (HEPES
6 O’clock position to avoid injury to the meiotic spin-
first polar body (Figs. 41.1A to C). buffered) after removing cumulus cells.
dles during microinjection.
• Injection pipette is moved close to oocyte.
• Sperms are moved up and down in the pipette before
injecting which helps to dispel PVP around the sperm.
• Sperm is brought near to beveled tip of injection
pipette which is then advanced through the zona and
ooplasm from 3 O’clock position.
• A little bit of cytoplasm is aspirated which helps in
oocyte activation. Also it makes sure that the injecting
A B C
needle is inside the ooplasm.
• Then sperm is slowly injected into the cytoplasm of
oocyte after which needle is gently taken out and
Figs. 41.1A to C: (A) Germinal vesicle (B) M I (C) M II. Fig. 41.2: Micromanipulator used for ICSI. oocyte is released from the holding pipette.
382 The Infertility Manual IVF, ICSI, IMSI, IVM 383
Physiological Intracytoplasmic • In IMSI prior to insemination, sperm morphology is • For optimum success, both cytoplasmic and nuclear
assessed with high power microscope. maturation should be synchronous.
Sperm Injection
• Once assembled, IMSI equipment can provide a mag- • In IVM, immature oocytes are aspirated before they are
• Spermatozoa bound to zona pellucida are considered nification of over 6000× which helps for a detailed eval- affected by endocrine and paracrine factors of growing
more optimal for ICSI. uation of sperm morphology. dominant follicle.
• Studies have shown that ZP bound sperm has a better • Calculation of magnification in IMSI: • Immature oocytes can be cultured in vitro in media
implantation and clinical pregnancy rate.13 with added FSH, LH, and various other growth factors
microscope video coupler video monitor
• Mature spermatozoa tend to bind to hyaluronic acid obtained from mature follicular fluid.
(HA) compared to immature sperms. magnification X magnification X magnification. • Studies have shown that addition of hypoxanthine to
• The HA bound sperm helps in selection of individual (150×) (1×) (television monitor 14’’—355 mm/8 mm culture media inhibits nuclear maturation without
mature sperm which will have low levels of aneuploi- CCD chip) affecting cytoplasmic maturation.22
dies. 150× X 1× X (355/8)× = 6600× • Once cytoplasmic maturity is satisfactory, hypoxan-
Fig. 41.3: Intracytoplasmic sperm injection procedure. • More studies are required in this field. thine can be withdrawn and oocytes can be transferred
• Large vacuoles seen in sperm nucleus during IMSI has
to media for maturation of nucleus.
shown negative impact on in vivo and IVF potential.19
• Cleavage rates are higher when immature oocytes are
• The membrane at injection site is normally seen as CHILDREN BORN AFTER • Cytoplasmic vacuoles show correlation with increased
pretreated with hypoxanthine.
funnel shaped, pointing in toward the center. INTRACYTOPLASMIC SPERM INJECTION DNA fragmentation and lower pregnancy rates but
• IVM reduces frequent monitoring which is required
• Oocytes are washed in culture medium once the pro- results are controversial.20
• Risk of congenital malformation in children born with in controlled ovarian hyperstimulation (COH) in the
cedure is over and then placed in the CO2 incubator. • Pooled data of IMSI cycles has shown better implanta- form of blood tests and scans.
ICSI is relatively low (4.2%) but pooled results from all
• Oocytes are assessed next morning for the presence of tion and pregnancy rate with reduction in miscarriage
published data suggest that children born from ART • IVM can be done in natural cycles or stimulated cycles.
pronuclei (Fig. 41.3). rate.21 • Gonadotropin stimulation improves maturation rate
are at an increased risk of birth defects compared to
• Spermatozoa selected with IMSI more often seem to
spontaneous conception (30–40% higher risk with and developmental potential of immature oocytes.23
Fertilization Assessment carry X chromosome and incidence of female embryos • In stimulated cycles, gonadotropins in the form of FSH
ART).14
is on a higher side after IMSI (Fig. 41.5).21 or HMG are given till follicles reach 11–12 mm size.
Fertilization check is done 16 hours after ICSI and 18 hours • Risk of congenital malformation is increased espe-
after IVF. Fertilized oocyte at this stage consists of: cially after ICSI with surgically retrieved sperms in • It is followed by human chorionic gonadotropin (hCG)
nonobstructive azoospermia.15 IN VITRO MATURATION IN ASSISTED trigger (10,000 IU) and oocytes are aspirated 36 hours
• Two pronuclei which are of similar size and centrally
• The prevalence of autism in ICSI group was higher REPRODUCTIVE TECHNIQUE post-hCG.
located with close approximation • The hCG stimulation helps to loosen cumulus cells
(3.4% vs. general population +/- 0.3%).16
• Two polar bodies • Oocyte maturation is the process by which oocyte
• ICSI and naturally conceived children show compara- and improves retrieval rate.
• Each pronucleus showing five to seven nucleolar pre- attains the ability to undergo fertilization and further
ble cognitive and motor development until the age of • Immature oocytes show better nuclear maturation rate
cursor bodies that are even sized, with similar distribu- embryogenesis. It involves maturation of both nucleus
8 years.17 after hCG trigger.
tions (Fig. 41.4).12 and cytoplasm. • Premature aspiration of oocytes leads to reduced
• Except for an increased risk for congenital malforma-
• Nuclear maturation is characterized by resumption exposure to endogenous estradiol which affects endo-
tion, most studies report that ICSI children as com-
of meiosis I and release of first polar body while cyto-
pared to natural conception have similar health and metrial preparation, so exogenous estradiol should be
plasmic maturation involves reorganization of the
development.17 started on the day of ovum pick-up (OPU).
cytoplasmic organelles and activation of metabolic • Estradiol valerate can be given daily as 6–10 mg orally
pathways. in divided doses.
INTRACYTOPLASMIC
• Also defective corpus luteum may affect endometrium
MORPHOLOGICALLY SELECTED in secretary phase, so progesterone support should
SPERM INJECTION be given starting from the day after OPU (600 mg/day
micronized progesterone vaginally).
• With ICSI ,which uses 200–400× magnification, though
• If endometrial growth is not satisfactory, it is better to
the embryologist selects the most good looking sperm,
freeze the embryos.
minute head defects are left undetected.
• As a result, sometimes sperms with chromosomal
abnormalities may be selected for fertilization in ICSI.
Indications for In Vitro Maturation
• Sperms with severe morphological abnormalities • PCOS patients who have a large cohort of immature
show lower fertilization, implantation, and clinical follicles developing in unfavorable endocrine environ-
Fig. 41.4: Fertilized oocyte- 2PN stage. pregnancy rate.18 Fig. 41.5: Sperms observed during ICSI Vs IMSI.
ment.24
384 The Infertility Manual IVF, ICSI, IMSI, IVM 385
• Patients at risk of ovarian hyperstimulation syndrome • It can be because of asynchrony between nuclear and outcome with ICSI for azoospermic men. Hum Reprod. 18. De Vos A, Van De Velde H, Joris H, Verheyen G, Devroey
(OHSS): IVM reduces the requirement of gonadotro- cytoplasmic maturation, out of phase endometrium, 1999;14:741-8. P, Van Steirteghem. Influence of sperm morphology on
7. Palermo GD, Cohen J, Rosenwaks Z. ICSI: a powerful fertilization and pregnancy outcome of intra-cytoplasmic
pins and risk of OHSS. suboptimal culture conditions, or incorrect timing of
tool to overcome fertilization failure. Fertil Steril. 1996;65: sperm injection Fertil Steril. 2003;79:42-8.
• Fertility preservation: Patients undergoing cancer insemination. 899-908. 19. Franco JG Jr, Baruffi RL, Mauri AL, Petersen CG, Oliveira
treatment in the form of chemoradiation can cryopre- 8. Vincent C, Pickering SJ, Johnson MH. The hardening effect JB, Vagnini L. Significance of large nuclear vacuoles
serve their immature oocytes which can undergo IVM Children born from In Vitro Maturation of DMSO on the mouse zona. J Reprod Fertil. 1990;89:253-9. in spermatozoa: implications for ICSI. Reprod BioMed
in later life.25 9. Mencaglia L, Falcone P, Lentini GM, Consigli S, Pisoni Online. 2008;17:42-5.
• Buckett, et al. reported on 48 IVM pregnancies with M, Lofiego V, et al. ICSI for treatment of HIV and HCV- 20. Cassuto NG, Hazout A, Hammoud I, Balet R, Bouret D,
• IVM for oocyte donor is patient friendly. It avoids the
overall healthy obstetric, neonatal, and infant out- sero-discordant couples with infected male partner. Hum Barak Y, et al. Correlation between DNA defect and sperm
risk of OHSS and reduces the duration of treatment. head morphology. Reprod BioMed Online. 2012;24:211-8.
come.26 Reprod. 2005;20:2242-6.
10. Varghese AC, Goldberg E, Agarwal A. Current & 21. Setti AS DP, de Almeida Ferreira Braga DP, Vingris L, de
• He also found no difference in perinatal morbidity, Cassia Savio. Predictive value of high magnification on ICSI
future perspectives on ICSI. Reprod Biomed Online.
Oocyte Aspiration in In Vitro Maturation birth weight, prematurity, or congenital abnormalities 2007;15(6):719-27. outcomes. J Assist Reprod Genet. 2012;29(11):1241-7.
among IVM, IVF, and ICSI cycles. 11. Strassburger D The cytogenetic constitution of embryos 22. Down SM (1995b). Hypoxanthine-principle inhibitor
• IVM aspiration needle is 17 gauge size. It can be single of immature oocyte maturation. Pro Natl Acad Sci.
lumen or double lumen. derived from MI, Fertil Steril. 2010;94:971-8.
To conclude, the standardization of IVM is ongoing 1985;82:454.
12. Istanbul consensus document, ESHRE special interest
• It has short bevel which prevents through and through and it is too early to offer IVM to all the infertility patients, group of embryology 2011.
23. Wynn P, Picton HM, Krapez JA, Rutherford AJ, Balen AH,
penetration of small follicles. It also prevents spilling of but it has a potential to develop into alternative type of Gosden RG. Pre-treatment with FSH hormone promotes
13. Black, Yoeli R, Ashkenazi J, Orvieto R, Levy T, Ben-Rafael
number of oocytes reaching M II in IVM. Hum Reprod.
small volume contents of immature follicles. assisted reproductive technology. Z, et al. comparison between conventional ICSI and ZP
1998;13(11):3132-8.
• It is more rigid so that it can easily poke through ovar- bound sperm for ICSI. Ferti Steril. 2010;93(2):672-4.
24. Trounson A, Wood C, Kaushe A. IVM and fertilization and
14. Barnes J, Sutcliffe AG, Kristoffersen I, Bonduelle M.
ian cortex and shorter so as to accumulate less volume. PROBABLE QUESTIONS Influence of ART on family functioning and children’s
developmental competence of oocytes recovered from
• Aspiration pressure is set at 50–60 mm Hg to avoid untreated PCO patients. Fertil Steril. 1994;62:353-62.
1.
Describe the process of fertilization. development. Hum Reprod. 2004;19(6):1480-7. 25. Oktay K, Buyuk E, Rodrigues-Wallberg KA, Sahin G. IVM
damage to the granulosa cells of immature oocytes. 15. Vernaeve V, Bonduelle M, Devroey P. Pregnancy outcome
2.
What are the indications of ICSI? improves oocyte or embryo cryopreservation outcome in
• Immature oocytes are more adherent to follicular wall after ICSI using testicular sperm in azoospermia. Hum
3.
Describe the procedure of ICSI. breast cancer patients undergoing stimulation for fertility
so frequent curetting and flushing is required during Reprod. 2003;18:2093-7. preservation. Reprod Biomed Online. 2010;20:634-8.
aspiration. 4.
What are the indications of IVM? 16. Williams JG, Higgins JP. Systemic review of prevalence of 26. Buckett WM, Chian RC, Barrington K, Dean K, Abdul-Jalil,
• Multiple punctures in the ovary leads to more bleeding 5.
Write a short note on physiological ICSI (PICSI). autism. Arch Dis Child. 2006;91:8-15. Tan SL. Obstetric, neonatal and infant outcome in babies
6.
What is the role of IMSI in ART? 17. Leunens L, Kristoffersen P. Cognitive and motor conceived by IVM; initial 5 years result 1998-2003. Fertil
during aspiration of immature oocytes.
7.
What are the advantages and disadvantages of IVF development after ICSI. Hum Reprod. 2006;21:2922-9. Steril. 2004:82:S133.
• Heparin (2–5 IU/mL) should be added to the buffered
media to prevent formation of blood clots. over ICSI?
• Identification of OCC is very important step of IVM. 8. Write a short note on IVM.
• OCC can be identified under microscope or special fil- 9. Write a short note on assessment of fertilization.
ters of pore size 50–70 µ can be used for oocyte separa- 10. Draw a diagram of mature oocyte (MII), MI oocyte,
tion. and germinal vesicle (GV).
• Immature oocytes should be observed every 12 hours
for maturity. REFERENCES
• IVM culture may extend from 22 to 72 hours. 1. First test tube baby born, BBC, 25th July 1978.
• A comparison of cultures at various time interval 2. Ng S-C, Bongso TA, Ratnam SS, Sathananthan, AH, Chan,
showed that ova which showed extrusion of first polar CLK, Wong, PC, et al. Pregnancy after transfer of multiple
body was mostly in 46-hour culture. sperms under zona. Lancet. 1988;ii:790.
• After that denudation is carried out to remove cumu- 3. Cha KY, Koo JJ, Choi DH, Han SY, Yoon TK. Pregnancy
after IVF of human follicular oocytes collected from
lus cells and to identify mature oocytes.
nonstimulated cycles, their culture in vitro and transfer.
• ICSI is preferred over IVF for fertilization because of Fertil Steril. 1991;55(1):109-13.
zona hardening due to longer duration of culture. 4. Bartoov, Berkovitz A, Eltes F, Kogosovsky A, Yagoda A,
• Embryos derived from IVM show higher incidence Lederman H, et al. Pregnancy following IMSI. Fertil Steril.
of cleavage arrest and most of them require D2 or D3 2003;80(6):1413-9.
5. Palermo GD, Cohen J, Alikani M, Adler A, Rosenwaks
transfer.
Z. ICSI: a novel treatment for all forms of male factor
• Assisted hatching can be done in these embryos. infertility. Fertil Steril. 1995;63:1231-40.
• Implantation and pregnancy rate are still on lower side 6. Palermo GD, Schlegel PN, Hariprasad JJ, Goldstein M,
after IVM. Menendez S, Zaninovic N, et al. fertilization and pregnancy
C HA PT E R
42
Assessment of Fertilization 387
Assessment of Fertilization
Arunima Halder, Dileep Kumar, Divyashree PS
Chapter Outline
• Sperm Oocyte Interaction • Pronuclear Scoring
• Events Occuring in the Immediate Post-fertilization Period • Abnormal Fertilization
• Limitations of Using the Pronuclear Stage Scoring • Cytoplasmic Morphology Assessment
• The Fertilization Check
SPERM OOCYTE INTERACTION Cortical Granulation
Fig. 42.2: Schematic diagram showing steps in oocyte activation.
Within the ooplasm are present vesicles that contain corti- Fig. 42.1: Schematic diagram showing the structure of M phase pro-
Activation of the Oocyte moting factor. PLCz: Phospholipase C zeta, PIP2: Phosphatidylinositol4,5bisphospha
cal granules. These granules were first observed by Austin te, IP3:Inositol triphosphate, DAG: diacyl glycerol, PKC: Protein kinase
The molecular, structural, and physiological changes CR.4 These are structures formed from the Golgi appara- C, ER: Endoplasmic reticulum)
brought about by the sperm ultimately causing the attenu- tus of the oocyte. Upon fusion of the single sperm with
ation of the meiosis II (MII) arrest and leading to the pro- the oocyte, these cortical granules undergo exocytosis
changing the characteristic of the zona pellucida, making cytoskeletal structures. These are the microtubule organ- Table 42.1. Implications of temporal abnormalities in
gression of the cell cycle within the oocyte is called as
it resistant to the entry of other sperms. (Thus preventing izing center (MTOC) which is responsible for initiation of the pronuclear development.
oocyte activation.1,2 mitosis and meiosis. These help in the movement of the
The MII arrest is brought about by the “M Phase Pro- polyspermy.) It has been suggested that calcium as well as Sl No. Abnormalities Implications
SNARE (soluble N-ethylmaleimide-sensitive factor attach- male nucleus toward the female pronucleus. During inter-
moting Factor” or MPF which is a combination of cyclin B phase, the centrosome splits into two, moves apart, such 1. Delayed nucleolar Failure of early pronu-
ment protein receptors) protein pathway play a role in precursor bodies (NPB) clear RNA synthesis
and cdc2 kinase (Fig. 42.1). that the microtubules extend between the male and the
membrane fusion leading to the cortical reaction.5 development (ideally, ultimately leading to
During oocyte activation, the cdc2 kinase causes the female pronuclei. beginning as early as abnormal DNA syn-
activation of cdc2, which ultimately leads to cell cycle pro-
Fusion of the Sperm and Defect in the centrosome function can lead to fertiliza- 4 hours and completing thesis
gression and spindle formation. tion failure. within 12 hours)7
Pronuclear Formation
2 Inherent defect in the Defect in juxtaposi-
Steps in Oocyte Activation In order to gain forward motility, the sperm loses its cyto-
EVENTS OCCURING IN THE IMMEDIATE
sperm derived cen- tion of pronuclei and
plasm and the nuclear material gets highly compacted. trosome to form micro- syngamy
tubules
Step 1: Depolarization of the Plasma Membrane Also, the nuclear membrane becomes devoid of pores as POST-FERTILIZATION PERIOD
there is no necessity of the nucleocytoplasmic transport at 3 Inability of the pronuclei Failure of cleavage and
When the sperm comes in contact with the oocyte • Establishment of the pronuclei to juxtapose zygote stage arrest
this time.6
membrane, it leads to the depolarization of the oocyte The sperm enters the cytoplasm with the midpiece and • Assembly of nucleolar precursor bodies that is depend-
4 Pronuclei of uneven size Mosaic embryos
membrane, by an efflux of potassium via calcium gated the tail. The nucleus becomes the male pronucleus and the ent on RNA synthesis and the ooplasmic milieu
• Cortical reaction 5 Asynchrony in the num- Abnormalities in the
channels. midpiece and the tail disintegrate.
• Rearrangement of cell organelles ber, size, or location of further embryo
Formation of the male pronucleus occurs in the fol- NBP development (could be
Step 2: Release of the Sperm-soluble Factor or lowing five steps:7 because of abnormal
gene expression)
the Phospholipase c Zeta 1. Disappearance of the outer pore less membrane Implications of Temporal Abnormalities
2. Disappearance of the sperm lamina
Phospholipase c Zeta (PLC) zeta causes the breakdown of in the Pronuclear Development
3. Decondensation of the highly compacted chromatin
phosphatidyl inositol 4,5 biphosphate to inositol triphos- 4. Development of the outer lamina with pores This is shown in Table 42.1. Timing: 12–16 hours after ICSI (it has been suggested
phate (IP3) and diacyl glycerol. IP3 then causes the acti- 5. Enlargement of the male pronucleus that fertilization occurs 1 hour later in case of IVF than in
case of ICSI).
vation of the cAMP pathway ultimately causing efflux of Soon after the oocyte activation, anaphase begins. This Observation of the Pronuclei in Human
calcium within the oocyte cytoplasm in an oscillatory man- As per the Istanbul consensus,8 17±1 hours postinsem-
is followed by extrusion of the polar body and formation of Zygotes During In Vitro Fertilization ination may be the correct time to visualize the pronuclear
ner. The primary surge of cytoplasmic free calcium begins the female pronucleus.
from the point where the sperm penetrates followed by a Once the sperm enters the ooplasm, it releases its cen- Instrument used: Hoffman modulation contrast optics embryo.
wave like expansion through the oocyte (Fig. 42.2).3 trosome. Around the centrioles develop asters that are
388 The Infertility Manual Assessment of Fertilization 389
Need for the Morphological Evaluation • Male and female pronuclei apposed to each other
• Distinct membranes of the pronuclei
of the Pronuclear Stage Embryo
• NPBs equal in size and number and arranged synchro-
• To choose the best embryo for embryo transfer at an nously at the equatorial region at the juxtaposition of
early stage. both nuclei.
• To choose the best embryo for cryopreservation. • Presence of a peripheral cytoplasmic halo (caused
• In countries where the freezing of embryos beyond the by peripronuclear redistribution of ooplasmic mito-
zygote stage may be prohibited, this could be an alter- chondriae leading to peripheral cytoplasmic clearing).
native. However, evidence to include it in the pronuclear scor-
• A combined zygote and cleavage stage embryo scoring ing is insufficient.8,9
could lead us to the best decision regarding the choice
of the embryos. Normal Variations Fig. 42.7: Schematic diagram of pronuclei showing scattered NPBs.
• Pronuclear stage embryo scoring could hint regarding 1. Axis of the pronuclei as compared to that of the polar
presence of chromosomal anomalies. bodies may be parallel or perpendicular (Figs. 42.5 Fig. 42.6: Axis of the pronuclei as compared to that of the polar bodies
and 42.6). may be perpendicular.
The NPBs develop early in the zygote formation and
LIMITATIONS OF USING THE depends upon an initial transcriptional activity within
PRONUCLEAR STAGE SCORING 2. Pronuclei showing scattered NPBs are shown in
the pronuclei. Moreover, ooplasmic factors may affect its
development. Hence, NPBs become an apt tool for mor-
• Pronuclear stage is a dynamic state and vitals features Figure 42.7.
phological assessment of the pronuclear stage.
may be elusive in the restricted time frame.8
• Pronuclear abnormalities may be affected by vari- PRONUCLEAR SCORING • Number: Ideally, each pronucleus contains three to
ous parameters like ovarian reserve, age, and sperm ten nucleoli. The maximum difference in the number
abnormalities which may be overcome eventually.
Nucleolar Precursor Bodies of nucleoli between the two pronuclei should be one
Nuclear polar bodies are electron dense granules that are to three.
• Usually NPBs are small in size. Following fusion their
THE FERTILIZATION CHECK (FIGS. 42.3 found in the pronuclei soon after they are formed. Orga-
nelles in the nucleus where preribosomal RNA is synthe- size increases but the number decreases.
AND 42.4) sized, which is essential for meiosis and cell cleavage. • Synchronization of the nucleoli: A synchronized fusion
• Round oocyte These particles coalesce to each other in time. Fusion and and polarization in both pronuclei is preferred.
polarization occurs in a synchronized manner in both
• Two polar bodies and their alignment
the pronuclei. In this process, the number of particles Tesarik and Greco in 199910 came forward with the fol-
becomes inversely proportional to the size of the particles. lowing classification (Table 42.2):
Fig. 42.4: Human embryo in the pronuclear stage with two symmetri-
cal pronuclei, equal number of nuclear precursor bodies (NPBs2) in
juxtaposition, and two polar bodies at 1 O’clock position.
Table 42.2. Pronuclear scoring by Tesarik and Greco.
Pattern Number of NPB Polarity of NPB Diagrammatic representation
P0 •• 3–7 Either polarized in both or nonpolar-
•• No difference >3 ized in both
P1 Difference in pronuclei >3 Either polarized in both or nonpolar-
ized in both
Fig 42.3: Schematic diagram representing structures to be noted at Fig. 42.5: Axis of the pronuclei as compared to that of the polar bodies
the fertilization check. may be parallel. Contd...
390 The Infertility Manual Assessment of Fertilization 391
Contd... Table 42.3. Z score
Table 42.2. Pronuclear scoring by Tesarik and Greco. Z SCORE Equality OF NPBs Alignment of the NPBs Schematic Representation
Pattern Number of NPB Polarity of NPB Diagrammatic representation Z1 Equal Aligned
P2 <7 in number Nonpolarized in at least one pronu-
cleus
Z2 Equal Nonaligned
P3 >7 in number Polarization in at least one pronucleus
Z3-1 1–2 large nucleoli Scattered NPBs on opposite
P4 <3 NPB in one pronucleus Both polarized or both nonpolarized side
Z3-2 Equal Aligned on one side scattered
P5 3–7 NBPs in each pronuclei Polarized distribution in one and non- on the other side
polarized in the other
Z3-3 Unequal in number with Either aligned or scattered
difference of >2 NPBs
Z3-4 Small pinpoint nucleoli >8 Scattered through the nucleus
in number
Z4 (abnormality in the nuclei Unequal Not aligned
may also be present)
Fig. 42.8: Human embryo in pronuclear stage, with two unequal pro- Fig. 42.9: Embryo in two symmetrical pronuclei with juxtaposed nu-
Table 42.4. The Istanbul consensus.
nuclei, and juxtaposed nuclear precursor bodies (NPBs) unequal in clear precursor bodies (NPBs) unequal in number. Two polar bodies
number. can be seen at the background. Category Rating Features
1 Symmetrical Equal number of NPBs either aligned in both the nuclei or scattered in both
(similar to Z1 and Z2)
The Z score was given by Lynette Scott in 200011 and
ABNORMAL FERTILIZATION 2 Nonsymmetrical Any other arrangement. This may include peripherally arranged nucleoli.
modified in 2003 (Table 42.3).12
3 Abnormal Pronuclei with ≤1 NPBs. “Ghost” pronuclei or “bull’s eye” pronuclei (Fig. 42.11)
Several other workers including Scott and Smith,13 Pronuclear Abnormalities
Senn, et al.,14 and Zollner, et al.15 had come out with sev-
eral pronuclear scoring system in the subsequent years 1 PN Mechanisms for the presence of a single pronucleus • Abnormal synchrony between the male and female
(Figs. 42.8 to 42.10). The development of both the pronuclei may take any-
(Fig. 42.12):18 pronuclear development
where from 3 to 20 hours.16 When evaluated at 16–17 hours, • Parthenogenetic activation: It may occur spontaneously • Early pronuclear fusion
The Istanbul Consensus8 approximately 5% of the zygotes are seen to have single (usually in postmature oocytes) or iatrogenically follow- • Failure of one of the two gametes to form the respec-
It is described in Table 42.4. pronuclei (Fig. 42.12).17,18 ing injection of calcium containing media during ICSI19 tive pronucleus. This could be because of abnormality
392 The Infertility Manual Assessment of Fertilization 393
Figs. 42.15A to E: Pronuclear morphology.
• Defect in oocyte decondensing factors which activate
the sperm chromatin decondensation by releasing
appropriate levels of glutathione.
Fig. 42.10: Two pronuclei unequal in size and two polar bodies at the Fig. 42.11: Abnormal zygote with single bulls-eye pronucleus (pronu- Fig. 42.14: Two pronuclei unequal in size and two polar bodies at • PNs normally appear to be equal in size, although the
9 and 11 O’clock position. cleus with single NPB) and two polar bodies. 9 and 11 O’clock position. female PN which is often located toward the second
polar body can be slightly smaller.
polar body which could ultimately lead to diploid Clinical Implications23
chromatin from the oocyte. • Little developmental potential
• Maternal factors: • High degree of aneuploidy
Abnormal oocyte quality that prevents meiosis
to occur. Pronuclear Morphology
Clinical implications of the 3 PN zygote: This is shown in Figures 42.15A to E.
The position of PNs has a relevant effect on the first
• Could be used as a prognostic tool for detection of a
cleavage plane that normally goes through the pronuclear
cohort of oocyte that is globally dysfunctional22
axis.24,25
• May indicate toward the need for modification of the
If the pronuclei are not centrally located, this could
stimulation protocols
mean an abnormality in the aster or the microtubule for-
• Usually ends up in an early miscarriage.
mation.23 Such embryos are not used for transfer.
Fig. 42.12: An unfertilized, parthenogenetically activated human Fig. 42.13: Abnormal zygote with three pronuclei. Pronuclear Size CYTOPLASMIC MORPHOLOGY
oocyte showing one prominent pronucleus and one of the two polar
bodies. Normally, the male and the female nuclei are equal in size. ASSESSMENT
Mechanism of development of unequal pronuclei
During the process of IVF and ICSI, damage may be caused
(Fig. 42.14)
to the cytoplasm. Since the cytoplasm has a vital role in the
in the sperm chromatin decondensation and centro- 3 PN • Defect in the replacement of the sperm protamines cell division, it is important to conduct a morphological
meric abnormality. assessment for the same (Table 42.5).
The 3 PN pronuclei may be seen approximately in 1% of all with the histones.
pregnancies.22 Usually, such cases end up in a miscarriage
Clinical Implications in the early pregnancy (Fig. 42.13).
Table 42.5. Cytoplasmic morphology assessment.
• Many a times, at the time of examination, the pronu-
Mechanism of development of the 3 PN zygote: Cytoplasmic
clei would have already fused giving the false impres- Sl No. abnormality Description Clinical implication
sion of 1 PN. On cytogenetic analysis, such embryos • Paternal origin: 1. Homogeneity Homogenous Expected finding
may be diploid and can result in a healthy pregnancy Polyspermic fertilization (rare in case of ICSI as
Heterogeneous Unknown clinical significance
and live birth, if transferred.20 only one sperm is injected)
2. Cytoplasmic Halo Unknown clinical significance
• One has to keep in mind, the chances of aneuploidy in A probable mechanism could be that certain sperm
these embryos which are higher than the 2PN zygotes.21 factors might prevent the extrusion of the second Contd...
394 The Infertility Manual Assessment of Fertilization 395
Contd... 17. Staessen C, Janssenwillen C, Devroey P, Van Steirteghem sperm injection outcomes. Journal of Assisted Reproduction
AC. Cytogenetic and morphological observations of single and Genetics. 2011;28(10):879-83.
Table 42.5. Cytoplasmic morphology assessment.
pronucleated human oocytes after in-vitro fertilization. 23. Scott L. Pronuclear scoring as a predictor of embryo
Cytoplasmic Human Reproduction. 1993;8:221-3. development. Reprod Biomed Online. 2003;6(2):201-14.
Sl No. abnormality Description Clinical implication 18. Noyes N. Embryo biopsy: the fate of abnormal 24. Scott L. Oocyte and embryo polarity. Semin Reprod Med.
3 Other minor Morpho- Debris in PVS Unknown clinical significance pronuclear embryos. Reproductive Biomedicine Online. 2001;18:171-83.
logical parameters 1993;17(6):782-8. 25. Edwards RG, Beard HK. Oocyte polarity and cell
19. Goud P, Van Oostveldt P, Van der Elst J, Dhont M. determination in early mammalian embryos. Mol Hum
Refractile bodies Unknown clinical significance
Fertilization abnormalities and pronucleus size asynchrony Reprod. 1997;3:863-905.
4 Granularity Severe granularity involving whole of Abnormal embryo developmental potential after intracytoplasmic sperm injection are related to oocyte 26. Otsuki J, Okada A, Morimoto K, Nagai Y, Kubo H. The
the zygote, small, or large area of the postmaturity. Fertil Steril. 1999;72(2):245-52. relationship between pregnancy outcome and smooth
zygote. 20. Feenan K, Herbert M. Can ‘abnormally’ fertilized zygotes give endoplasmic reticulum clusters in MII human oocytes.
5 Organelle clustering Severe abnormality that is likely to Decreased intracytoplasmic pH rise to viable embryos? Human Fertility. 2006;9(3):157-69. Hum Reprod. 2004;19:1591-7.
repeat in consequent cycles. May Decreased ATP content of the zygote 21. Yan J, Li Y, Shi Y, Feng HL, Gao S, Chenz Z. Assessment 27. Papale L, Fiorentino A, Montag M, Tomasi G. Atlas of
give normal appearing cleavage stage Aneuploidy, hypohaploidy of sex chromosomes of human embryos arising from Human Embryology. ESHRE.
embryos. Poor predictor of pregnancy and implanta- monopronucleus zygotes in in vitro fertilization and 28. Li M, Zhao W, Xue X, Zhang S, Shi W, Shi J. Three pro-
tion rates intracytoplasmic sperm injection cycles of Chinese women. nuclei (3PN) incidence factors and clinical outcomes: a
6 Vacuoles Small No specific biological significance Gynecol Obstet Invest. 2010;69:20-3. retrospective study from the fresh embryo transfer of in
22. Figueira RCS, Setti AS, Braga DPAF, Iaconelli A, Borges E. vitro fertilization with donor sperm (IVF-D). Int J Clin Exp
Large >14 µm Obstruction to the cleavage planes. Not Prognostic value of triploid zygotes on intracytoplasmic Med. 2015;8(8):13997-4003.
good for transfer
7 SER disc Deleterious clinical outcome26-28
PROBABLE QUESTIONS 8. Alpha Scientists in Reproductive Medicine and ESHRE
Special Interest Group of Embryology; The Istanbul consensus
1. Describe the process of oocyte activation. workshop on embryo assessment: proceedings of an expert
2. What is fertilization check and when is it performed? meeting. Human Reproduction. 2011;26(6):1270-83. [online]
Available from https://doi.org/10.1093/humrep/der037.
3. How is pronuclear scoring done? Mention the istan-
9. Bavister BD, Squirrell JM. Mitochondrial distribution and
bul grading of the pronuclear stage? function in oocytes and early embryos. Hum Reprod.
4. What are the various pronuclear stage abnormalities? 2000;15:189-98.
10. Tesarik J, Greco E. The probability of abnormal
preimplantation development can be predicted by a single
REFERENCES static observation on pronuclear stage morphology. Hum
1. Ramadan, Balaban B, Brison D, Calderon, G. Oocyte Reprod. 1999;14:1318-23.
activation and phospholipase C zeta (PLCζ): diagnostic 11. Scott L, Alvero R, Leondires M, Miller B. The morphology
and therapeutic implications for assisted reproductive of human pronuclear embryos is positively related to
technology. Cell Communication and Signaling. 2012;10:12. blastocyst development and implantation. Hum Reprod.
2. Elders K, Dale B. In Vitro fertilization, 2nd edition. 2000;15:2394-403.
Melbourne: Cambridge University Press; 2000. 12. Scott L. Pronuclear score as a predictor of embryo
3. Swann K, Larman MG, Saunders CM, Lai FA. The cytosolic development. Reprod BioMed Online. 2003;6:201-14.
sperm factor that triggers Ca2+oscillations and egg 13. Scott L, Smith S. The successful use of pronuclear embryo
activation in mammals is a novel phospholipase C: PLCζ. transfers the day following oocyte retrieval. Hum Reprod.
Reproduction. 2004;127:431-9. 1998;13:1003-13.
4. Austin CR. Cortical granules in hamster eggs. Exp Cell Res. 14. Senn A, Urner F, Chanson A, Primi MP, Wirthner D,
1956;10(2):533-40. Germond M. Morphological scoring of human pronuclear
5. Liu M. The biology and dynamics of mammalian cortical zygotes for prediction of pregnancy outcome. Hum Reprod.
granules. Reproductive Biology and Endocrinology: RB&E. 2006;21:234-9.
2011;9:149. 15. Zollner U, Zollner KP, Hartl G, Dietl J, Steck T. The use
6. Poccia D, Collas P. Nuclear envelope dynamics of a detailed zygote score after IVF/ICSI to obtain good
during pronuclear development. Dev Growth Differ. quality blastocysts: the German experience. Hum Reprod.
1997;39(5):541-50. 2002;17:1327-33.
7. Tesarik J, Kopecny V. Development of human male 16. Balakier H, MacLusky N, Casper R. Characterization of
pronucleus: ultrastructure and timing. Gamete Res. the first cell cycle in human zygotes implications for
1989;24:135-49. cryopreservation. Fertil Steril. 1993;59:359-65.
C HA PT E R
Culture Systems
43 •
•
A cleavage stage embryo uses pyruvate till the eight cell
stage, and for the later development and subsequent
cleavages, the zygote utilizes glucose as the energy
substrate.11-13
The key anabolic precursor required for the synthesis
•
ylic acid in equimolecular volume.24
Culture Systems 397
L-glutamine (Gln), breaks down rapidly forming the
byproducts of ammonium and pyrrolidine-5-carbox-
Also any culture medium should have enough amounts
of sulfur containing amino acids which will minimize
Anuja Kamath
of triacylglycerols and phospholipids is glucose. Glu- apoptosis thus reducing the instances of mono-zygote
cose also acts as a precursor for various glycoproteins twinning.25
Chapter Outline
and complex sugars.
• Culture Media
• Pentose phosphate pathway (PPP) metabolizes glu-
Ethylenediaminetetraacetic Acid
cose, thereby generating moieties of ribose which is
required for nucleic acid synthesis.14 • The EDTA is a divalent cation. It acts as a ligand
INTRODUCTION Serum albumin aids in the micromanipulation of the gam-
• Additionally, glucose participates in important cellu- and a chelating agent for metal ions. Bound metal
etes and embryos, preventing them from sticking to the
Human embryos are known to be metabolically dynamic, lar functions apart from being an energy source. This ions exhibit less reactivity although present in the
surface of pipette and dish. The most basic media used
their requirement of nutrients changes on day to day basis. is supported by the finding that mouse blastocysts, solution.
during the initial days was based on HTF.7 It mimics the
Due to the ongoing research of last 30 years, it is possible to which were obtained in the absence of glucose in the • The addition of EDTA to culture media helps to over-
develop more physiological culture media which is com- composition in human Fallopian tubes.
culture medium, had impaired developmental com- come the two-cell block in mice embryos.26
petent to maintain the growth and viability of embryo. Sub- petence after transfer, in comparison to blastocysts • Also, premature utilization of glycolysis by cleavage
optimal culture conditions impaired embryo development Components of Culture Media which developed in the presence of glucose.15 stage embryo is inhibited by addition of EDTA, thus
and decreased the implantation rates of the embryos.1 The
Composition of culture media differs largely between having a positive effect on embryos till compaction.27
objective of a successful in vitro fertilization (IVF) cycle is
to produce viable embryos with good implantation poten-
companies manufacturing them. The key components Amino Acids • EDTA demonstrates biphasic effect; it inhibits cyto-
remain the same, they are: solic kinases in glycolytic pathway in cleavage embryo.
tial and finally a healthy take home baby and the key to
• Amino acids benefit the development of the preim- ICM uses glycolysis as its main energy pathway; hence,
successful embryo culture is to minimize perturbations in • Carbohydrates plantation embryo. They are multifunctional as they EDTA presence has adverse effect on ICM cell number
the microenvironment around the embryo. IVF outcomes, • Amino acids act as: and later fetal development.28
preimplantation and postimplantation development of an • Ethylenediaminetetraacetic acid (EDTA)
embryo, and the future health of the offspring, are related Precursors of biosynthesis • An EDTA concentration of 0.005–0.01 mmol/L does
• Macromolecules Energy sources along with pyruvate and lactate
to culture conditions, as shown by large evidences.2-5 not have a deleterious effect on murine preimplanta-
• Antibiotics Regulators of energy metabolism tion or postimplantation embryo development.29
Osmolytes which counteract the toxic effect in the
CULTURE MEDIA Carbohydrates presence of high levels of inorganic ions
One of the more important factors in any culture system Buffers for maintaining pHi
Macromolecules
• The levels of glucose, lactate, and pyruvate varies
is the culture media. Culture media have evolved from Chelators of heavy metals16 • Proteins present in the culture media have various
simple salt solutions, which were used during the initial within the female reproductive system and also varies
intracycle.8 • Specific changes in the nitrogen requirements of an functions:
years of assisted reproductive technique (ART) to a more embryo are also noted.17,18 Reducing embryo toxicity
complex media, which was produced due to continuous • High concentrations of pyruvate and lactate, and low
concentrations of glucose are found in the oviduct. • Growth of early cleavage stage embryo is stimulated by Act as a source for basic nutritive requirements of
research. The different types of culture media that have
Whereas, relatively low concentrations of pyruvate, the seven nonessential amino acids.19 early embryos
been used are:
lactate, and higher concentrations of glucose are found • An inhibitory effect on blastocyst development and its Act as a source of growth factors promoting
• Simple salt solutions with added energy substrates: viability was also noted if the 13 essential Amino acids embryonic development directly or indirectly via
in the uterus.
Earle’s, KSOM, human tubal fluid (HTF) were present during the early stages of embryo growth.20 cumulus-cell proliferation.30-32
• Embryos up to the compaction stage demonstrate
• Complex tissue culture media – Ham’s F 10 (contain • Both essential as well as nonessential amino acids act • Serum is the commonly used protein supplement in
simple physiology, wherein lower levels of oxidative
amino acids, vitamin, nucleic acid precursors, transi- as a stimulant to the development of inner cell mass of culture medium, due to its multiple beneficial factors
metabolism is maintained.
tional metals) blastocysts. In addition, the nonessential amino acids which includes amino acids and vitamins. But the pre-
• Embryos after compacting, behaves like a somatic cell,
• Simplex optimized media—Computer program to gen- along with glutamine are responsible for the formation implantation embryo in vivo is never exposed to serum.
and utilizes many nutrients, has a higher biosynthetic
erate successive media based on response to mouse of trophectoderm and later stimulate the hatching of • Serum when present in the culture media causes
activity, higher respiratory capacity, and also it has a
embryos in culture—KSOMAA blastocyst from the zona pellucida.21,22 detrimental metabolic, genetic, and morphological
capability to utilize glucose as an energy source.9,10
• Sequential media. • Amino acids undergo spontaneous breakdown, releas- changes in the blastocyst.33,34
• This occurs during the postcompaction stage, when
ing the byproduct ammonium in the culture medium. • Serum is also found to be embryo toxic, and difficult
In 1984, Menezo, et al. developed a special media with the embryonic genome gets activated wherein a shift
The concentration of ammonium is time dependent. to standardize due to variations in the levels of metab-
addition of serum albumin as a source of amino acids.6 in the energy requirement is also noted.
• It is known that embryo viability is reduced due to olites, growth factors, hormones, and protein from
the toxicity of ammonium.23 The highly unstable batch-to-batch.35
398 The Infertility Manual Culture Systems 399
• Nowadays, serum albumin, a most abundantly found designed based on the composition of oviducal fluids Table 43.1. Comparison of single step medium and sequential medium.
macromolecule in the female reproductive tract, is used and uterine fluids. Various patterns of metabolism of the
Factors Sequential medium Single step medium
to supplement media. It has various benefits such as: embryos are also studied. There are two types of culture
media devised to culture the embryos from zygote stage to Change of medium Required on D3 Can be used till D5/D6
It negates the toxic effects associated with colloidal
blastocyst stage, they are: Energy substrates Change on D3 No change
osmotic pressure.
It alters the surface tension of the media thus pre- EDTA Blastocyst medium does not contain Contains EDTA
• Single step: Wherein the embryo is cultured in a single
venting the gametes from sticking to the culture Effect of essential Amino Acid Cleavage medium does not contain Contains essential amino acids
medium throughout till blastocyst stage.
vessel and aids their manipulation.36 • Sequential: Wherein the embryo is cultured in two dif- pH Cleavage medium: 7.20–7.25 pH is similar throughout, unless CO2 is
Sperm capacitation. Blastocyst medium: 7.30–7.35 altered on D3
ferent media, the first media is used till the precom-
• With the availability of recombinant human serum paction stage and then a second media is used from
albumin (HSA), the inherent problems associated with postcompaction to blastocyst stage. The composition
blood derived products have minimized thus leading of both the media is different. Table 43.2. Comparison of various characteristics.
to standardization of the culture media.37 Single medium Single medium
• Synthetic polymers such as polyvinyl alcohol (PVA) Characteristics (nonrenewed) (renewed) Sequential medium
and polyvinyl pyrrolidone (PVP) has also been used Single Step Media Leaves embryos undisturbed Yes No No
in some cases38 but none of them can be considered a • Single step media is based on the theory of “let the Accumulated endogenous growth factor Left in place Lost Lost
physiological alternative to protein.39 embryo choose”. It essentially has a single media for- Replacement of essential nutrients No Yes Yes
• Glycosaminoglycan hyaluronate (hyaluronic acid, mulated for the entire preimplantation period. Accumulated toxins Left in place Removed Removed
hyaluronan) can be used as a physiological alternative • Medium is developed from the simplex optimization
Environmental stress to embryos Low Moderate High
to albumin. The human embryo expresses the recep- approach. The embryo will utilize the required optimal
tor for it throughout preimplantation development.40 Requires quality control One medium One medium Two media
components, and adapt to the medium.44
While hyaluronate could not only replace serum • The advantages of single step media are: Relative labor intensity Low Moderate High
albumin in culture, it increased the implantation rate Reducing stress to the embryo Relative cost Low Low High
of resultant mouse embryo blastocysts.41 Therefore, Avoids osmotic shock
hyaluronate can replace albumin as a sole macro- Maintains paracrine or autocrine factors
molecule in an embryo transfer medium and in some Less media used • In this type of culture system, interaction and exchange available ART culture media have a lower osmolarity
infertile patients it can improve ongoing pregnancy Labor efficient. of gases with media happens rapidly, with both advan- at about 250–300 mOsmol.
rates.42 tages and disadvantages. Advantage being rapid equi- • Embryo development is severely impaired when
Sequential Culture Media libration of culture media and the disadvantage is the sodium chloride concentration goes up to 290
Antibiotics rapid change in pH when out of the incubator. mOsmol.47
• Sequential culture media system is based on the the- • In closed system, the culture media is overlaid with oil, • Extracellular amino acids like glycine, betaine, pro-
• Antibiotics are routinely added to the embryo culture ory of “back to nature”. which acts as a barrier between the embryos and the line, alanine, and hypotaurine can be added to the
media, to prevent bacterial contamination.43 • The sequential system essentially mirrors the environ- ambient atmosphere media. These protect the preimplantation embryo
• Most commonly used antibiotics are penicillin ment of the female reproductive tract. • It delays gas diffusion, media evaporation, and filters against hypertonicity and increases embryo develop-
(-lactam; 100 U/mL), streptomycin (aminoglycoside; • The first phase (D1–D3) precompaction, where the any air borne particles. It thus maintains pH, osmolar- ment.48
100 g/mL), and gentamicin (aminoglycoside; 50 g/mL). media is composed on the lines of the oviduct fluid and ity, and temperature better than an open system.
• Penicillin is known to be antibacterial. It disrupts the the second phase (D4–D5) postcompaction, where the
cell wall integrity by inhibiting the synthesis of pepti- pH and Buffers
doglycan.
media is based on the uterine fluid.45 Culture Conditions
• The pH range for embryo culture media is usually
• Penicillin has no direct toxic effects on the preimplan- A comparison between Single step medium and There are different culture conditions which affects cul- between pH 7.4 and 7.2.
tation embryo. Sequential medium is shown in Table 43.1. ture media.
• Important ions which affects pH are sodium, potas-
• Streptomycin and gentamycin disturb bacterial pro- Table 43.2 shows comparisons of various characteris-
sium, magnesium, chloride lactate, and AA glycine
tein synthesis. However, the aminoglycosides show tics between single step and sequential culture medium.44 Osmolality which acts as an intracellular zwitterionic buffer.49
more toxic effects. • The balance of CO2 concentration of incubator and the
• The most common problem faced by cells in culture is
Open and Closed Systems the maintenance of intracellular and extracellular per- bicarbonate ions concentration in the culture media
Culture Systems • In an open system, the culture media is not overlaid turbations occurring in the osmotic environment. regulates the pH.
Numerous culture media have been structured for the cul- with oil, thus the media comes in direct contact with • The osmotic pressure of oviduct fluid is greater than • The pHi of human cleavage stage embryo is 7.250 and
ture of human embryos in the laboratory. These have been the incubator gases. 360 mOsmol.46 However, the most commercially pH is necessary to maintain intracellular homeostasis
400 The Infertility Manual Culture Systems 401
• The pHi of morulae and blastocyst are slightly higher, perinatal outcome: findings after transfer of fresh and and induction of fetal retardation and exencephaly by 36. Gardner DK. Dissection of culture media for embryos:
i.e., 7.4, also they have better control on pHi due to the cryopreserved embryos. Hum Reprod. 2012;27:1966-76. ammonium ions. J Reprod Fertil. 1994;102:305-12. the most important and less important components and
4. El Hajj N, Haaf T. Epigenetic disturbances in in vitro 21. Lane M, Gardner DK. Non-essential amino acids and characteristics. Reprod Fertil Dev. 2008;20:9-18.
formation of tight junction between cells.51
cultured gametes and embryos: implications for human glutamine decrease the time of the first three cleavage 37. Bungum M, Bungum L, Humaidan P, Yding Andersen
• Buffers play an important role in maintaining the pH assisted reproduction. Fertil Steril. 2013;99:632-41. divisions and increase compaction of mouse zygotes C. Day 3 versus day 5 embryo transfer: a prospective
of the media. There are two commonly used buffers— 5. Mantikou E, Youssef MAFM, van Wely M, van der Veen F, in vitro. J Assist Reprod Genet. 1997;14:398-403. randomized study. Reprod Biomed Online. 2003;7(1):
HEPES and MOPS. Al-Inany HG, Repping S, et al. Embryo culture media and 22. Lane M, Gardner DK. Differential regulation of mouse 98-104.
• HEPES [4-(2-hydroxyethyl)-1-piperazineethanesul- IVF/ICSI success rates: a systematic review. Hum Reprod embryo developments and viability by amino acids. 38. Bavister BD. Culture of preimplantation embryos: facts and
Update. 2013;19:210-20. J Reprod Fertil. 1997;109:153-64.
phonic acid] is used in a concentration of 21 mmol/L. artefacts. Hum Reprod Update. 1995;1:91-148.
6. Quinn P, Pool T. Development of modern culture media. In: 23. Gardner DK, Lane M. The 2-cell block in CF1 mouse
• MOPS [3-(N-morpholino)-propanesulfonic acid] is the 39. Gardner DK. Development of serum-free media for
Flemings, Cooke (eds). Textbook of Assisted Reproduction embryos is associated with an increase in glycolysis and
the culture and transfer of human blastocysts. Hum
other common buffer. for Scientists in Reproductive Technology. Freemantle: a decrease in tricarboxylic acid (TCA) cycle activity:
alleviation of the 2-cell block is associated with the Reprod. 1998;13:218–25.
• Both buffers have a pKa value of 7.2, closest of the zwit- Vivid Publishing; 2009. 40. Campbell S, Swann HR, Aplin JD, Seif MW, Kimber SJ,
restoration of in vivo metabolic pathway activities. Biol
terionic buffers to the pHi of embryos of 7.12. 7. Quinn P, Kerin JF, Warnes GM. Improved pregnancy rate Elstein M. CD44 is expressed throughout pre-implantation
Reprod. 1993;49(suppl):152.
• In the presence of optimal culture conditions, such as in human in vitro fertilization with the use of a medium human embryo development. Hum Reprod. 1995;10:
24. Winitz M. Chemistry of the Amino Acids. New York: John
based on the composition of human tubal fluid. Fertil 425-30.
pH, gas concentration, osmolarity, etc. HEPES is able Wiley and Sons, Inc.; 1961:1929-54.
Steril. 1985;44:493-8. 25. Cassuto G, Chavrier M, Menezo Y. Culture conditions 41. Gardner DK, Lane M, Rodriguez-Martinez H. Fetal
to support mammalian embryo development and can 8. Leese HJ, Hooper MAK, Edwards RG, Ashwood-Smith MJ. and not prolonged culture time are responsible for development after transfer is increased by replacing
also act as a chelator of heavy metals such as copper.52 Uptake of pyruvate by early human embryos determined by monozygotic twinning in human in vitro fertilization. Fertil protein with the glycosaminoglycan hyaluronate for
a non-invasive technique. Hum Reprod. 1986;1(3):181-2. Steril. 2003;80:462-3. embryo culture. HR Update. 1997:380.
9. Gardner DK. Changes in requirements and utilization of
CONCLUSION nutrients during mammalian preimplantation embryo
26. Abramczuk J, Solter D, Koprowski H. The beneficial effect 42. Valojerdi MR, Karimian L, Yazid PE, Gilani MAS, Madani
of EDTA on development of mouse one-cell embryos in T, Baghestani AR. Efficacy of a human embryo transfer
Human embryos have the potential to grow in vitro, in dif- development and their significance in embryo culture. chemically defined medium. Dev Biol. 1977;61:378-83. medium: a prospective, randomized clinical trial study.
ferent types of media right from the basic systems to the Theriogenology. 1998;49:83-102. 27. Gardner DK, Lane M. The 2-cell block in CF1 mouse J Assist Reprod Genet. 2006;23:207-12.
more complex sequential media. The culture media used 10. Rieger D. Relationship between energy metabolism embryos is associated with an increase in glycolysis and 43. Borland RM, Biggers JD, Lechene CP, et al. Elemantal
and development of the early embryo. Theriogenology. a decrease in tricarboxylic acid (TCA) cycle activity:
in vitro, usually contains only a small subset of parts which composition of fluid in the human Fallopian tube. J Reprod
1992;37:75-93. alleviation of the 2-cell block is associated with the
are found under in vivo conditions. Hence, embryos cul- Fertil. 1980;58:479-82.
11. Leese HJ, Barton AM. Pyruvate and glucose uptake by restoration of in vivo metabolic pathway activities. Biol
tured in vitro are always exposed to constant stress which 44. Biggers JD, Summers MC. Choosing a culture medium:
mouse ova and preimplantation embryos. J Reprod Fertil. Reprod. 1993;49(suppl):152.
is not natural. Embryos undergo several adaptations to making informed choices. Fertil steril. 2008;90(3):473-83.
1984;72:9-13. 28. Gardner DK, Lane MW, Lane M. Bovine blastocyst cell
counter the suboptimal culture conditions, and thus may number is increased by culture with EDTA for the first 72 h of 45. Ledger W, Tan Sl, Bahathiq A (eds). The fallopian tube
12. Leese HJ, Biggers JD, Mroz EA, Lechene C. Nucleotides in a
lead to lower pregnancy and higher miscarriage rates. development from the zygote. Theriogenology. 1997;47:278. in infertility and IVF practice. UK: Cambridge University
single mammalian ovum or preimplantation embryo. Anal
29. Biggers JD, McGinnis LK, Lawitts JA. One-step versus two- Press; 2010.
Therefore, it is essential that a high level of quality con- Biochem. 1984;140:443-8.
step culture of mouse preimplantation embryos: is there a 46. Borland RM, Biggers JD, Lechene CP, Tymor ML. Elemental
trol exists in the laboratory, and it is suggested that further 13. Leese HJ, Conaghan J, Martin KL, Hardy K. Early human
difference? Hum Reprod. 2005;20:3376-84. composition of fluid in the human Fallopian tube. J Reprod
embryo metabolism. Bio Essays. 1993;15:259-64.
investigations are necessary to optimize environmental con- 30. Choi YH, Lee BC, Lim JM, Kang SK, Hwang WS. Fertil. 1980;58:479-82.
14. Reitzer LJ, Wice BM, Kennell D. The pentose cycle; control
ditions in which the preimplantation embryo can evolve in. and essential fuction in HeLa cell nucleic acid synthesis. Optimization of culture medium for cloned bovine 47. Biggers JD, Lawitts JA, Lechene CP. The protective
J Biol Chem. 1980;255:5616-26. embryos and its influence on pregnancy and delivery action of betaine on the deleterious effects of NaCl on
outcome. Theriogenology. 2002;58:1187-97. preimplantation mouse embryos in vitro. Mol Reprod
PROBABLE QUESTIONS 15. Gardner DK. Environment of the preimplantation human
31. Lonergan P, Carolan C, Van LA, Donnay I, Khatir Dev. 1993;34:380-90.
embryo in-vivo: metabolic analysis of oviduct and uterine
1. What are the different types of culture media? H, Mermillod M. Role of epidermal growth factor in 48. Van Winkel LJ, Haghighat N, Campione AL. Glycine protects
fluids and metabolism of cumulus cells. Fertil Steril.
bovine oocyte maturation and preimplantation embryo preimplantation mouse conceptuses from a detrimental
2. What are the components of culture media? 1996;65:345-53.
development in vitro. Biol Reprod. 1996;54:1420-9. effect on development of the inorganic ions in oviductal
3. What are the different types of culture systems? 16. Gardner DK, Pool TB, Lane M. Embryo nutrition and
32. Srisathien S, Hernandez-Fonseca HJ, Brackett BG. fluid. J Exp Zool. 1990;253:215-9.
energy metabolism and its relationship to embryo
4. How does different culture conditions affect the cul- Influences of epidermal growth and insulin-like growth 49. Campbell S, Swann HR, Aplin JD, Seif MW, Kimber SJ,
growth, differentiation, and viability. Semin Reprod Med. factor-I on bovine blastocyst development in vitro. Anim
ture media? 2000;18(2):205-18. Elstein M. CD44 is expressed throughout pre-implantation
Reprod Sci. 2003;77:21-32.
17. Leese HJ. Metabolism of the preimplantation mammalian human embryo development. Hum Reprod. 1995;10:
33. Gardiner CS, Reed DJ. Status of glutathione during oxidant-
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Rieger D. Relationship between energy metabolism
induced oxidative stress in the preimplantation mouse
50. Phillips KP, Leveille MC, Claman P, Baltz J. Intracellular
embryo. Biol Reprod. 1994;51:1307-14.
1. Bowman P, Maclaren A. cleavage rate of mouse embryos and development of the early embryo. Theriogenology. 34. Thompson JG, Gardner DK, Pugh PA, McMillan WH, pH regulation in human preimplantation embryos. Hum
in vivo and in vitro. J Embryol Exp Morphol. 1970;24:203-7. 1992;37:75-93. Tervit HR. Lamb birth weight is affected by culture system Reprod. 2000;15:896-904.
2. Dumoulin J, Land J, Van Montfoort A, Nelissen E, Coonen E, 19. Gardner DK, Lane M. Amino acids and ammonium utilized during in vitro pre-elongation development of 51. Edwards LJ, Williams DA, Gardner DK. Intracellular pH of
Derhaag J, et al. Effect of in vitro culture of human embryos regulate the development of mouse embryos in culture. ovine embryos. Biol Reprod. 1995;53:1385-91. the preimplantation mouse embryo: effects of extracellular
on birthweight of newborns. Hum Reprod. 2010;25:605-12. Biol Reprod. 1993;48:377-85. 35. Maurer HR. Towards serum-free, chemically defined pH and weak acids. Mol Rerpdo Dev. 1998b;50:434-42.
3. Nelissen E, Van Montfoort A, Coonen E, Derhaag J, Geraedts 20. Lane M, Gardner DK. Increase in postimplantation media for mammalian cell culture. In: Freshney RI (ed). 52. Mash HE, Chin YP, Sigg L, Hari R, Xue H. Complexation of
J, Smits L, et al. Further evidence that culture media affect development of cultured mouse embryos by amino acids Animal Cell Culture: A Practical Approach. Oxford: Oxford copper by zwitterionic aminosulfonic (good) buffers. Anal
University Press; 1992:15-46. Chem. 2003;75:671-7.
C HA PT E R
44
Cryopreservation 403
remove the spermatozoa with disturbed plasma mem- Patients with Cancer
brane.27
Cryopreservation • A magnetic cell sorting technique (MACS) can be used
for separation of cells not vulnerable to apoptosis acti-
• Cryopreservation of sperm is recommended for those
men diagnosed with cancer and wants to retain their
vation. fertility as the treatment affects the sperm quality.
Mohammed Ashraf • Most of such patients tend to have sufficient viable
Chapter Outline Cryoprotectants sperms that can be frozen before start of chemo
therapy.
• Sperm Cryopreservation • Ovarian Tissue Cryopreservation • There are two groups of cryoprotectants used in rou-
• Oocyte and Embryo Cryopreservation • Before they begin their treatment, regardless of the
tine cryogenic work:
stage of malignancy, sperm cryopreservation should
1. Permeating cryoprotectants, viz. glycerol, dimethyl
be recommended for all pubescent boys and men.
INTRODUCTION • All these injures the sperm plasma membrane by sulfoxide (DMSO)
• Boys who need to undergo gonadotoxic therapy and
changing the lipid composition and its location.4-6 2. Nonpermeating cryoprotectants, viz. polyhydroxy-
Cryobiology is the branch of biology involving the study ethyl starch, glycine, and sugars like sucrose, raffi- who do not yet have sperm in their ejaculate, testicular
• Such changes affecting the plasma membrane leads to
of the effects of low temperatures on organisms. It is the nose, etc. tissue cryopreservation becomes a fertility preserva-
cell leakage of many intercellular components which
core of all fertility cryopreservation. Cryopreservation • For sperm cryopreservation, glycerol is most widely tion method.30
consequently leads to reduced sperm metabolic activ-
was first performed on animal sperm cells to store them used. • In boys who already have sperm in their semen, it
ities. This in turn causes the elimination of cytoplasmic
for use in artificial insemination. Similarly, human • Along with cryoprotectants, natural extenders viz. egg would be best to freeze it.31,32
and membrane bound proteins and enzymes. Hence,
fertility cryopreservation began with sperm cells and then yolk, or synthetic extenders, viz. zwitter ion buffers are • In prepubertal boys, where spermatogenesis is not
the membranes loses its fluidity and integrity,7-9 lipid
went on to be applied to embryo, oocyte, and gonadal used for better recovery of the motile cells. fully active, it is recommended to preserve the sper-
cryopreservation. peroxidation is caused by oxidative stress,10,11 DNA
matogonia and adjacent cells as an intact tissue.33
fragmentation,12-14 and cytoskeletal modification.15
• Microtubule containing structures which are suscep- Indications for Sperm Cryopreservation • Preserving Sertoli and maintaining intercellular con-
SPERM CRYOPRESERVATION tible to low temperatures are affected by the freezing
tact in testicular tissue is vital for the maturation of
Obstructive Azoospermia spermatogonia.34
• In 1776, Lazaro Spallanzani was the first to reportedly process.16-19
• The sperm frozen unprepared from seminal fluid • Patients with obstructive azoospermia undergo sperm
observe the effects of low temperature on spermatozoa.1
appears to be more resistant to cryoinjuries than are retrieval techniques like Microsurgical epididymal Various Techniques of Sperm Freezing
• Only with the discovery of the glycerol’s cryoprotectant
frozen sperm prepared either by percoll density cen- sperm aspiration (MESA), testicular sperm extraction
properties in 1949, sperm cryopreservation became a • Vapor freezing method35,36
trifugation or direct swim up method followed by (TESE), and Percutaneous epididymal sperm aspira-
reality.2 • Pellet method37
freezing in seminal plasma. tion (PESA) for various assisted reproductive tech-
• The first human birth from a cryopreserved semen • Dry-shipper freezing technique38
nique (ART) procedures.
through artificial insemination were performed in • On thawing sperm frozen in seminal plasma, an • Freezing in cold ethanol39
• These retrieval techniques allow the extraction of sper-
1953 by Bunge and Sherman.3 increase in the motility and the DNA integrity of sperm
matozoa from the epididymis or testicular tissues. Among these methods, the vapor freezing method is
cells is observed.
• Since only a portion of the extracted spermatozoa more widely used. In this method, before plunging the
General Characteristics of Sperm • Increased cholesterol levels in the human sperm mem-
is required for IVF, the rest can be cryopreserved for semen into liquid nitrogen, it is placed in plastic straw and
branes are known to stabilize membrane while cooling.20
Cryopreservation • There may be a decrease in the fertilizing capability
future use.28 placed above liquid nitrogen at a predetermined distance
• On the completion of TESA, testicular tissue can be
• Human spermatozoa has a high membrane fluid- due to the activation of apoptosis signal transduc- for several minutes.34,35
cryopreserved after dividing it into several aliquots.
ity resulting from unsaturated fatty acids in the lipid tion caused by cryopreservation and thawing in trace
bilayer. Thereby, making it less sensitive to damages amounts of spermatozoa.21-23
Nonobstructive Azoospermia (NOA) Freezing Process
due to rapid initial cooling. • The translocation of phosphatidyl serine from the This process is given in Figure 44.1.
• They tend to be more resistant than other cells to cryo- inner to the outer leaflet of the sperm plasma mem- • In contrast to obstructive azoospermia, nonobstructive
preservation damage due to the low water content. brane affects the integrity of the sperm membrane and azoospermia (NOA) patients may pose a challenge.
• The factors responsible for cryoinjury in living cells is seen as one of the early signs of terminal phase of • In the event that no or immotile sperm is obtained, it Thawing Process
undergoing cryopreservation are: apoptosis.24,25 is recommended to have a fresh surgery or use donor It is shown in Figure 44.2.
Low temperature • The specific binding of annexin V to phosphatidyl ser- sperm.
Crystallization of the intracellular and the extracel- • It is not recommended to use immotile sperm obtained % of motility of post thaw specimen
ine can be used for the detection of the translocation26 Cryosurvival =
lular water content. on thawing.29 % of motility of prefreeze specimen
and then a magnetic separation can be performed to
404 The Infertility Manual Cryopreservation 405
Table 44.1. Differences between slow freezing
and vitrification.
Slow freezing Vitrification
Low levels of cryoprotectants High levels of cryoprotectants
Slow controlled rates of Very fast cooling rates
cooling (0.3°C/min) (~20,000°C/min)
Slow dehydration to mini- Fast cooling rate result in
mize ice crystal formation solidification of solution into
glass like structure
Need of programmable No programmable freezers
freezers required
Take hours Takes only seconds
Fig. 44.1: Freezing process. Fig. 44.2: Thawing process.
• Cryoprotectants with a higher permeability are used as
the exposure time is reduced due to its rapid permea-
tion, decreases cryoinjuries, and the osmotic swelling
Historical Background that develops while it is being removed.
• While it is impossible to entirely eliminate the toxic and
Important dates to remember:
osmotic injuries caused by CPA on oocytes and embryos,
• 1983: First pregnancy after freezing human embryo the following can minimize its negative effects:
• 1984: First baby after freezing human embryo Fig. 44.4: Schematic presentation of an embryo (circle) before cooling, Use of a highly permeating CPA
during cooling, and in liquid nitrogen in slow freezing, conventional
• 1986: Freezing of human oocytes Keep the concentration to the lowest necessary
straw vitrification, and ultrarapid vitrification. White circles represent
ice crystals. Keep the exposure time to the minimum
Maintain a high temperature: Permeation is tem-
Basic Principles of Cryopreservation perature dependent. High temperatures acceler-
During thawing, the embryo/oocyte is warmed and
Cryopreservation of reproductive cell is described in ates the rate of permeation.
then unloaded from the cryodevice into a series of solu-
Figure 44.3.
tions with reducing values of osmolarities. These solu-
Nonpermeating Cryoprotective Agents
tions consist of sucrose which is used for rehydrating the
Slow-freezing versus Vitrification oocytes/embryo and removal of the cryoprotective agents The CPA toxicity can be possibly reduced by substitut-
Fig. 44.3: Basic principles of cryopreservation. (CPA). ing the part of permeable CPA with nonpermeating CPAs
In slow-freezing, the extracellular water is crystallized into ice comprising of polymers like galactose, sucrose, and poly-
such that intracellular water is driven out in a slow-controlled mers like polyvinylpyrrolidone, polyethylene glycol (PEG),
manner. This results in excessive increase in the viscosity or Cryoprotective Agents ficoll, dexran, and polyvinyl alcohol (PVA).49
solidification without forming intracellular ice crystals.42 In vitrification, commonly used CPAs consists of permeat-
OOCYTE AND EMBRYO
Vitrification is a term applied to refer the physical ing and nonpermeating agents. Carrier Devices for Vitrification
CRYOPRESERVATION phenomenon where the solidification of water or water- • The intracellular water in the cell is displaced by the Vitrification of oocytes/embryos are generally carried out
based solutions into a glass-like amorphous liquid state
Introduction permeating CPA and takes its place by osmosis. with carrier systems like electron microscopy (EM) grid,
(called vitreous state) occurs, due to extreme elevation • An osmotic gradient is created throughout the cell open pulled straw (OPS), Cryotop, Cryoleaf, and Cryotip
Great scientific efforts are being made to discover more in viscosity during cooling, without the formation of ice membrane when the extracellular osmolarity is successfully. However in human ART, Cryotop with its
logical, ethical, and economical procedures in order to crystals (Fig. 44.4).43,44 increased due to the extracellular CPA. derivatives like Hemi-Straw System (HSS),50 cryolock,51 cry-
maintain and advance the success of assisted reproduc- • It also prevents the water from rapidly entering the cell oleaf, cryoloop, EM grid, and OPS are more widely used.52
tion techniques.
Differences between Slow Freezing after thawing during rehydrating.45-47
Cryopreservation of human oocytes, zygotes, cleav- Technical Features of Human Oocyte
and Vitrification
age stage embryos, and blastocysts is one of the necessary
Permeating Cryoprotective Agents Cryopreservation
procedures.40,41 It is given in Table 44.1.
Ethylene glycol (EG), DMSO, 1, 2 propendiol (PROH), and • The metaphase II (MII) oocyte is extremely fragile
glycerol are the four most widely used permeating CPAs and sensitive to low temperature due to its large size
for oocyte and embryo cryopreservation.42,48 (human oocyte is 130 μm), low surface to volume ratio,
406 The Infertility Manual Cryopreservation 407
water content, chromosomal arrangement, and the 2. Determine the number of embryos required for types, posing a challenge for its preservation due to its
presence of the spindle.53 vitrification. uneven cooling rates and diminished heat transfer.61
• The intracellular ice crystals formed during freezing 3. Transfer the embryo(s) with the least volume of cul- • For prepubescent girls about to undergo gonadotoxic
and thawing injures the spindle apparatus.54 ture medium to the drop of equilibration solution treatment, ovarian tissue cryopreservation is the only
• Spindle microtubule depolarization which occurs dur- (ES) for 5–15 minutes (the embryo will shrink and option.61
ing cryopreservation is a reversible feature. With time then gradually re-expand to its original size indicating
and optimum temperature the metaphase spindle the completion of the equilibration process). Cryopreservation of Cortical Strips
microtubule can repolymerize and form functional 4. Then transfer the embryo(s) from ES to the center
spindles postcryopreservation. of the first drop of vitrification solution 1 (VS1) for • The technique does not require pretreatment and can
• Zona pellucida hardening hinders the fertilization 5 seconds. be performed from one day to another independently
process.55 5. Immediately transfer the embryo(s) to the center of where the patient is in the menstrual cycle.
of the second drop of VS2 for 5 seconds. • It is a small surgical procedure and the patients are
often dismissed on the day of surgery.62
Technical Features of Human Embryo 6. Then, transfer the embryo(s) from VS2 to the center of
• Ovarian cortical strips are acquired from small 5 mm
the VS3 drop for 10 seconds.
Cryopreservation 7. Finally, transfer the embryo(s) from VS3 to the center biopsies or in 1–3 mm strips via laparoscopy.63
• The rate of survival of embryos increases with the of the VS4. • The immature follicles have decreased water content,
increase in the cell numbers.56 8. Carefully transfer the embryo(s) in less than 1 μL Fig. 44.6: Laboratory procedure for warming. an increased surface to volume ratio, and an absent
• The implantation rate can be improved by employing of VS solution from VS4 into the vitrification device zona pellucida, making it less vulnerable to the dam-
assisted hatching techniques like partial zona dissec- (Fig. 44.5). ages that can be caused during cryopreservation.64
tion, zona drilling, and laser assisted hatching. • In the cryopreservation of ovarian cortical strips, slow
4. Leave the embryos in the solution for 1 minute. The freezing and vitrification can be employed.
The Laboratory Procedure for Warming embryo will shrink and float to the upper part of the
Blastocyst Cryopreservation • Ovarian cortical strips can be autotransplanted either
1. Fill the reservoir with liquid nitrogen to completely drop. into the pelvis (orthotopic) or outside of the pelvis
and its Advantages submerge the cryolock or goblet. 5. Then, transfer the embryos to the first drop of 0.5 M (heterotopic).63
The activation of the embryonic genome takes place after 2. Remove the carrier device from the storage tank and WS for 2 minutes and then to second drop of 0.5 M • In orthotopic transplantation, the thawed ovarian cor-
the 8-cell stage. (Braude et al., 1988) and blastocyst culture quickly transfer them to the liquid nitrogen reservoir. WS for 2 minutes. tex is transplanted onto the remaining ovary or into a
allows the embryos to be transferred when it has an acti- 3. Remove the cryolock from the container and sub- 6. Aspirate the embryos from the liquid and transfer them peritoneal window in the pelvis.
vated embryonic genome. merge it instantly (within 2 seconds) into 1 M warm- into the first of the three drops of each 20 μL MOPS solu- • The first live birth resulting from the transplantation of
• Cryoinjuries will be compensated when the num- ing solution (WS). tion for a duration of 3 minutes. Repeat the same for the cryopreserved ovarian tissue was in 2004.65
ber of cells are high which aid in rapid recovery and second and third droplet for the same duration each. • However, there is a risk of metastasis when dealing
enhances the viability. 7. Finally, transfer the embryos to a dish of pre- with the cryopreservation of tissues from patients with
• The surface volume ratio is high due to the low cyto- equilibrated appropriate culture media and incubate malignancy and their subsequent reimplantation.66
plasmic volume of cells which aids in rapid penetra- in a CO2 incubator at 37°C for 3–4 hours (Fig. 44.6).
tion of cryoprotectants. Cryopreservation of Whole Ovary
• Reducing blastocele volume reduces the intracellular OVARIAN TISSUE CRYOPRESERVATION • Follicular loss results from the cryopreservation and
ice crystal formation. Therefore, by reducing the blas-
subsequent reimplantation of cortical strips second-
tocele volume before vitrification can increase the sur- Introduction ary to ischemia.
vival rates.
Ovarian tissue cryopreservation is an emerging area of fer- • Less follicular loss may result from the reimplantation
• The survival rates and pregnancy rates can be improved tility preservation. Cryopreservation of ovarian tissue and of a whole ovary due to the rapid establishment of the
in humans by mechanically reducing the blastocele by its subsequent transplantation enables the steroidogenic vascular supply.67
puncturing it.57 and gametogenic functions to be restored in an ovary. • The drawback of this technique is the diffusion of cryo-
• The blastocele when collapsed by puncturing a hole This helps in the restoration of the quality of life in those protectant into the entire organ and intravascular ice
into the trophectoderm by a laser beam can produce a women faced with premature ovarian failure.59,60
formation which will result in vascular injury.
survival rate of 98% and an implantation rate of 49%.58 Ovarian tissue can be cryopreserved as fragments,
• Also to preserve the vascular pedicle as well as to
slices, hemiovaries, or whole ovaries with a vascular pedi-
establish the vascular reanastomosis.
The Laboratory Procedure for Vitrification cle for future vascular anastomosis.
• The vascular anastomosis is an important concern
1. Fill the reservoir with liquid nitrogen to completely • The ovarian tissues have a dense structure with an intri- because if anastomosis fails, it will result in the loss of
submerge the cryolock or goblet. Fig. 44.5: Laboratory procedure for vitrification. cate vascular system, and are composed of diverse cell the whole ovary.
408 The Infertility Manual Cryopreservation 409
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high security straws, and cryobags.
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analysis of human spermatozoa and seminal plasma by vitro after cryopreservation at the germinal vesicle stage. 32. Kliesch S, Behre H, Jurgens H, Nieschlag E. Cryopreservation
Vitrification Techniques Employed MALDITOF mass spectrometry and NMR spectroscopy— Fertil Steril. 1997;68:920-6. of semen from adolescent patients with malignancies. Med
effects of freezing and thawing. Chem Phys Lipids. 19. Zenzes MT, Bielecki R, Casper RF, Leibo SP. Effects of Pediatr Oncol. 1996;26:20-7.
1. Direct cover vitrification (DCV):68 This method allows 2000;106:145-56. chilling to 0 degrees C on the morphology of meiotic 33. Ehmcke J, Hubner K, Scholer HR, Schlatt S. Spermatogonia:
the ovarian tissues to be directly exposed to liquid 5. Schuffner A, Morshedi M, Oehninger S. Cryopreservation spindles in human metaphase II oocytes. Fertil Steril. Origin, physiology and prospects for conservation and
nitrogen and also employs the use of cryoprotectants of fractionated, highly motile human spermatozoa: effect 2001;75:769-77. manipulation of the male germ line. Reprod fertil Dev.
on membrane phosphatidylserine externalization and lipid 20. Drobnis EZ, Crowe LM, Berger T, Anchordoguy TJ, 2006;18:7-12.
at a low concentration.
peroxidation. Hum Reprod. 2001;16:2148-53. Overstreet JW, Crowe JH. Cold shock damage is due to 34. Ogawa T, Ohmura M, Ohbo K. The niche for spermatogonial
2. Needle immersion vitrification (NIV):69 This method 6. Duru NK, Morshedi MS, Schuffner A, Oehninger S. lipid phase transitions in cell membrane: a demonstration stem cells in the mammalian testis. Int J Hematol.
involves the use of acupuncture needle (0.18 mm Cryopreservation—thawing of fractionated human sper- using sperm as a model. J Exp Zool. 1993;265(4): 2005;82:381-8.
diameter and 20 mm in length) for holding the tissues matozoa is associated with membrane phosphatidylser- 432-7. 35. Roussel JD, Kellgren HC, Patrick TE. Bovine semen frozen
and to directly place it into the liquid nitrogen after ine externalization and not DNA fragmentation. J Androl. 21. Paasch U, Sharma RK, Gupta AK, Grunewald S, Mascha EJ, in liquid nitrogen vapour. J Dairy Sci. 1964;47(12):1403-6.
treating it with equilibration solution and vitrification 2001;22:646-51. Thomas AJ Jr, et al. Cryopreservation and thawing is 36. Sherman JK. Improved methods of preservation of human
7. Hazel JR. Thermal adaptation in biological membranes: associated with varying extend of activation of apoptotic spermatozoa by freezing and freeze drying. Fertil Steril.
solution. Thereby, maximizing the cooling rate and
is homeoviscous adaptation the explanation? Annu Rev machinery in subsets of ejaculated human spermatozoa. 1963;14:49-64.
avoiding cryoinjury. Physiol. 1995;57:19-42. Biol. Reprod. 2004;71:1828-37. 37. Gibson CD, Graham EF. The relationship between fertility
3. Solid surface vitrification:70 A method in which a metal 8. Holt WV, North RD. Effects of temperature and restoration 22. Peris SI, Morrier A, Dufour M, Bailey JL. Cryopreservation and post freeze motility of bull spermatozoa (by pellet
surface floating on liquid nitrogen vitrifies samples of osmotic equilibrium during thawing on the induction of ram semen facilitates sperm DNA damage: relationship freezing) without glycerol. J Reprod Fertil. 1969;20(1):
when it comes into contact to its surface. This metal of plasma membrane damage in cryopreserved ram between sperm andrological parameters and the sperm 155-7.
spermatozoa. Biol Reprod. 1994;5:414-24. chromatin structure assay. J Androl. 2004;25:224-33. 38. Roth TL, Bush LM, Wildt DE, Weiss RB. Scimitar-horned oryx
surface will be at -196°C.
9. Quinn PJ. Effects of temperature on cell membranes. Symp 23. Sakkas D, Urner F, Bizzaro D, Manicardi G, Bianchi PG, (Oryx dammah) spermatozoa are functionally competent
4. The Ohio-Cryo method:71 It is performed using Ohio- Soc Exp Biol. 1988;42:237-58. Shoukir Y, et al. Sperm nuclear DNA damage and altered in a heterologous bovine in vitro fertilization system after
Cryo device. The device is designed to accommodate 10. Chatterjee S, Gagnon C. Production of reactive oxygen chromatin structure: effect on fertilization and embryo cryopreservation on dry ice, in a dry shipper, or over liquid
one processed hemiovary per device. species by spermatozoa undergoing cooling, freezing, and development. Hum Reprod. 1998;13(Suppl):11-9. nitrogen vapour. Biol Reprod. 1999;60(2):493-8.
thawing. Mol Reprod Dev. 2001;59:451-8. 24. Glander HJ, Schaller J. Binding of annexin-V to plasma 39. Saroff J, Mixner JP. The relationship of egg yolk and
11. Mazzilli F, Rossi T, Sabatini L, Pulcinelli FM, Rapone S, membranes of human spermatozoa: a rapid assay for glycerol content of diluters and glycerol equilibration
PROBABLE QUESTIONS Dondero F, et al. Human sperm cryopreservation and detection of membrane changes after cryostorage. Mol time to survival of bull spermatozoa after low temperature
reactive oxygen species (ROS) production. Acta Eur Fertil. Hum Reprod. 1999;5:109-15. freezing. J Dairy Sci. 1955;38(3):292-7.
1. Explain the vapor freezing technique for freezing 1995;26:145-8. 25. Hinkovska-Galcheva V, Petkova D, Koumanov K. Changes 40. Trounson A, Mohr L. Human pregnancy following
semen sample. 12. Chohan KR, Griffin JT, Carrell DT. Evaluation of chromatin in the phospholipid composition and phospholipid cryopreservation, thawing and transfer of an eight-cell
2. Write a short note on magnetic cell-sorting technique. integrity in human sperm using acridine orange staining asymmetry of ram sperm plasma membrane after embryo. Nature. 1983;305:707-9.
3. Briefly describe the difference situations involved in a with different fixatives and after cryopreservation. cryopreservation. Cryobiology. 1989;26:70-5. 41. Zeilmaker GH, Alberda AT, Gent I, Rijkmans CM,
Andrologia. 2004;36:321-6. 26. Paasch U, Grunewald S, Fitzl G, Glander HJ. Deterioration Drogendijk AC. Two pregnancies following transfer of
cryopreservation of sperm.
13. Peris SI, Morrier A, Dufour M, Bailey JL. Cryopreservation of plasma membrane is associated with activation of intact frozen–thawed embryo. Fertil Steril. 1984;42:293-6.
4. Distinguish between slow freezing and vitrification. of ram semen facilitates sperm DNA damage: relationship caspaces in human spermatozoa. J Androl. 2003;24:246-52. 42. Gupta MK. Cryopreservation of oocytes and embryos by
5. Define cryoprotective agents and mention their uses between sperm andrological parameters and the 27. Shen HM, Dai J, Chia SE, Lim A, Ong CN. Detection vitrification. Korean J Reprod Med. 2010;37(4):267-91.
6. Explain the technical aspects of oocyte cryopre-serva- sperm chromatin structure assay. J Androl. 2004;25: of apoptotic alterations in sperm in subfertile patients 43. Fahy GM, MacFarlane DR, Angell CA, Meryman HT.
tion. 224-33. and their correlations with sperm quality. Hum Reprod. Vitrification as an approach to cryopreservation.
7. Explain the advantages of blastocyst cryopreservation. 14. Linfor JJ, Meyers SA. Detection of DNA damage in response 2002;17:1266-73. Cryobiology. 1984;21:407-26.
to cooling injury in equine spermatozoa using single-cell 28. Park YS, Lee SH, Song SJ, Jun JH, Koong MK, Seo JT. 44. Vajta G, Nagy ZP, Cobo A, Conceicao J, Yovich J. Vitrification
8. Explain the laboratory procedure for vitrification and
gel electrophoresis. J Androl. 2002;23:107-13. Influence of motility on the outcome of in vitro fertilization/ in assisted reproduction: myths, mistakes, disbeliefs and
thawing. 15. Holt WV, North RD. Cryopreservation, actin localization intracytoplasmic sperm injection with fresh vs. frozen confusion. Reprod Biomed Online. 2009;19 (Suppl 3):1-7.
9. Write a short note on ovarian tissue cryopreservation and thermotropic phase transitions in ram spermatozoa. testicular sperm from men with obstructive azoospermia. 45. Liebermann J, Tucker MJ. Effect of carrier system on
and narrate the various vitrification techniques. J Reprod Fertil. 1991;91:451-61. Fertil Steril. 2003;80:526-30. the yield of human oocytes and embryos as assessed by
C HA PTE R
45
410 The Infertility Manual
survival and development potential after vitrification.
Reproduction. 2002;124:483-9.
ovarian tissues increases follicle viability and pregnancy
capability in mice. Hum Reprod. 2006;21(11):2794-800.
PolScope, Embryoscope and
46. Liebermann J. Potential importance of vitrification in
reproductive medicine Biol Reprod. 2002;67:1671-80.
60. Sugimoto M, Maeda S, Manabe N, Miyamoto H.
Development of infantile rat ovaries autotransplanted Assisted Hatching
47. Liebermann J, Nawroth F, Isachenko V. Recent developments after cryopreservation by vitrification. Theriogenology.
in human oocyte, embryo and blastocyst vitrification: 2000;53(5):1093-103.
Mir Jaffar, Waseem Andrabi
where are we now? Reprod Biomed Online. 2003;7: 61. Courbiere B, Caquant L, Mazoyer C, Franck M, Lornage J,
623-33. Salle B. Difficulties improving ovarian functional recovery
48. Kasai M, Mukaida T. Cryopreservation of animal and by microvascular transplantation and whole ovary Chapter Outline
human embryos by vitrification. Reprod Biomed Online. vitrification. Fertil Steril. 2009;91(6):2697-706. • PolScope • Assisted Hatching
2004;9:164-70. 62. Rosendahl M, Andersen CY, Ernst E, Westergaard LG, • Embryoscope
49. Shaw JM, Oranratnachai A, Trounson AO. Cryopreservation Rasmussen. Ovarian function after removal of an entire
of oocytes and embryos. In: Trounson AO, Gardner DK ovary for cryopreservation of pieces of cortex prior to
(Eds). Handbook of In Vitro Fertilization, 2nd edition. Boca gonadotoxic treatment: a follow-up study. Hum. Reprod. POLSCOPE zona pellucid of oocyte having orderly arranged filamen-
Raton: CRC Press; 2000. pp. 373-412. 2008a;23:2475-83. tous, they refract the light ray known as birefringence.12
50. Vanderzwalmen P, Bertin G, Debauche C, Standaert V, 63. Oktay K, Buyuk E, Rosenwaks Z, Rucinski J. A technique Introduction Birefringence: Birefringence is a property of any mate-
Schoysman R. In vitro survival of metaphase II oocytes and for transplantation of ovarian cortical strips to the forearm.
rial having almost nonrandom arrangement of molecules
blastocysts after vitrification in a Hemi-Straw (HS) system. Fertil Steril. 2003;80(1):193-8. Over the years, results in assisted reproductive programs
Fertil Steril. 2000;74(Suppl):215. 64. Sonmezer M, Oktay K. Fertility preservation in female like crystal or mitotic spindle.
have improved in terms of pregnancy rates but birth rates
51. Lucena E, Bernal DP, Lucena C, Rojas A, Moran A, Lucena A. patients. Hum Reprod Update. 2004;10(3):251-66. If the oocyte is subjected to polarized light, the light
are still on lower side. To improve the pregnancy rates,
Successful ongoing pregnancies after vitrification of 65. Donnez J, Dolmans MM, Demylle D. Live birth after parallel to the axis of mitotic spindle experiences a higher
many centers transfer multiple embryos leading to multi-
oocytes. Fertil Steril. 2006;85:108-11. orthotopic transplantation of cryopreserved ovarian tissue. refractive index compared to the light parallel to the spin-
52. Vajta G, Nagy ZP, Cobo A, Conceicao J, Yovich J. Vitrification Lancet. 2004;364(9443):1405-10. ple pregnancies.1
dle axis. This difference in the refractive index is called
in assisted reproduction: myths, mistakes, disbeliefs and 66. Courbiere B, Prebet T, Mozziconacci MJ, Metzler- To avoid the complication of multiple pregnancies,
confusion. Reprod Biomed Online. 2009;19(Suppl 3):1-7. Guillemain C, Saias-Magnan J, Gamerre M. Tumor cell birefringence and the material is known to be birefringent.
single embryo transfer has to be implemented, provided
53. Bernard A, Fuller BJ. Cryopreservation of human oocytes: a contamination in ovarian tissue cryopreserved before an accurate assessment of embryo quality is followed. • A healthy oocyte spindle retardance is known to be
review of current problems and perspectives. Hum Reprod gonadotoxic treatment: should we systematically exclude
At the time of ovulation, oocytes are arrested in met- 3–5 nm and the spindle diameter to be about 30–40 µm.
Update. 1996;2:193-207. ovarian autograft in a cancer survivor? Bone Marrow
54. Shaw JM, Oranratnachai A, Trounson AO. Fundamental Transplant. 2010;45(7):1247-8. aphase of second meiosis (MII). At this stage, a normal • To minimize any unwanted birefringence effect, glass bot-
cryobiology of mammalian oocytes and ovarian tissue. 67. Jeremias E, Bedaiwy MA, Gurunluoglu R, Biscotti CV, meiotic spindle is unipolar barrel shaped formed by the tom dishes are by dishes are preferred over plastic dishes.
Theriogenology. 2000;53:59-72. Siemionow M, Falcone T. Heterotopic autotransplantation bundles of filaments. For a proper division, an intact and
55. Matson PL, Graefling J, Junk SM, Yovich JL, Edirisinghe WR. of the ovary with microvascular anastomosis: A novel healthy spindle is vital for the alignment and separation of Device
Cryopreservation of oocytes and embryos: use of a mouse surgical technique. Fertil Steril. 2002;77(6):1278-82. chromosomes.2 Any abnormality in meiotic spindle at this
model to investigate effects upon zona hardness and 68. Chen SU, Chien CL, Wu MY, Chen TH, Lai SM, Lin CW, et al. The polScope system can be added to the preexisting
formulate treatment strategies in an in vitro fertilization Novel direct cover vitrification for cryopreservation of
particular stage leads to unequal division of chromosomes
inverted microscope having mainly two polarizers and
programme. Hum Reprod. 1997;12:1550-3. ovarian tissues increases follicle viability and pregnancy and subsequently results to aneuploidy.3-5 Aneuploidy can
computer to process and analyze the images (Fig. 45.1).
56. El-Danasouri I, Selman H. Successful pregnancies and capability in mice. Hum Reprod. 2006;21(11):2794-800. result in poor or abnormal fertilization, poor embryo qual-
deliveries after a simple vitrification protocol for day 3 69. Wang Y, Xiao Z, Li L, Fan W, Li SW. Novel needle immersed ity or arrest, decreased or failed implantation rates, and
human embryos. Fertil Steril. 2001;76(2):400-2. vitrification: a practical and convenient method with spontaneous pregnancy loss.
Spindle Imaging
57. Hiraoka K, Kinutani M, Kinutani K. Blastocoele collapse by potential advantages in mouse and human ovarian tissue
Initially, spindle integrity and morphology was studied The role of the meiotic spindle:
micropipetting prior to vitrification gives excellent survival cryopreservation. Hum Reprod. 2008;23(10):2256-65.
and pregnancy outcomes for human day 5 and 6 expanded 70. Santos RR, Tharasanit T, Van Haeften T, Figueiredo JR, from fixed oocytes by using conventional immunofluores-
• During the first meiotic division, microtubules o
rganize
blastocysts. Hum Reprod. 2004;19:2884-8. Silva JR, Van den Hurk R. Vitrification of goat preantral cence microscopy.6-10 Since this conventional method was
into an orderly placed filaments forming thick bundles.
58. Mukaida T, Oka C, Goto T, Takahashi K. Artificial shrinkage follicles enclosed in ovarian tissue by using conventional invasive and did not reveal dynamic nature of spindles and
• This structure is responsible for proper chromosomal
of blastocoeles using either a micro-needle or a laser pulse and solid-surface vitrification methods. Cell Tissue Res. therefore not suitable for clinical use.
prior to the cooling steps of vitrification improves survival 2007;327(1):167-76. segregation throughout both meiotic division and
More recently, with the development in the field of image
rate and pregnancy outcome of vitrified human blastocysts. 71. Kader A, Biscotti C, Falcone T. Analysis of post-warming activation.
Hum Reprod. 2006;21:3246-52. degeneration & apoptosis following porcine ovarian tissue
processing, it has been possible to visualize the meiotic spin-
• Microtubules forming the spindle are dynamic, form-
59. Chen SU, Chien CL, Wu MY, Chen TH, Lai SM, Lin CW, et vitrification using the Ohio-Cryo device. Hum Reprod. dle by a noninvasive method with the help of polarized light
ing a polymerization and depolymerization chain in
al. Novel direct cover vitrification for cryopreservation of 2008;23(Suppl 1):147-11. microscopy, without affecting the viability of the cell.11
rapid turnover.
• The spindle is highly sensitive to environmental
Principle changes, such as temperature and pH.
The polScope basically works on the principle of refraction. • Abnormalities in the spindle have been linked to failed
When two orthogonally polarized light rays are made to or abnormal fertilization, poor embryo development,
pass through any dense structure like meiotic spindle and and failed implantation.
412 The Infertility Manual PolScope, Embryoscope and Assisted Hatching 413
• Spindle length: Shortened spindle correlates with the • Selection of sperm for ICSI: Birefringence associated • Single-step media is used from fertilization through
lowest pronuclei (PN) score and lower development with nucleoprotein filaments are useful in selecting day 5/6 without disturbing the incubator.
rate to blastocyst.13 the sperm. • With the help of software, embryologist can review,
• Spindle orientation: Normal smooth barrel-shaped annotate, and compare development of selected
spindle yields better results than the irregular-shaped Limitations of Spindle Imaging embryos using data files acquired by the time-lapse
spindle.14 system and information can be accessed from a distant
• Meiotic spindle is highly sensitive to nonphysiological place too.
• Screening for anaphase/telophase MI oocytes: MI
temperature and pH. Any fluctuation is the values will
anaphase and telophase oocytes already exhibit an
affect the retardance measurement.19
extruded polar body.15 Performing intracytoplasmic
• Timing of spindle imaging is very important because
Time-lapse Technologies in
sperm injection (ICSI) at this stage will lead to abnor-
the spindles are dynamic structures and also orienta- In Vitro Fertilization
mal fertilization mostly with three PN (two female and
tion dependant. The different TLS present in the market are:
one male), so it is advised to extend the culture of such
• Spindle retardance measurements vary during certain
oocytes till they attain metaphase-II. • EmbryoScope (Fertilitech)
physiological events like activation.20
• Spindle positive and negative oocytes: Approximately • Primo Vision (Vitrolife)
20% of oocytes lack a visible spindle in spite of first Claudia Peterson carried a meta-analysis which indi- • Eeva (Auxogyn, inc)
polar body in perivitelline space. Such oocytes are less cates that the presence of a birefringent meiotic spindle in • MIRI® TL (Esco Biotech)
viable and yield poor results compared to spindle posi- oocytes is a better indicator of oocyte quality and subse-
quent outcomes.13
tive oocytes.16 Features of Time-lapse Monitoring
• Angle of deviation of the meiotic spindle: MII oocytes
• Provide pictures which are suitable for diagnosis of key
show varying degree of polar body deviation with EMBRYOSCOPE
respect to meiotic spindle. Performing ICSI by using milestones
To select the developmentally most competent embryo for • Monitoring of multiple embryos at any given time with
polar body as the marker is a crude method and may
transfer into uterus, morphological evaluation has been proper identification of each embryo
damage the spindle in case of deviation.17
preferred method since the beginning of in vitro fertili- • Undisturbed culture throughout the incubation
• Zona pellucida: A thicker inner zona retardance cor-
zation (IVF). Currently one of the major challenges is to • Provide picture at any given point of time as per the
relates with better embryo development and greater
find the optimal solution for the selection of most compe- need
Fig. 45.1: Schematic representation of light field LC-PolScope probability of conception.18
including the optical set-up with image acquisition and processing
tent embryo to achieve the highest pregnancy rates with • Assemble all the pictures in a time-lapse video mode
components. minimal number of embryos for transfer to avoid multiple and also enable morphometric analysis of individual
Additional Applications of PolScope pregnancies. embryo.
• Laboratory quality assessment: When more than 30% of Time-lapse monitoring system (TLS) can either be
Application of PolScope oocytes lack spindles in oocytes from multiple patients installed into an existing incubator by placing the camera
Embryo Selection Parameters
of varied ages and case histories, there is a nonbiologi- inside it or a exclusively designed incubator with inbuilt
Mean Spindle Retardance cal reason (temperature, pH, osmolarity, and stress camera for digital images of embryos at set time inter- • Timing of PN breakdown
vals. The images of developing embryos at different stages • Early cleavage
The oocytes having high-degree of spindle molecular during manual handling of oocytes).
are utilized for embryo selection without disturbing the • Synchrony of cell division
order measures higher retardance, is scored better than • Nuclear transfer
embryos. Some TLS also offer a semiquantitative com- • Fragmentation history
the oocyte with lower retardance (Figs. 45.2A and B). • Correct timing of ICSI after thawing of oocytes and in
puter assisted assessment to select the most competitive • Appearance of nuclei after division
For example: vitro maturation (IVM) cultured oocytes
embryo.21 • Detection of multinucleation
• Duration of cleavage cell cycles
Imaging System • Cleavage planes.
• This system uses a conventional incubator used in rou-
tine IVF laboratory with inbuilt camera which emits
Timing of PNB (Pronuclei Breakdown)
low intensity red light to minimize light exposure to It was found in a study that PNB times was associated with
cultured embryos. live birth rates; the PNB time of zygotes resulted signifi-
• A modified Hoffmann contrast with a 20X specialty cantly higher (p = 0.022) live birth (23 h 10 min ± 35 min)
A objective is used. than the PNB time (23 h 10 min ± 23 min) with no birth
B
• Images of each embryo are automatically recorded in group. In fact no live birth was obtained with PNB earlier
Figs. 45.2A and B: (A) With high birefringence; (B) With lower birefringence. seven focal planes at present time intervals. than 20 hours and 45 minutes after fertilization.22,23
414 The Infertility Manual PolScope, Embryoscope and Assisted Hatching 415
Early Cleavage program, the results are similar because of embryo trans- Methods of Assisted Hatching B. Noncontact laser system:
fer in nonhyperstimulated cycle.27-29 • In this system, microscope objective is used to
It was reported that shorter time an embryo spends in Mechanical Partial Zona Dissection: deliver the laser beam without any direct contact
2-cell, 3-cell, 4-cell, and 5-cell stage have better implanta- with the embryo.
• In this technique, the embryo is held in position by
tion potential. Most importantly if a 2-cell embryo cleaves ASSISTED HATCHING • Laser beam produces super heating effect at the
gentle suction from the holding pipette and a sharp
before 25–27 hours postfertilization, the outcomes are
better. Introduction dissecting micropipette is passed through the ZP at the point of laser delivery (200°) dissolving the ZP on
largest perivitelline space and advanced tangentially. the axis.
Failure of embryo to implant may have several factors. • A slit in the ZP is created by gently rubbing the holding • The laser has options of three different intensi-
Synchrony Failure of embryo to hatch after the transfer may be one of pipette over the small part of ZP trapped against the ties, low, medium, and high that can be delivered
the reasons for implantation. micropipettes. in a single 25 ms pulse.
It has been reported that synchronous appearance of
• This method is preferred for D2 embryos. • These options are preferred as per the size of
nuclei after first division and synchrony of cell division is
significantly associated with pregnancy and can be con- What is Hatching? Chemical Zona Drilling:
desired defect.
sidered as an indicator of embryo quality.24 The main function of zona pellucida (ZP) is the protec- • In this technique, embryo is stabilized at 9 O’clock
tion of the embryo and the maintenance of its integrity.30 by holding pipette and another pipette oriented at Key Issues
Fragmentation History During the initial stages of cleavage, the blastomeres are 3 O’clock containing Tyrodes solution. Although improved clinical implantation and pregnancy
loosely attached without having tight junctions between • By using controlled delivery system, acid Tyrode is
Studies have shown reduced viability with the history of rates have been reported after AH, but a recently pub-
the adjacent blastomeres, so for holding these blasto- blown on the external surface of embryo creating a
fragmentation and uneven sized embryos.25 lished meta-analysis by Cochrane did not support the reg-
meres in close approximation for protection and assist in 30-µm diameter defect and subsequently washed in ular use of AH. They stressed that, although the live birth
compaction, zona pellucida is essential. Once blastomeres Tyrode free media. rate should be considered the primary outcome, there is
Multinucleation have developed tight junctions, which normally happens • This technique is generally preferred for D3 embryos insufficient evidence about the effects of AH on live birth
Multinucleation impairs embryo viability. Some studies after 8-cell stage, ZP is not essential except its protective because of unstretched and thick ZP. rates.31,39 Thereby, the conclusions drawn regarding the
documented that even a single multinucleate blastomere role against hostile uterine factors.
Enzymatic Zona Softening or Complete Removal with benefits are as follows:
may be detrimental for the viability of the embryo.26 For the implantation to occur, the transferred embryo
Pronase:
should come out of ZP known as hatching to allow the • AH does not improve the outcomes in the patients
Among all above, highest correlation was found for the
interaction between trophectoderm and endometrial cells • In this technique, the embryo is transferred to a diluted undergoing first attempt of embryo transfer.
timing for PNB, fully hatched blastocyst, and first nuclei
for proper implantation. solution of Pronase (10 IU/mL Pronase diluted 10× by • AH enhances the outcomes in the patients who have
disappearance followed by second nuclei appearance
G2 media) in G2 medium under oil for ∼60 seconds for previously failed implantation.
after first division.
initial stretching and softening of ZP. • It was believed that patients with advanced age,
There is almost perfect agreement for all cleavage Assisted Hatching • The embryo is quickly examined on the heated stage embryos with thick zona, or frozen thawed embryos
stages, with highest precision for the first division.
Assisted hatching (AH) is one of the proposed methods to of an inverted microscope to observe if the zona is may benefit with AH, but the evidence does not sup-
Moreover, direct cleavage from 1 to 3 along with
improve implantation in which ZP is disrupted artificially expanded in size. If not, then embryo is further incu- port this.
unevenness and multinucleation at the 2-cell stage have
by micromanipulation or laser AH. bated with Pronase for an additional 30–60 seconds. • More robust data with adequate evidence is needed to
been reported to display a strong negative correlation to
• Embryo is transferred to G2 after repeated washing of investigate the role of AH.
pregnancy.24
embryo. • Currently the data is insufficient about the recommen-
Indications for Assisted Hatching
• Complete zona removal should not be done for dation of AH as routine practice.
Safety of Time-lapse Monitoring System • Recurrent implantation failure (two to three cycles)31,32 precompacting embryo.
• Periodic exposure of embryos to bright light could be • Thick ZP (>18 µm)31,32
Laser-assisted Hatching:
• Advanced maternal age (>37 years)33 PROBABLE QUESTIONS
potentially harmful to the embryos. The application of laser on the ZP for AH results in photoa-
• Unlike any conventional incubator, TLS is dynamic • Elevated basal follicle-stimulating hormone (FSH)
blation of the ZP. 1. PolScope, its principle, application, and limitations of
having multiple motors working during the movement levels34
A. Contact laser spindle imaging.
of dished toward camera, the possibility of generation • Cryopreservation cycles35
B. Noncontact laser 2. What is the role of meiotic spindle and how does pol-
of heat, lubrication fumes, and electromagnetic field • Extended in vitro culture (e.g. IVM, D6/7)36,37
• Fragment removal.38 A. Contact laser: Scope help to improve the outcomes?
may be detrimental to the embryos.
• Use of this method was reported in 1991. 3. Briefly describe embryoscope and elaborate on
To conclude with, TLS helps in selecting the best • It necessitates the contact between laser deliver- embryo selection parameters and safety of TLS.
embryo out of the present pool to minimize the time to
Contraindications for Assisted Hatching ing pipette and ZP. 4. Assisted hatching, indications, and contraindications
pregnancy rather than cumulative pregnancy rate. Also • If a traumatic embryo transfer is expected • Maintaining the sterility is a real challenge for assisted hatching. Describe different methods of
the evidence suggests that with a good cryopreservation • If blastomeres do not appear to be in interphase. because of contact procedure. assisted hatching.
416 The Infertility Manual PolScope, Embryoscope and Assisted Hatching 417
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2. Edwards RG, Brody SA. Principles and Practice of Assisted 18. Rama Raju GA, Prakash GJ, Krishna KM, Madan K. Meiotic Implantation enhancement by selective assisted hatching
Human Reproduction. Philadelphia: WB Saunders; 1995. using zona drilling of human embryos with poor prognosis. 37. Zhang X, Rutledge J, Armstrong DT. Studies on zona
spindle and zona pellucida characteristics as predictors
3. Moore RM, Crosby IM. Temperature-induced abnormalities Hum Reprod. 1992;7:685-91. hardening in rat oocytes that are matured in vitro in a
of embryonic development: a preliminary study using
in sheep oocytes during maturation. J Reprod Fertil. PolScope imaging. Reproductive BioMedicine Online. 34. Stein A, Rufas O, Amit S, Avrech O, Pinkas H, Ovadia J, et al. serum-free medium. Mol Reprod Dev. 1991;28:292-6.
1985;75:476-83. 2007;14:166-74. Assisted hatching by partial zona dissection of human pre- 38. Geber S, Bossi R, Panteliades M, Sampaio M. Influence
4. Aman RR, Parks JE. Effects of cooling and re-warming on embryos in patients with recurrent implantation failure of fragment removal from embryos in pregnancy rates of
19. Shen Y, Betzendahl I, Sun F. Non-invasive method to
the meiotic spindle and chromosomes of in vitro-matured after in vitro fertilization. Fertil Steril. 1995;63:838-41.
assess genotoxicity of nocodozole interfering with spindle patients submitted to human assisted reproduction. Jornal
bovine oocytes. Biol Reprod. 1994;50:103-10. 35. Balaban B, Urman B, Kayhan Yakin K, Isiklar A. Laser
formation in mammalian oocytes. Reprod Toxicol. Brasileiro de Reproducao Assistida. 2009;13(2):13-7.
assisted hatching increases pregnancy and implantation
5. Pickering SJ, Johnson MH. The influence of cooling on the 2005;19(4):459-71. 39. Das S, Blake D, Farquhar C, Seif MMW. Assisted hatching
rates in cryopreserved embryos that were allowed to cleave
organization of the meiotic spindle of the mouse oocytes. 20. Navarro PA, Liu L, Trimarchi JR, Ferriani RA, in vitro after thawing: a prospective randomized study. on assisted conception (IVF and ICSI). Cochrane review.
Hum Reprod. 1987;2:207-16. Keefe DL. Noninvasive imaging of spindle dynamics during Human Reprod. 2006;2136-40. The Cochrane Library. 2009;(4).
6. Battaglia DE, Goodwin P, Klein NA, Soules MR. Fertilization mammalian oocyte activation. Fertil Steril. 2005;83(Supple 1):
and early embryology: influence of maternal age on 1197-205.
meiotic spindle assembly oocytes from naturally cycling 21. Conaghan J. Chen AA, Willman SP, Ivani K, Chenette PE,
women. Human Reprod. 1996;11(10):2217-22. Boostanfar. Improving embryo selection using a computer-
7. Hardarson T, Lundin K, Hamberger L. The position of the automated time-lapse image analysis test plus day 3
metaphase II spindle cannot be predicted by the location of morphology: results from a prospective multicenter trial.
the first polar body in the human oocyte. Human Reprod. Fertil Steril. 2013;100:412-9.
2000;15:1372-6; 11:2217-22. 22. Azzarello A, Hoest T, Mikkelson AL. The impact of pronuclear
8. Cooke S, Tyler JPP, Driscoll GL. Meiotic spindle location and morphology and dynamicity on live birth outcome after
identification and its effect on embryonic cleavage plane time-lapse culture. Human Reprod. 2012;27:2649-57.
and early development. Human Reprod. 2003;18:2397-405. 23. Meseguer M, Herrero J, Tejera A, Hilligsoe KM, Ramsing NB,
9. Eichenlaub-Ritter U, Shen Y, Tinneberg H-R. Manipulation Remohi J. The use of morphokinetics as a predictor of
of the oocyte: possible damage to the spindle apparatus. embryo implantation. Hum Reprod. 2011;26:2658-71.
Reproductive BioMedicine Online. 2002;5:117-24. 24. Ritter UE, Shen Y, Stalf T, Mehnert C. Light retardance by
10. Varghese AC, Goldberg E, Agarwal A. Current and future human oocyte spindle is positively related to pronuclear
perspectives on intracytoplasmic sperm injection: a score after ICSI. Reproductive BioMedicine Online.
critical commentary. Reproductive BioMedicine Online. 2006;737-51
2007;15:719-27. 25. Neuber E, Rinaudo P, Trimarchi JR, Sakkas. Sequential
11. Cohen Y, Malcov M, Schwartz T, Mey-Raz N, Carmon A, assessment of individually cultured human embryos
Cohen T, et al. Spindle imaging: a new marker for optimal as an indicator of subsequent good quality blastocyst
timing of ICSI. Human Reprod. 2004;19:649-54. development. Human Reprod. 20013;18(6):1307-12.
12. Sato H, Ellis GW, Inoué S. Microtubular origin of mitotic 26. Borini A, Lagalla C, Cattoli M, Sereni E, Sciajno R, Flamigni C,
spindle form birefringence. Demonstration of the et al. Predictive factors for embryo implantation potential.
applicability of Wiener’s equation. Journal of Cell Biology. Reproductive BioMedicine Online 2005;15:653-68.
1975;67:501-17. 27. Roberts S, McGowan L, Hirst W, Vail A, Lieberman B. Towards
13. Petersen CG, Oliveira JBA, Mauri AL, Massaro FC. single embryo transfer? Modelling clinical outcomes of
Relationship between visualization of meiotic spindle potential treatment choices using multiple data sources:
in human oocytes and ICSI outcomes: a meta-analysis. predictive models and patient perspectives. Health Technol
Reproductive BioMedicine Online. 2009;235-43. Assess. 2010;14:1-237.
14. Wang WH, Keefe DL. Prediction of chromosome 28. Mastenbroek S, van der Veen F, Aflatoonian A, Shapiro B,
misalignment among in-vitro matured human oocytes Bossuyt P, Repping S. Embryo selection in IVF. Human
by spindle imaging with the PolScope. Fertil Steril. Reprod. 2011;26:964-6.
2002;78:1077-81. 29. Wong KM, Mastenbroek S, Repping S. Cryopreservation of
15. Fang C, Mandy T, Li T. Visualization of meiotic spindle and human embryos and its contribution to in vitro fertilization
subsequent embryonic development in in-vitro and in-vivo success rates. Fertil Steril. 2014;102:19-26.
matured human oocytes. Journal of Assisted Reproduction 30. Cohen J. Assisted hatching of human embryos. J In vitro
and Genetics. 2007;24:547-51. Fertil Embryo Transf. 1991;8:179-90.
16. Wang WH, Meng L, Hackett RJ, Keefe DL. Developmental 31. Stein A, Rufas O, Amit S. Assisted hatching by partial
ability of human oocytes with or without birefringent zona dissection of human pre-embryos in patients with
spindles imaged by PolScope before insemination. Human recurrent implantation failure after in vitro fertilization.
Reprod. 2001a;16:1464-8. Fertility and Sterility. 1995.pp.838-41.
CH A PT E R
Embryology Instruments
46 Table 46.1. IUI catheter.
Order number
131181
Rigidity
Hard
Catheter
length (mm)
180
Catheter OD (mm)
2.0
Volume
minus hub (mL)
0.03
Embryology Instruments 419
Quantity (pcs)
50
Snehal Dhobale-Kohale 131182 Intermediate 180 2.0 0.10 50
131184 Soft 180 1.7 0.10 50
Chapter Outline
140198 Stylet Cannula – – 0.03 50
• Intrauterine Insemination Instruments • Oocyte and Embryo Cryopreservation
• Instruments for Oocyte Retrieval • In Vitro Fertilization Laboratory Disinfectants
• Embryo Transfer Catheters • Instruments for Laboratory Quality Control
• Culture Media • IVF Workstation: Biological Safety Cabinet
• Plastic Wares in In Vitro Fertilization Laboratory • Andrology Instruments
• Micromanipulation
The success of assisted reproductive technology is based on the “quality control” maintained by the coordinated efforts of
clinicians, embryologists, IVF nurses, and IVF counsellors.
INTRAUTERINE INSEMINATION Sterile Container with Lid (Fig. 46.2)
INSTRUMENTS Sterile, wide mouth, graduated container (110 mL) made
Intrauterine Insemination Trolley up of polypropylene for semen collection.
• Cusco’s pelvic speculum
• Powder free surgical gloves—one pair Round-bottomed BD Falcon Tube
• 1 cc sterile syringe (Fig. 46.3)
• Allis forceps/tenaculum
• Gauge • Made up of polystyrene/polyethylene
Fig. 46.2: Sterile container with lid. Fig. 46.3: Round bottomed BD falcon tube.
• Sponge holder • Dual snap cap enables both culturing and storage in
• Saline the same tube
• IUI catheter. • Used for semen preparation, storage of media, and for • Available in 5 mL, 8 mL, 14 mL, 16 mL, and 19 mL
collecting follicular fluid
Intrauterine Insemination (IUI) Catheter • For aerobic culturing, the cap is left loose while keep-
(Figs. 46.1A and B) Conical BD Falcon Tube (Fig. 46.4)
ing sample clean and sterile
• Made up of polystyrene/polyethylene
This is described in Table 46.1.
• Screw cap, double-threaded allowing one-handed
manipulation and reducing the chance of cross threading
• Conical shape helps in formation of discrete pellet for
easy removal
• Application: Centrifugation, multiple aliqoting, stor-
age of media
A • Multiple aliqoting of media helps prevent ph change
due to multiple openings
• Density gradient sperm preparation: Conical tube is
preferred as purest of sperms settles at the cone, most
Fig. 46.4: Conical BD falcon tube.
efficient wash
• Sterilized by Gamma irradiation
• It has thermal resistance, chemical stability, and high- • Nonpyrogenic at less than 0.25 endotoxin units/mL
optical clarity • Mouse embryo assay (MEA): Day 5 more than 80%
B
• High strength to withstand high centrifugal force blast
Figs. 46.1A and B: IUI catheter.
• Sterilized by Gamma irradiation • Human sperm survival assay (HSSA) ≥ 70%
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420 The Infertility Manual Embryology Instruments 421
• Available in 15 mL, 50 mL, 175 mL, and 225 mL Remi Centrifuge (Fig. 46.5) • Head and part of tail should be counted as one sperm A single lumen needle is provided with a blunt can-
• Number of sperm in 10 squares of the grid is consid- nula which can be used for flishing.
• Sperm preparation for IUI, in vitro fertilization (IVF), ered as “sperm density” in millions/ML • Quality control:
and intracytoplasmic sperm injection (ICSI) • If no sperm seen in any grid but seen outside the grid MEA: Two cell survival more than 80%
• Can accommodate 16 tubes of 15 mL capacity at the then it is reported as “occasional motile sperms”. LAL: Endotoxin level is less than 0.5 EU/mL
time of centrifugation ■■ Mouse embryo assay: It involves the culture of
• Maximum speed: 5,250 rotations per minute
• Maximum relative centrifugal force is 3,600 ‘g’
INSTRUMENTS FOR OOCYTE RETRIEVAL mouse embryos in medium maintained in an
incubator from early stages to stages just prior
• Power supply: 220–240V Ultrasound Machine to implantation. Quantization is generally in
• It is best to choose centrifuge with built in tachometer the form of a percentage of the starting embryos
to check its speed. • Frequency: 5–7 MHz: sufficient penetration depth and
that achieve the advanced stage used for scor-
enough resolution
ing. Extreme variability in results is often asso-
• Transducer: Long (total length 40 cm): allows wide
Makler Chamber (Figs. 46.6A and B) ciated with lack of standardization.
range of movements while doing procedure. Its shape
• 10 µL semen sample to be added in center of chamber. ■■ Limulus amebocyte lysate test: It is used for
allows easy use of a slim sterile probe cover. It also
Objective used is 20X. the detection and quantification of bacterial
should have an attachment for “Needle Guide”.
• Motile sperm in 20 squares are counted. If the count is endotoxins. It is based on the reaction between
less than 10 million/mL, then all 100 squares are to be Probe Covers (Figs. 46.7A and B) limulus amebocyte lysate (LAL)—an aqueous
Fig. 46.5: Remi centrifuge. counted. extract of blood cells (amebocytes) from the
• Polyethylene probe covers: Designed with a welded horseshoe crab—(Limulus polyphemus) and
seam along its lengths to inhibit accidental breakage bacterial endotoxin or a membrane component
• Latex condoms: Nonembryotoxic, washed before use. of Gram negative bacteria—lipopolysaccharide
21 cm length and 4.5 cm wide (LPS).
• These should be nonlubricated and nonmedicated
• Probe covers are recommended as part of the infection
Single Lumen Oocyte Retrieval Needle
control process, but they do not replace the need for a
high-level disinfection. (Fig. 46.8)
• Single lumen aspiration needle with bevel (orientation
Oocyte Retrieval Needle indicated by thumb notch), 17 G , 30–35 cm in length
• Purpose: Aspiration of oocytes from ovarian follicles. • Rotation of needle during aspiration is facilitated by
• Description: appropriate “handle design”.
Material: Surgical grade stainless steel (AISA 304) • Proximal hub for syringe having “Luer lock”.
Consists of tip and body • Visualization of needle tip and placement of the nee-
Vary in length from 30 to 40 cm dle is improved by “echo tip”.
A Attachments: Aspiration tubings and silicone rub- • Teflon tubings prevent temperature changes of follicu-
ber cork lar fluid.
B
A B
B
Figs. 46.6A and B: Makler chamber. Figs. 46.7A and B: Probe covers.
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422 The Infertility Manual Embryology Instruments 423
Fig. 46.9: Parts of single lumen oocyte retrieval needle. (1) Needle
length; (2) Aspiration lining; (3) Silicone Bung; (4) Tubing from cork to
Fig. 46.8: Single lumen oocyte retrieval needle. Fig. 46.12: Immature ovum aspiration needle. Fig. 46.13: Needle tip markings.
pump; (5) Luer Connector; (6) Tip.
• According to Poiseuille Law, the rate of flow is depend-
ent upon many variables, such as:
Differential height between the needle tip and the
collection test tube
Inner diameter of the needle
Aspiration pressure (vacuum)
Fig. 46.14: Bevel.
Total length of the system.
• Important points for an optimal oocyte recovery rate
and minimal damage to the oocyte cumulus complex
Bevel and zona pellucida:
• Noncoring tip to prevent tissue aspiration into needle Vacuum pressure should not be raised more than
lumen 100–120 mm Hg
• A-Bevel: 12 degree point (for small follicles) Flow rate should be 20–25 mL/min (a flow rate of
Fig. 46.11: Parts of double lumen oocyte retrieval needle. (1) Needle • B-Bevel: 15 to 18 degree point (for routine egg collection) 20–25 mL/min equates to 24–30 seconds to aspirate
length; (2) Flushing line; (3) Aspiration lining; (4) Silicone bung; (5) Tub-
• C-Bevel: 30 degree point (Fig. 46.14). 10 mL of water).
Fig. 46.10: Double lumen oocyte retrieval needle. ing from cork to pump; (6) Luer connector; (7) Tip.
Aspiration time for each 20 mm follicle should
Gauge ideally be 8–10 seconds to create sufficient
turbulence to optimize release of oocyte cumulus
Parts (Fig. 46.9) Immature Ovum Aspiration Needle • Gauge numbers describe outer diameters of needle
complex.
(smaller the gauge number larger is the outer diam-
(Fig. 46.12)
Double Lumen Oocyte Retrieval Needle eter)
(Fig. 46.10) • Suitable for aspiration of immature oocytes from • Needle gauge decides: Flow rate, pain, bleeding dura- Dead Space
smaller ovarian follicles tion of procedure
• Dead space volume of the needle and its tubing is
• Purpose: Aspiration and flushing of oocytes • “Guide Needle” is for puncturing vaginal wall (17 gauge, • Gauge and wall thickness will determine the inner
approximately 1 mL, and the oocyte may thus move
• Two lumens are provided for aspiration and simulta- 30 cm). It also adds rigidity to the “aspiration needle” diameter of the needle.
backward and forward within this dead space during
neous or intermittent flushing. (20 gauge, 35 cm).
aspiration and flushing in single lumen needle.
• The needle is 16 G, 30 and 35 cm in length. Flow Rate • The design of the double-lumen needle eliminates this
• The aspiration line length: 75 cm Needle Tip Markings (Fig. 46.13) • Optimum turbulence can be achieved either by problem, since the aspirating channel and the flushing
• The flushing line length:100 cm.
• Dual Laser markings: stable and excellent vision increasing flow rate or flushing of follicles thus opti- channel are separated, ensuring a unidirectional flow
Parts (Fig. 46.11) • Embossed markings mizing the number of oocytes retrieved. in the aspirating channel.
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424 The Infertility Manual Embryology Instruments 425
Fig. 46.15: Teflon tubing. Fig. 46.16: Blunt cannula. Fig. 46.18: Craft suction unit. (1) Illuminated power switch (On/Off ); Fig. 46.19: Rocket pump.
(2) Vacuum display mmHg—1; (3) High vacuum (440 mm Hg -1)—
control button; (4) Vacuum control: Clockwise to increase and anti-
clockwise to decrease the set value; (5) Water trap connection port;
• The size of the translucent tubing should be the same (6) Water trap set comprising glass bottle, bung, angled connectors,
• Overflow reservoir: To accommodate excess volume of
size as the needle which ensures the safe recovery of and silicone tube set; (7) Footswitch connection port; (8) Medium
vacuum (standard) 50–250 mm Hg—1 footswitch; (9) Silicone tube set follicular fluid
oocyte. with metal Luer connector. • It is equipped with “fluid trap” to prevent inadvertent
• Contamination of aspiration unit is prevented by fluid aspiration in to the unit.
“hydrophobic filter lines” which are used to connect
the needle to aspiration unit.
• Length of aspiration line: Double lumen needle:
Test Tube Heater (Fig. 46.20)
100 cm, single lumen needle: 60–90 cm. • It is the portable unit which reduces temperature-
induced damage to the oocytes by controlling the tem-
perature of test tubes and the follicular fluid. Operat-
Blunt Cannula (Fig. 46.16) ing temperature is preset to 36.9°C. It is provided with
The single lumen needle is supplied with a blunt cannula temperature controller preventing overheating.
of 4 cm length and 17 Gauge for flushing of the follicle, if • Capacity: 6 Falcon® 2001 test tubes
desired. • The front panel is transparent so the follicular fluid can
Fig. 46.17: Cook aspiration unit. be continuously observed.
Cook Aspiration Unit (Fig. 46.17) • Decontamination of the unit is possible as the panel is
easily removable.
Principle of Double Lumen • It is a regulated vacuum pump specially designed for • When the Amber light flashes, unit is at optimal oper-
oocyte retrieval providing low flow vacuum from -10 to ating temperature.
• Constant infusion of oocyte collection media into the
-500 mm Hg for general suction which is indicated by • When the power cable is connected or disconnected,
follicle at the same time as the follicular fluid is being
LED display (either mm Hg or kPa). short alarm sounds.
removed Fig. 46.20: Test tube heater.
• When the foot pedal (allowing hands free operation) is • Table stand of the unit is made up of polycarbonate.
• Increases the turbulence within the follicle
activated, it gives immediate suction response (to the
• Assist in dislodging the oocyte-cumulus complex from
adjusted prefixed level) at the needle tip and it holds
the follicle wall
this very accurately for long periods.
Trouble Shooting in Ovum Pick-Up
• Increase the chances of oocyte collection.
• It has the boost button on the front panel. Vacuum
Craft Suction Unit (Fig. 46.18) If Suction stops or decreases:
pressure is rapidly goes to -530 mm of Hg by using it • The craft: Rocket pump provides a predetermined • Ensure that the suction pump is turned on and that the
Teflon Tubing (Fig. 46.15) which can be used to clear blockages in the needle. smooth low volume vacuum on activation of foot suction pedal is functioning.
• The length of the teflon tubing between the needle and • It is vibration free unit. When the foot pedal is activated switch. • Check that all connections of tubing between the
the collecting test tube should be minimal to avoid vacuum application is indicated by volume adjustable • Rocket pump: Green zone for safe pressure of aspira- aspiration tube and the pump are tightly connected.
unnecessary cooling of the oocytes. tone. tion (Fig. 46.19) • Exclude any cracks in the aspiration test tube.
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426 The Infertility Manual Embryology Instruments 427
• Ensure that the collection tubing is not kinked or • Embryologist brings the loaded inner catheter which
damaged. is then gently introduced into the outer catheter and
• Rotate the needle within the follicle to ensure that it is advanced in the miduterine cavity and stopped from 1
not blocked by follicular wall tissue. to 2 cm short of the fundus.
• If still no suction, remove the needle and perform a • Catheter is left in situ for 30–60 sec
“retrograde flush” to clear any blockage. • Pressure is kept on the plunger of the syringe while
• Before reinserting the needle, recheck by aspirating slowly withdrawing the catheter out avoiding negative
some culture medium. pressure.
EMBRYO TRANSFER CATHETERS Advantage of after Loading
Quality Control Tests • It allows slow, gentle, and atraumatic passage of cath-
eter through the endocervix
1. One cell mouse embryo test (75% or greater blasto- • Less exposure of embryos to outside environment
cyst rate) • Embryos in the inner sheath not exposed to the mucus
2. LAL test (endotoxin level 20 EUs or less). in the cervix Fig. 46.22: Classic and sure Wallace catheters. Fig. 46.23: Standard and sure view Wallace.
• Decreased mucus contamination of the catheter.
Classification
(Mucus causes retention and displacement of the
• Rigid/Soft/Intermediate embryos. It contaminates the intrauterine environment
• Brands-Wallace/Gynetics/Cooks/Labotect thus reducing chances of successful implantation.)
• Transcervical/Transmyometrial
• Length: 19 cm/23 cm Wallace Catheter
• Side opening/Front opening
Four types (Fig. 46.21)
• Single(Frydman)/Double coaxial.
Classic, SurePro, Sure ultra, SureView
Components of Catheter
Difference between Classic and Sure Wallace
1. Outer sheath or Outer catheter or Guiding catheter: A
Catheters (Fig. 46.22)
hollow plastic tube with markings having bulb at the
tip and movable stopper (outer diameter—2.5 mm) Sure view catheter
• Bulb: Helps for easy and nontraumatic entry
• Outer catheter: 19 cm long, firm, made of teflon
through the endocervical crypts of cervical canal
• Inner catheter: 23 cm long, made of polyethylene
• Stopper: For depth adjustment A B
• Markings: For easy and accurate positioning. Figs. 46.24A and B: Cooks catheter.
2. Obturator or Stylet or Mandrel: It fits snugly within the
outer sheath allows to preshape the outer sheath thus
can negotiate the curvature of the endocervix espe-
cially for difficult ET (outer diameter—1 mm). • It is an open ended catheter with outer diameter of Cooks Catheter (Figs. 46.24A and B)
3. Inner sheath or transfer or loading catheter: It is a soft 1.6 mm.
• A double lumen catheter set
tube where the embryologist loads embryos. It has
• The “guiding catheter” is 19 cm long “outer catheter”
markings at distal end. Luer lock at the handle ensures Difference between Standard and
having a polycarbonate hub and a bulbar tip providing
secure syringe connection (outer diameter—1.6 mm) Sure View Wallace (Fig. 46.23)
• Total volume: 0.2 mL atraumatic passage through cervix. Its distal end is
• Sterilized with Gamma radiation. Small air bubbles present in the wall of inner sure view angled to facilitate the insertion.
catheter which has following advantages: • The “transfer catheter” is 23 cm long “inner catheter”.
Procedure • Visible under ultrasound from hub to tip Its base has attachment for 1.0 mL plastic syringe. Its
• The outer ET catheter is introduced through the cervix to • Easy tracking of direction of the catheter in the uterus tip is size 2.8 French.
stop short of the internal os under ultrasonography (USG) • Accurate placement of catheter and thus embryos in • “Microvol® technology” is for less medium volume
Fig. 46.21: Wallace catheter. the uterus. required for embryo transfer.
guidance, stabilized with help of hypothenar eminence
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428 The Infertility Manual Embryology Instruments 429
A B A B
Figs. 46.25A and B: Gynetics catheters. Figs. 46.28A and B: Towakos transmyometrial needle.
B
Figs. 46.29A and B: Embryo-loading techniques.
Fig. 46.26: Labotect catheters. Fig. 46.27: Frydman rigid catheter.
Gynetics Catheters (Figs. 46.25A and B) • Its unique curved stiff outer sheet with a ball-shaped Towakos Transmyometrial Needle Insulin Syringe (Fig. 46.31)
• It consists of a solid cervical catheter and a soft, flexible
spherical tip allows negotiation with difficult more (Figs. 46.28A and B) • Inner piston made of rubber can release hydrocarbons
easily compared to other soft ET catheters.
intrauterine catheter. Embryo-loading Techniques which can be toxic to embryos.
• Types: • Syringe requires flushing with 1 mL media to decrease
Frydman Rigid Catheter (Fig. 46.27) (Figs. 46.29A and B)
EMTRAC: Combination of soft and rigid catheters toxicity.
SEMTRAC: Soft catheters • The Frydman catheter is a polyethylene open ended • More controlled pressure application.
catheter with an outer diameter of 1.6 mm.
Nontoxic Syringe (Fig. 46.30)
TULIP: Soft catheters with bulb at tip.
• Its distal portion is soft (4.5 cm) and proximal portion • Nontoxic, two-piece syringe, 1 mL volume CULTURE MEDIA (FIG. 46.32)
is rigid (12.5 cm). It is graduated at 5.5 and 6.5 cm dis- • Quality control with MEA and LAL tests
Labotect Catheters (Fig. 46.26) tances from the tip. • Attached to embryo transfer catheter.
Types of Media
• Available in three lengths: 15 cm, 19 cm, and 23 cm • Disadvantages of rigid catheter: Bleeding , trauma, • Contains no natural rubber latex
• It is an intermediate catheter. uterine contractions, mucous plugging, and retained • Less chances of retained embryos 1. Flushing media
• It can be inserted without a malleable stylet embryos • Pressure less controlled. 2. Fertilization media
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430 The Infertility Manual Embryology Instruments 431
Table 46.2. Dish preparation: Day 0: ovum pick–up day.
Sr. No Dish No Media Oil overlay Use
1. Centre well (60 × 15 mm) 2 Fertilization media (1 mL per dish) No Incubation of oocvtes prior
to insemination (1–2 hour for
IVF/3–4 hour for ICSI)
2. Centre well with Lid 1 0.1 mL Hylase+1–2 mL MOPS in No Denudation f/b washing
centre well MOPS drops in lid
3. ICSI Dish (50 × 9 mm) 1 7% PVP, MOPS–8–10 μL drops Yes ICSI
4. Centre well or Nunc 4 1 0.5–1 mL fertilization media Yes Conventional iVF
well Dish
5. Culture Dish (30 × 15 mm) 1 Cleavage media 30–40 μL each Yes Post ICSI incubation, Fertilized
drop oocytes in IVF
Fig. 46.30: Nontoxic syringe. Fig. 46.31: Insulin syringe.
Fig. 46.33: Dish change.
After that it acts as an effective buffer to maintain pH • Hyalase 0.5–1 mL and 7% polyvinylpyrrolidone (PVP)
for up to 10 minutes (Conaghan, 2008). for both CO2 equilibration not required only tempera-
ture at 37°C 1 hour before the procedure (Table 46.3).
Dish Preparation
Day 0: Ovum Pick-Up Day (Table 46.2)
Table 46.3. Laboratory preparation.
Dish Change Flushing 8 mL–Clinician
Media 2 mL–Oocyte collection
• Day 1: Culture dish (35 × 10 mm) with cleavage media (15 mL) 3 mL–Sperm Washing
Fig. 46.32: Culture media. 30–40 μL drop size with oil overlay 0.9 to 1 mL–Denudation with Hylase
• Day 3 (or Morning Day 4): Culture dish (35 × 10 mm) 0.1 mL–ICSI dish
with blastocyst media 30–40 μL drop size with oil over- Fertilization 2 mL–2 Centre Well Dish (oocyte
3. Cleavage media
Oil lay (Fig. 46.33). Media (5 mL) preincubation)
4. Blastocyst media 1 mL–Semen preparation–Overlay
• Paraffin oil or silicon oil (dimethyl polysiloxane)
1–2 mL–Conventional IVF–Centre Well or
• All medias are supplemented with: • Nowadays ready to use washed, sterilized oil is Laboratory Preparation 4 well dish
Human serum albumin (5 mg/mL) available
• Mineral oil: 10 mL per patient Cleavage Culture dish post ICSI
Buffer • When the oil is exposed to direct sunlight for about Media
• Flushing medium: 15 mL per patient
Gentamicin as antibacterial agent (0.01 mg/ 4 hours, it turns highly embryotoxic. (Provo and Herr, (0.3–0.7 mL)
• Fertilization medium: 3–5 mL per patient
mL) 1998) Oil (10 mL) Culture dish, ICSI dish, Conventional IVF
• Cleavage medium: 0.3–0.7 mL per patient.
Cleavage and blastocyst media contain hyalu- • Once equilibrated, the oil acts as to maintain stable pH centre well or 4 well dish
ronan under normal culture conditions; there is a require- Pre-equilibrate overnight incubation at 37°C, 5–6% CO2 (pre incubation of oocytes and denudation
• Storage: At dark place 2–8 degree ment of minimum 8 hours of equilibration time for oil. (as per instructions in kit inserts) without oil overlay at our center)
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432 The Infertility Manual Embryology Instruments 433
Hyalase Three to four oocytes with 1,00,000 motile sperms
• 0.1 mL hyalase diluted with 0.9 mL of MOPS
[(3-(N-morpholino)propanesulfonic acid)] buffer: • Incubation for 17–18 hours
80 IU hylase (not more than 1 minute)
• 600–300 (Pasteur Pipette ) and 175–140 µm (Flexi
Pipette) • Day 1: Denudation and Fertilization Check
• Denudation is done after initial oocyte incubation for Without hylasme—with Flexi pipettes serially with
2–4 hours diameter 300 μm-200 μm-175 μm-140 μm
• Purpose: Transfer to culture dish (35 × 10 mm) cleav-
Assessment of nuclear maturity of the oocyte age media with oil overlay three to four fertilized
Proper handling during micromanipulation oocytes per drop (droplet culture)
• Denudation procedure: • Day 3 (or morning day 4): Transfer to blastocyst media
culture dish.
1. Culture dish: One drop of hyaluronidase and five A B
wash drops MOPS buffered medium, with an oil
Figs. 46.34A and B: Flexi pipettes.
overlay PLASTIC WARES IN IN VITRO
2. 30–60 minutes incubation at 37°C
FERTILIZATION LABORATORY
3. One to four oocytes are stirred together (< 1 min)
into the enzyme drop gently till the cells start to • Nonembryotoxic, nonpyrogenic, noncytotoxic properties
dissociate • Tissue culture treated for a consistent hydrophilic s urface
4. They are washed by transferring them through at • Sterilization by gamma irradiation
least five drops of culture medium and changed to • MEA, LAL, HSSA tested
a fine-bore tip (600–140 µm hand-drawn Pasteur • Polypropylene: Very strong, tough, resistant to chemi-
pipette) for in and out aspiration so as to remove all cals and temperature
of the coronal cells. • Polystyrene: High gloss and clarity, temperature sensi-
tive, e.g. culture dish
• Polyethylene: Inert to acid or alkali, ideal for handling
Conventional Insemination chemicals, e.g. pipettes
• Each oocyte incubated with 25,000–1,00,000 motile • Polycarbonate: Tough with optical clarity but porous
sperms: leads to solution loss, unbreakable.
For 12–18 h
At 37ºC Flexi Pipettes (Figs. 46.34A and B)
In 5% CO2 Fig. 46.35: Pippetter. Fig. 46.36: Pasteur pipette.
• Made up of polycarbonate
At 98% relative humidity
• They are flexible and resistant to breaks and scratches.
Fertilization medium
• Use: Denudation, gamete, and embryo manipulation • It gives the fine control required for oocyte, embryo, • Larger volume of fluid can be aliquoted compared to
Centre well dish or 4 well dish.
• Pipettes—120, 130, 140, and 170 µm inner diameter and/or blastomere manipulation. flexipet
• Blastomere or polar body manipulation: 80 µm diameter • Accessories: One small O-ring, one large O-ring, For semen liquefaction, semen analysis
Practical Steps in Conventional IVF • Manipulation of oocyte-cumulus complex: 600 µm spacer, and a collet For oil overlay, droplet culture, oocyte washing
diameter • Rotate the plunger clockwise to decrease aspiration • Have stems and bulbs in the form of a single piece
OCC preincubated for 1–2 hours in fertilization media in
centre well dish in CO2 incubator (without oil overlay). • Denudation: 135–140 µm diameter volume made of plastic (polystyrene/polyethylene).
• D3 embryo handling: 175 µm diameter • Rotate the plunger counterclockwise to increase aspi-
• Blastocyst handling : 270–300 µm diameter. ration volume (Table 46.4).
Table 46.4. Adjustment chart.
• Nunc 4 well dish: 0.5 mL fertilization media with oil
overlay per well
Pippetter (Fig. 46.35) Pasteur Pipette (Fig. 46.36) Total Capacity 3.0 μL
360 Rotation 0.2 μL
One oocyte with 25,000–1,00,000 motile sperms • It is adjustable handle used with all sizes of Flexipet for • Plastic Pasteur pipettes, also referred to as transfer
180 Rotation 0.1 μL
• Centre well dish: 0.5–1 mL fertilization media with oil denudation and manipulations of gametes/embryos. pipettes, to transfer or remove small amounts of liquid
overlay • Aspiration volume can be preset from 0.25 to 3.0 µL. from an experiment 90 Rotation 0.05 μL
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Fig. 46.37: Center well dish. Fig. 46.38: Round well dish. Fig. 46.39: Well dish. Fig. 46.40: ICSI dish.
• The plastic Pasteur pipettes come in 1–25 mL sizes • Purpose: To keep the dish in the incubator for at least 20–30 min-
• Glass pipette to handle oocyte cumulus complex for Denudation of oocytes utes (lids tightly fixed if CO2 incubator).
better visibility. Incubation of oocytes
For fertilization (IVF) Preparing ICSI Dish
For embryo culture
Petri Dish Cryoprocedures and washing embryos • Arrangement: PVP in the center and the droplets in
• Crystal-grade polystyrene for optical clarity • To be placed in CO2 incubator for equilibration with oil a circle or in parallel groups so as to allow quick and
• Hydrophobic polystyrene is rendered hydrophilic to overlay easy differentiation between sperm and oocyte drop-
• Advantage: Easy to screen oocytes as all in center, also lets. Number marking is done at the bottom of the dish.
support cell attachment and spreading by vacuum gas
saves media. • Droplets are not too close to the edge of the dish so that
plasma treatments (i.e. corona)
• Dishes are designed for optional gas exchange. manipulation becomes easy.
• Accidental lid opening of smaller dishes is prevented Round Well Dish (Fig. 46.38) • The proper distance is maintained between oocyte
droplets, sperm, and PVP to avoid mixing (Fig. 46.41).
by easy grip design • 35 × 10 mm, capacity—3 mL, growth area—11.78 cm2
• Sterilized by gamma irradiation • 60 × 15 mm, capacity—7 mL, growth area—21.29 cm2
• Easy gripping ring for convenience and secure For oocyte screening
Fig. 46.41: Preparing ICSI dish. MICROMANIPULATION
handling Rinsing of oocyte cumulus complex with culture
• Size selection depends upon primary purpose of media • Reduction and translation of coarse hand movement
culture Oocyte cumulus complex harvesting from follicular to microscopic movement at the level of egg or embryo
Embryo Culture
• Plastic dishes are cheap, disposable, and make lab less fluid • Goal: Efficient, smooth, and confident translation of
Cryoprocedure
labor intensive as no investment into autoclaves for Fertilization hand movement to the clearly visualized specimen
resterilization and special equipment for washing cul- Embryo culture, embryo washing (as level of precision needed cannot be achieved by
Intracytoplasmic Sperm Injection Dish unaided human hand)
ture glass ware. TESE/PESA sample collection
Cryoprocedures. (Fig. 46.40) • Five critical pieces of good micromanipulation system:
• 50 × 9 mm Petri dish (Falcon 1006) 1. Inverted microscope (clear visualization of micro-
Center Well Dish (Fig. 46.37) Well Dish (Fig. 46.39) • Central 7% PVP droplet of 4–5 μL is placed procedure to be attempted)
• Round with a flat bottom made up of polystyrene • Depth of microwell (1.45–1.55 mm); dimensions, • Central droplet can be straight or round (MOPS drops 2. Micromanipulator (smooth translation of movement)
• Size 60 × 15 mm 17 × 11 mm; volume, 1.85 mL below PVP in severe OATS) 3. Microscopic glass tools
• Center well 19 mm × 4 mm • Uses: • Peripheral eight droplets of 8–10 μL of MOPS media 4. Stereomicroscope (prepare eggs and embryos for
• Capacity: 2.5 mL Denudation are placed which contain oocytes manipulation)
• Growth area: 2.89 cm2 Insemination • Oil overlay (small volumes of media evaporate quickly). 5. Environment control (temperature/pH).
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Fig. 46.42: Holding pipette. Fig. 46.43: Injection pipette. Fig. 46.45: Polar body biopsy pipette. Fig. 46.46: Blastomere aspiration pipette.
A B
Fig. 46.47: Micropipette holder. Fig. 46.48: Compound microscope.
Figs. 46.44A and B: 35° angle at the distal end of microtools.
Blastomere Aspiration Pipette (Fig. 46.46) • Counting motile spermatozoa in final sperm prepara-
tion.
• Used for PGD to aspirate blastomeres and trophecto-
Microtools • There is bent at 35°angle at the distal end of these
derm cells
microtools. This allows in horizontal positioning Stereomicroscope (Fig. 46.49)
1. Holding pipettes: Used for to holding and immobiliza- • It has straight and smooth tip (flame-polished).
of microtools within culture dishes helping easy
tion the oocyte • Internal diameter: 30/35 μm, external diameter: • To see fertilization check
manipulations (Figs. 46.44A and B).
• Outer diameter: 0.080–0.150 mm 42/49 μm • Denudation
• Inner diameter: 0.018–0.025 mm • 35°angle at the distal end. • Dish change
• Its aperture is fire-polished (Fig. 46.42)
Polar Body Biopsy Pipette (Fig. 46.45) • Screening of oocytes.
2. Injection pipettes: Used for immobilization, aspira- • Used for preimplantation genetic diagnosis (PGD) to Micropipette Holder (Fig. 46.47)
tion, and injection of the sperm aspirate the polar body Inverted Microscope (Fig. 46.50)
• Outer diameter: 0.0068–0.0078 mm • It has beveled spiked tip for easy penetration through Microscopes
• Inner diameter: 0.0048–0.0056 mm zona pellucida. Micromanipulation
• It has bevelled tip. (Some have the spike at the tip.) • Internal diameter 20 μm, external diameter 28 μm and
Compound Microscope (Fig. 46.48)
“Reduction and translation of coarse hand movement to
(Fig. 46.43) 35°angle at the distal end • For semen analysis microscopic movement at the level of egg or embryo.”
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438 The Infertility Manual Embryology Instruments 439
Fig. 46.49: Stereomicroscope. Fig. 46.50: Inverted microscope. Fig. 46.52: RI integra micromanipulator. Fig. 46.53: Eppendorf micromanipulator.
Controller unit Contain one or more manual control Table 46.5. Incubators.
Temperature Humidity Contamination
Drive/receiver unit Contain microtool holder which is Gas type CO2 sensor O2 sensor controla Designb control controla,c
observed under high magnification optical lens of micro-
CO2–only Infrared Zirconium Air jacket Benchtop Yesd Heat
scope
Low O2–mixer Thermal Galvanic Water jacket Two-chamber No UV
conductivity (fuel-cell)
Micromanipulator Equipments
Low O2–permixed Direct heat Multichamber H2O2
• Inverted microscope with 4X ,10X, 20X, and 40X objec- cylinder
tives (integra micromanipulator (RI)—4X spacer for Other (i.e. time- Copper alloy
alignment) lapse imaging)
• Optical microscopy technique to enhance contrast: Small box External HEPA
Hoffman modulation contrast microscopy: To visu- Large box
alize the cells on plastic Petri dishes
Nomarski interference contrast (NIC) or Differen-
Fig. 46.51: Narishige micromanipulator. tial interference contrast (DIC): Uses polarized light Incubators (Table 46.5) • Decontamination cycles not possible
and glass dishes • Gas phase recovery compromised
Goal: Efficient, smooth, and confident translation of • Two coarse manipulators and two fine joysticks Types: • Maintenance of water jacket challenging.
hand movement to the clearly visualized specimen (as • Micro-syringes • Conventional box type-air jacketed, water jacketed, Air-jacketed incubators:
level of precision needed cannot be achieved by unaided • Camera attachment to optical outlet of the microscope CO2, or triple gas incubators • Warm up quickly but do not retain heat for long
human hand). for training purpose • Bench top incubators (Minc, K-systems) periods
• Mounted on anti-vibration table or anti-vibration base • Time lapse • Preservation of chamber temperature—not satisfactory
Five Critical Pieces of Good plate. • Transport incubators. • Decontamination cycles possible
Micromanipulation System Narishige micromanipulator Water-jacketed incubators: • Smaller in size
• Retain heat longer in case of incubator opening or • Maintenance easy
1. Inverted microscope (clear visualization of micropro- • Hydraulic and mechanical (Fig. 46.51)
power failure • Less contamination.
cedure to be attempted)
2. Micromanipulator (smooth translation of movement)
RI integra micromanipulator • Heavy Box type (Hera Cell) incubator (Figs. 46.54 and 46.55):
3. Microscopic glass tools • Mechanical (Fig. 46.52) • Higher power consumption which may burden emer- • Uses:
4. Stereomicroscope (prepare eggs and embryos for gency power supplies For media and oil equilibration
manipulation)
Eppendorf micromanipulator (Fig. 46.53) • Contamination may originate from inside the water Sperm and oocytes incubation
5. Environment control (Temperature/pH). • Motorized jacket PVP—heating for at least 1 hour.
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During this routine, a moist and wet atmosphere with
highly decontaminating effect is created for 9 hours at a
temperature of 90°C.
Bench top incubator (K system) (Fig. 46.56):
Bench top incubator (Minc) (Fig. 46.57):
• Rapidly equilibrate temperature and pH
• Heated chamber base plate and lid
• Compact size
• Digital record system for temperature and gas flow
(24 hour)
• Time-stamped alarms
• Physiological culture environment is maintained by
minimum premixed gas.
Advantages of bench top incubators: Fig. 46.56: Bench top incubator (K system). Fig. 46.57: Bench top incubator (Minc).
Fig. 46.54: Box type (Hera Cell) incubator—different capacities (150 L,
240 L). • Easy sterilization
• Less power consumption
• Cheaper to run than tri-gas big-box incubators
Embryoscope
ContraCon:
• Better embryo quality as compared to tri-gas big-box • It is tri-gas benchtop incubator incorporated camera
The ContraCon decontamination routine is used to decon-
incubators as it can achieve low pO2 culture easily. • It provides high quality images of developing embryo
taminate the complete work space including all installed
components and sensors. Time lapse imaging (Fig. 46.58): at all stages (Table 46.6).
OOCYTE AND EMBRYO
CRYOPRESERVATION
Vitrification Kit (Fig. 46.59)
• 1.5 mL basic solution (BS—1 vial—only for oocyte
vitrification)
• 1.5 mL equilibration solution (ES—1 vial)
Fig. 46.58: Time lapse imaging. • 1.5 mL vitrification solution (VS—2 vials).
Table 46.6. Embryoscope.
Embryoscope Primovision EEVA(Early embryo viability assay)
Integrated incubator Yes No No
Optics Bright field Bright field Dark field
Frequency of images Every 10 mins Every 10 Mins Every 5 mins
Focal planes 7 1 1
Capacity of PTS 6/System 1/Camera 1/Camera
No: Embryos/PT 12 Individual culture 16 Group culture 12 Group culture
Automatic DX tool No No Yes
Operator dependent Yes Yes No
Time consuming analysis Yes Yes Automatic
Selection algorythm User defined User defined Yes
Blastocyst prediction on Day 3 No No Yes (Wong et al 2010)
Fig. 46.55: Box type (Hera Cell) incubator. Implantation prediction on Day 3 Yes (Meseguer et al 2011) No No
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442 The Infertility Manual Embryology Instruments 443
Composition
• HEPES within basic culture medium
• Ethylene glycol
• Dimethyl sulfoxide
• Trehalose Fig. 46.62: Open and closed cooling.
• Hydroxypropyl cellulose
• Gentamicin.
Open Systems (Figs. 46.60 to 46.62)
Cryotop (Figs. 46.63A and B) Fig. 46.61: Open and closed cooling.
• Cryotop is the special vitrification container made
up of a fine, thin film strip attached to a hard plastic
handle.
Fig. 46.59: Vitrification kit. • It works on the principle of “minimum volume cooling”
to ensure highest cooling and warming rates providing
• Four Cryotops (one Cryotop can store four oocytes or almost 99% post-thaw survival.
embryos) • Polypropylene strips allow vitrification of oocytes and
• Two Repro plate. embryos with very small volume.
• The strip has a hard plastic cover which protects the
Storage oocytes and embryos from physical damage and virus
• Temperature: 4–8ºC for solutions and 15–30ºC for contamination during storage in liquid nitrogen. Fig. 46.64: Cryolock.
Cryotop and Repro plate • It is easy and safe universal system for vitrification.
B
Figs. 46.63A and B: Cryotop.
Cryolock (Fig. 46.64)
Fig. 46.65: Cryoloop.
Cryoloop (Fig. 46.65)
Cryoleaf (Fig. 46.66)
• Recommended to store infectious samples to prevent
Closed Systems cross contamination
• Made of ionomeric resin
High security straw (Fig. 46.67)
• Hundreds of straws can be stored in plastic goblets
• Storage and preservation of sperm • Length: 133 mm (after sealing, it becomes 130 mm)
• Safer than cryovials; do not crack or become brittle at • Sterilized by gamma radiation
Fig. 46.60: Vitrification protocol (embryo). subzero temp • Disadvantage:
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Fig. 46.66: Cryoleaf. Fig. 46.67: High security straw. Fig. 46.69: CVM ring fiber plug. Fig. 46.70: Rapid I.
• Tubes have a white marking area and can be color
coded with a CAPINSERT.
• They can be used along with most of the storage sys-
tems.
• Only the round bottom vials can be centrifuged and up
to 17,000 g.
• Sterilization by gamma radiation
• Packing: Tamperproof, resealable, safety-lock bags of
100.
Aluminum clips (Figs. 46.72A and B)
• To carry cryovials
• Disadvantage: Aluminum clips may give way and vials
may fall off the holder.
Fig. 46.68: Cryostraws. Fig. 46.71: Cryovials. Cryovial workstation rack (Fig. 46.73)
• Material: Polypropylene with three colors
• Easily autoclavable, compact, stackable rack provided
Capacity only 0.5 mL with filling difficulties • Sealing of straws: By ID-sticks, glass balls, or by using • A leak proof seal is maintained by a specially designed with antiskid rubber feet
Overfilled straw prone to expel powder sealing heat or ultrasonic sealing techniques. “lip” inside the cap. • Capacity: Up to 50 cryogenic vials. Size 10 cm ×
plugs into liquid nitrogen • As the lid and vials are both made up of “polypropyl- 20 cm × 2.5 cm
CVM ring fiber plug (Fig. 46.69) ene” with the same coefficient of expansion, the seal is • The vials are properly fitted in each well such that they
Difficult to label
Exposure to environmental temperature can result Rapid I (Fig. 46.70) secured equally both at room temperature and at low will not turn and the lid can be easily unscrewed with
in “warming shock damage” easily due to its high cryogenic temperatures. one hand.
Cryovials (Fig. 46.71) • An alpha-numeric index identifies location of each
surface to volume ratio. • Screw top vials are not liquid nitrogen proof. During
• Used for storage of biological material or cells, at tem- thawing, liquid nitrogen trapped in the vials expands Cryovial in the rack.
Cryostraws (Fig. 46.68)
peratures as low as -196°C which might lead to explosion of vials.
Capinsert for cryovial (Fig. 46.74)
• 0.25cc; length, 90 mm and 133 mm • They should be used only in the gas phase of liquid • They have thick wall and low surface to volume ratio
• For oocyte and embryo freezing nitrogen due to which there are high chances of damage even if • Used for color coding of Cryovials
• Made of clear PVC and presterilized by gamma radiation • Stores nearly 1.5 mL of semen plus cytoprotectant mix- transient exposure to environmental temperature and • Material: Polypropylene
• Can fit to the Cryologic as well as the Planer freezer ture. also it takes more time to reach to a critical temperature. • Easy and perfect fit on the cap of the Cryovial
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446 The Infertility Manual Embryology Instruments 447
A B A
Figs. 46.72A and B: Aluminum clips. Fig. 46.76: Cryo-gloves.
Fig. 46.77: Water bath.
Fig. 46.73: Cryovial workstation rack. Fig. 46.74: Capinsert for Cryovial.
• It does not stain but can dissolve dried blood stains.
• For use: 1:100 water dilution
• Shelf life: 3 years shelf life
Goblets and Visotubes IN VITRO FERTILIZATION • 3–6% H2O2 can also be used for disinfection.
(Figs. 46.75A to C) LABORATORY DISINFECTANTS
INSTRUMENTS FOR LABORATORY
• Microbiological efficacy: Bactericidal, fungicidal, and
Cryo-Gloves (Fig. 46.76) selective virucidal [human immunodeficiency virus QUALITY CONTROL
C
• Wrist, mid arm, elbow length, shoulder length (HIV), Hepatitis B virus (HBV)].
Figs. 46.75A to C: Cryo storage—goblets, visotubes & liquid nitrogen
Sensor for O2 measurement (Fig. 46.78)
tanks.
Water Bath (Fig. 46.77) Oosafe Flat Tape Sensor PT1000 (Fig. 46.79)
• A water bath is necessary for warming of media • It is nonvolatile, nonirritant “quaternary ammonium • It is odorless solution compatible to metal, rubber, and • For temperature measurement inside bench top incu-
• Water bath should be adjusted to 37°C and 56°C. compound” without alcohol and aldehyde. plastic bators
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Fig. 46.78: Sensor for O2 measurement. Fig. 46.79: Flat tape sensor PT1000. Fig. 46.82: Hot plate. Fig. 46.83: CO2 changer.
Fig. 46.80: Surface sensor for temperature measurement. Fig. 46.81: pH meter.
Fig. 46.84: CO2 regulator. Fig. 46.85: CODA tower.
Surface Sensor for Temperature CO2 Changer (Fig. 46.83)
Measurement (Fig. 46.80) • Automatically changes from A-cylinder to B-cylinder First valve—tank pressure
Second valve—pressure going to incubator (10 pascals).
Gas in Line Filter (Fig. 46.87)
when one becomes empty
pH Meter (Fig. 46.81) • CO2 gas indication in percentages • For the decontamination of the gases entering into the
• LED and BEEP sound indication, if any cylinders gets
CODA Tower (Carbon Activated Gas incubator
Hot Plate (Fig. 46.82) Filtration) (Fig. 46.85) • All organic contaminants of the gases are almost com-
empty.
• Temperature control between 27 and 45°C with sepa- pletely removed by activated carbon filters
rate control unit
CODA Unit (Fig. 46.86) • Filter unit has polyethylene layer providing dust and
• It provides planar surface for adequate heat transfer
CO2 Regulator (Fig. 46.84) • Remove Volatile organic compounds (VOCs) and particle free filtration
to heating blocks and culture dishes with homogene- • Double safety pressure value chemical air contaminants (CACs) presents in any • Compatible with all cell culture incubators
ous temperature distribution and short heating up • Pressure gauges with PSI/BAR readings: Helps to set incubator or laboratory • Replacement of the filter unit is recommended every
time. accurate pressure • Mounted into access hole of incubator. 3 months.
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450 The Infertility Manual Embryology Instruments 451
A B
Figs. 46.89A and B: Class I biosafety cabinet. (a) Front opening; (b) Sash; (c) Exhaust high-efficiency particulate air filter; (d) Exhaust plenum.
Fig. 46.86: CODA unit. Fig. 46.87: Gas in line filter.
Air Quality Analyzer (Fig. 46.88) • It provides personnel and environmental protection,
but not product protection.
• It can be used for the work involving low-to-moderate-
risk agents.
Class II (Fig. 46.90)
• It is open-front, ventilated cabinet.
• This cabinet provides a HEPA-filtered, recirculated
mass airflow within the work space.
• The exhaust air from the cabinet is also filtered by
HEPA filter.
• HEPA filters trap particulates and infectious agents effec-
tively but cannot filter out volatile chemicals or gases.
• It provides personnel, environment, and product pro-
tection. Fig. 46.90: Class II biosafety cabinet. Fig. 46.91: Class III biosafety cabinet.
• It is used for the safe manipulation of low-, moderate-,
and high-risk microorganism.
Fig. 46.88: Air quality analyzer.
• Divided into four types (Types A1, A2, B1, and B2).
Class III (Fig. 46.91) ANDROLOGY INSTRUMENTS
IVF WORKSTATION: BIOLOGICAL • Class III cabinet is a totally enclosed ventilated cabinet
Orchidometer (Fig. 46.92)
of gas-tight construction with negative air pressure of
SAFETY CABINET at least 0.5 inches water gauge. • To measure the volume of the testicles
Class I (Figs. 46.89A and B) • It is provided with attached rubber gloves for the • Discovered by Prof. Dr. HC Andrea Prader (Pediatric
manipulations. Endocrinologist) in 1966
• The class I biosafety cabinet is negative pressure cabi- • The cabinet exhaust air is filtered by two HEPA filters • It is made up of 12 wooden or plastic beads of increas-
net. The front portion is open. which are installed in series and then expelled outside ing size from about 1–25 mm
• High-efficiency particulate air (HEPA) filters are used through separate exhaust fan. • Prepubertal sizes are 1–3 mL
to filter exhaust air from the cabinet. The incoming air • It provides the highest level of personnel, environmen- Pubertal sizes 4 mL
is not filtered. tal, and product protection. Fig. 46.92: Orchidometer. Adult sizes are 12–25 mL.
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C HA PTE R
47
452 The Infertility Manual
Recent Advances in Infertility
Sachin A Narvekar
Chapter Outline
• Advances in Clinical Aspects • In Vitro Fertilization Laboratory Advancements
• Pharmaceutical Advancements • Advances in Other Aspects of Infertility
INTRODUCTION PHARMACEUTICAL ADVANCEMENTS
A B
• There have been a number of technological advances Long Acting Gonadotropin
in infertility managemet especially in the field of IVF.
for Ovarian Stimulation
Figs. 46.93A and B: Vacuum erection devices. • The newly introduced long acting gonadotropins
(Corifollitropin alfa) ensures better patient compli- • The concurrent use of gonadotropins along with gon-
ance but is contraindicated in hyperresponders. adotropin-releasing hormone (GnRH) agonists or
• The current benefit of in vitro maturation is unclear. antagonists for controlled ovarian hyperstimulation
• Time lapse imaging systems helps in assessment of has significantly increased the oocyte retrieval and
hence success rates per cycle.
morphokinetic behaviour of an embryo with mini-
• Recombinant gonadotropins have greater batch-to-
mal disturbance in its environment which helps in
batch consistency and a more predictable follicular
determining the best embryo for transfer. However its
growth.
cost and lack of universally adoptable algorithms for
• Corifollitropin alfa is one of the most recently intro-
embryo selection limits its use in routine practice.
duced classes of recombinant gonadotropin drug for
• Preimplantation genetic screening using CGH has
stimulation. This new molecule is a combination mol-
potential to improve efficiency of IVF by improving
ecule of human follicle stimulating hormone and the
implantation, decreasing miscarriage and multiple
carboxy-end of human chorionic gonadotropin. The
pregnancy rates.
pharmacologic activity of this molecule is similar to
• Technologies like microfuidics and metabolomics are
recombinant follicle-stimulating hormone (FSH). It
still in research phase but have a great potential in
gets absorbed slowly and has a longer half-life (2X)
future. than recombinant FSH (rFSH). It has no luteinizing
• Uterine transplantation is also in trial stage but has a hormone (LH) activity (Fig. 47.1).
Fig. 46.94: Vibrect vibrator. Fig. 46.95: Electroejaculator. potential to benefit women with absolute uterine fac- • It can sustain follicle growth for 1 week so a single
tor infertility. injection equivalent to 7 days of stimulation. The safety
profile of this drug has been confirmed in the clinical
Vacuum Erection Devices (Figs. 46.93A and B) • It is provided with “vibrating Softpads” for skin contact
ADVANCES IN CLINICAL ASPECTS trials. The dose depends on the weight of the patient.
delivering stimulation to the superficial nerve recep- It is 100 µg for women weighing less than 60 kg and
• Pump sucks out the air from the container to create a
tors located on dorsal and ventral surfaces of the penis. In the recent decades there have been number of advances 150 µg for women more than 60 kg.
vacuum, causing the blood to be drawn into specific
in the field of infertility particularly in the field of in vitro • Cochrane review1 of six RCTs involving 3,753 par-
tissues of the penis, causing erection.
fertilization. ticipants showed that this drug had similar live birth,
• Rubber band is then placed at the base of the erected Electroejaculator (Fig. 46.95) Since the birth of Lousie Brown about four decades clinical pregnancy, ongoing pregnancy, miscarriage,
penis to maintain the erection (must be removed
• A probe is inserted into the rectum next to prostate, and back, research has been focused on development of tech- and ovarian hyperstimulation syndrome (OHSS) rates,
within 30 minutes).
electric current delivered through the probe stimulates nologies to improve the outcome and safety of in vitro when compared to daily FSH.
the nerves causing the contraction of pelvic floor mus- fertilization. While some of these are being used in clinical • It was also noted that among women on corifollitropin,
Vibrect Vibrator (Fig. 46.94) cles leads to emission of semen practice, others are still in the trial phase. those who used lower doses (60–120 µg) had signifi-
• It is a hand-held tool for psychological and situational • 5–10 V, 200 mA current This chapter aims to review some of these latest cantly lower live birth rates as compared to medium
ejaculatory failure or erection difficulty. • For neurogenic causes of ejaculatory dysfunction. advances in this field. rates (150–180 µg).
Kamini_Ch46.indd 452 21-12-2017 20:32:38
454 The Infertility Manual Recent Advances in Infertility 455
including priming with hCG, FSH, and/or LH and • Fluctuations in temperature and pH secondary to correlated. Patient characteristics, stimulation meth-
specific changes in IVM oocyte culture media. These repeated displacement of embryos from the incubator ods, culture conditions, O2 concentration, and so on
modifications have resulted in several relatively small can be detrimental to the embryo. can all influence embryo development and as a result
trials that reported IVM pregnancy rates approaching, • Also, embryos are exposed to large quanta of light that the kinetic markers. Therefore, when one group rec-
though still lower than IVF cycles. IVM does not appear is harmful. ommends an algorithm as a model to predict implan-
to introduce more risks, such as imprinting defects, as • A qualitative morphology observation of an individual tation or pregnancy, a different group in a different
compared to IVF cycles. embryo along with monitoring of the dynamics in the patient population using different culture conditions
• The chief benefit of IVM is often cited as a technol- embryo development is referred to as morphokinetic may not find the results helpful. In order to accept and
ogy that could eliminate the risk of OHSS in polycystic study. The kinetic behaviour of a normal and an abnor- introduce an algorithm in routine practice, it has to be
ovary syndrome (PCOS) patients. However, the recent mal embryo is different. This can be spotted with time tested in a different clinical setting and should be able
introduction of GnRH antagonist cycles utilizing a lapse technology. to reproduce the expected results. Currently a univer-
GnRH agonist trigger and the increased use of mild • Time lapse technology has enabled the embryolo- sally acceptable algorithm is yet to be developed.
gonadotropin stimulation protocols offer the ability to gist to study embryo development in more detail One of the earliest models was proposed by Meseg-
reduce or eliminate the risk of OHSS in otherwise con- and select the embryo with the highest implantation uer et al.5
ventional IVF cycles. potential. Here magnified images of embryos are taken
Fig. 47.1: Treatment regimen of corifollitropin alfa in comparison to Below is the summary of the Meseguer model:
• Other investigators have put forth IVM as a possi- at set time intervals, which can be played back as a TL
daily recombinant follicle-stimulating hormone. • Parameters correlated with improved implantation rates:
ble treatment for women who have had suboptimal sequence to study the development of the embryo.
responses to traditional IVF cycles. The benefit of IVM Early appearance of 2 PN: 5–17 hours post-ICSI
Benefits of Corifollitropin Alfa in today’s current clinical environment is unclear and Time Lapse Imaging systems currently available Early disappearance 20–25 hours post-ICSI
will need to be further explored. • Embryoscope Time to three cells (t3) between 35.2 and 40.5 hours
• One injection replaces seven daily injections and time to become five cells (T5) 48.5–56.6 hours
• Fertilitech Inc
• Patient friendly treatment, better compliance
• Lower potential for medical errors. Technologies Evaluating Embryos • Primovision system (Vitrolife) T5 was the best single parameter
• Eeva. First cleavage before 32 hours; second and third
A significant challenge in ART has been and continues to cleavage before 42 hours
Drawbacks be determining which embryos in a given IVF cycle are Potential advantages of time lapse imaging Second cell cycle (cc2 = t3–t2 represents duration of the
• It is contraindicated in high responders as there is high optimal for uterine transfer. Traditionally, embryo mor- • Important milestones such as pronuclear formation, period as two-blastomere embryo) less than 12 hours
risk of OHSS. phology has been the most utilized method of determining syngamy, cleavage events, synchronicity of cell divi- S2 represents transition from two to four blasto-
• Dosage adjustment cannot be made in the first 7 days embryo quality, but morphology alone has been shown to sion, cell cycle intervals, and initiation of blastulation mere embryo (S2 = t4–t3) less than 0.67 hours
in case the response is poor or higher. be a suboptimal indicator of determining which embryos can be studied. These can assist in selecting the best Direct cleavage from zygote to three blastomere
Available as prefilled syringes with 100 and 150 µg. have normal chromosomal status (euploidy) or optimal embryo for transfer. Conventional morphology assess- embryo (cc2 = t3–t2 <5 hours)
implantation potential. For this reason, different modali- ment can sometimes miss the early PN disappearance • Parameters correlated with poor implantation rates:
IN VITRO FERTILIZATION LABORATORY ties have been developed to assess the embryo quality which can lead to discard of diploid zygote. TL can Uneven blastomere size at two cell stage during
both noninvasively and invasively.
ADVANCEMENTS identify this and avoid this mistake. interphase where nuclei are visible and multinu-
• Time lapse improves the sensitivity for detecting mult- cleation at four cell stage.
Probably, the single most significant factor in the dra- Noninvasive Embryo Selection inucleation which is a transient occurrence.
matic improvement in IVF pregnancy rates over the past • Time lapse (TL) imaging • The kinetic and morphologic parameters obtained
10–15 years has been the technological modifications • Secretomics and metabolomics. through TL could be used to build algorithms that can Definitions of kinetic TL parameters.
in the embryology laboratory. Some of the important t0: time of insemination during IVF or mid-time of injection of the
help to choose the fittest embryo for transfer.
advances are discussed below: Time Lapse Imaging oocyte cohort during ICSI
Embryo selection for transfer is the most critical step dur- Time lapse-based algorithms and their clinical utilities ICSI: intra-cytoplasmic sperm injection
In Vitro Maturation • Many morphokinetic parameters have been tested in rela-
tPNf: time to pronuclear fading
ing an IVF procedure. t2, t3 . . . t8: time to the 2-, 3- . . . 8-cell stage
• The ability to mature oocytes in a laboratory environ- tion to a variety of laboratory (e.g. blastocyst development)
Drawbacks of conventional methods tx-ty: time between the x-cell to y-cell stage
ment, in vitro maturation (IVM), is a technology that is and clinical (implantation and livebirth rate) outcomes.
of embryo assessment S1: time from appearance of the cleavage furrow to the completion
being currently perfected by many centers. of first cell division
Researchers have attempted to build algorithms using
• The IVM is the practice of retrieving immature human • These are based on the static observations and assess- S2: time from the 3- to 4-cell stage
the TL morphokinetic parameters so as to identify the
oocytes which then complete the transition from pro- ment of morphologic parameters at specific times dur- S3: time from the 5- to 8-cell stage
embryos with a higher chance to implant.
phase I to metaphase II, including extrusion of the ing the day. tM: time to morula stage
first polar body, in vitro.2-4 Over the past decade or so, • These assessments are subjective, and have large flaws • However, there are many potential confounders when tSB: time to start of blastulation
there have been many modifications in IVM protocols such as inter- and intraobserver variation. morphokinetic parameters and clinical outcome are tEB: time to expanded Blastocyst
456 The Infertility Manual Recent Advances in Infertility 457
External validation of the algorithms diseases while PGS is used as a screening test to look Advantages of Comparative transfer was compared to double untested embryo
for abnormalities in chromosome number or size. The transfer. But it was noted that obstetrical outcomes
• Kahraman et al.6 tested the Meseguer hierarchical
human reproduction is highly inefficient, producing
Genomic Hybridization in the former group were better as it avoided multiple
model (based on t5, S2, CC2) in a small randomized
high percentages of aneuploid embryos even at young • Entire chromosome set can be analyzed without the pregnancies. Another retrospective study18 concurred
controlled trial (RCT) among good prognosis patients.
ages.13,14 These aneuploid embryos are frequently non- need for cell fixation. with this study.
No difference with elective single embryo transfer
viable, leading to implantation failure, miscarriages, or • The CGH may be able to detect whole chromosome • Subsequently, two meta-analyses19,20 of the stud-
using either conventional or TL incubators was noted.
congenital abnormalities. These aneuploid embryos abnormalities as well as unbalanced translocations of ies demonstrated that adoption of CCS-based PGS
• Rubio et al.7 found lower miscarriage rate and signifi-
can be picked up by PGS. at least 10–20 Mb in size. resulted in a higher implantation rate per transfer, as
cantly improved ongoing pregnancy rate in the TL arm
• In the initial years, PGS was done with blastomere • Array comparative genomic hybridization (aCGH) can well as lower miscarriage rate than controls.
using the same model.
aspiration of embryos on the 3rd day after fertilization, be completed within 10–18 hours. • Although these studies support the use of PGS, it should
• Other studies8,9 could not find any difference in the pro-
followed by fluorescence in situ hybridization (FISH) • These are now the methods of choice for most clinics be pointed that the RCTs published were mostly done
portion of the good quality day 2 embryos and clinical
analysis of the aspirated blastomere(s). PGS helped to undertaking PGS. Another method, next generation in a patient with good prognosis.
outcomes when the embryos were grown in TL incuba-
identify embryos with aneuploidy and allowed transfer sequencing (NGS) may offer more potential advan- • Multicentre RCTs comparing PGS to the standard care,
tors when compared to conventional incubators.
of only euploid embryos thus eliminating pregnancy tages including lower cost, reduced time, and higher whose primary outcome should be the delivery rate per
Time lapse and aneuploidy prediction models loss due to aneuploidy. With this underlying principle, chromosomal analysis resolution. intention to treat, are still eagerly needed in this field.
• Campbell et al.10 showed that late kinetic events (time PGS was adopted by IVF centers worldwide with the
The group of patients that have been suggested to
to start of compaction, time to start of blastocyst for- hope that it would increase the live birth rates.
potentially benefit from PGS were: Summary of Evidence
mation, and time to blastocyst formation) were all • Conversely, ensuing RCTs showed that instead of
enhanced IVF outcomes, FISH based PGS decreased • Infertile women of advanced maternal age (AMA; usu- • FISH-based PGS on day 3 embryos is not recom-
delayed in aneuploid embryos. Early markers, how- mended as it is associated with decreased live birth
probability of ongoing pregnancy when compared ally defined as ≥ 35 years)
ever, failed to correlate with genetic health. rate
with IVF without PGS. • History of recurrent pregnancy loss (RPL; usually at
• In a subsequent study, they tested the predictive • PGS is a testing approach. It does not modify the
least three previous miscarriages)
ability of time intervals (time to start of blastulation
Limitations of Fluorescence In Situ • History of repeated implantation failure (RIF; three or genetic composition of the embryos. PGS as such
(96.2 hours) and time to full blastocyst formation does not improve live birth rate per retrieval but has
(122.9 hours) and proposed low-, medium-, and high- Hybridization Analysis more failed embryo transfers)
• Severe male factor a potential to increase efficiency of IVF by improving
risk categories for aneuploidy based on them. • The generally used FISH analysis can only scrutinize embryo selection and thereby increase implantation
• Basile et.al.11 found t5–t2 and t5–t3 as the most reli- 9–12 chromosomes. Therefore, abnormalities linked to The embryo biopsy is now routinely being done on rate, decrease miscarriage rate, and multiple preg-
able parameters to predict euploidy and built a model the nontested chromosomes (25–30%) will be missed day 5 as damage to the embryo is more with day 3 embryo. nancy rates.
using in and out of ranges of these parameters to pre- and thus lowering the chance of live birth. Also day 5 embryo has lower incidence of mosaicism • Although PGS for patients of AMA has been shown to
dict healthy chromosome content. • Technical limitations are cell fixation and overlapping as low-quality embryos with post-zygotic/mitotic chro- improve in vitro fertilization outcomes in some stud-
signals. mosome errors will not develop to the blastocyst stage.
Summary of evidence ies, to our knowledge, there is not sufficient evidence
• This has led to the introduction of a novel approach Instead of one to two blastomere(s), three to six trophec- to use AMA as the sole indication for PGS. At the same
• Currently there is no good evidence that TL system toderm cells are removed. Thus currently instead of using
recently that involves trophectoderm biopsy at the time, PGS is not recommended for recurrent implanta-
improves live birth rates, safety, and cost effectiveness. 9- to 12-chromosome FISH, a 24-chromosome detection
blastocyst stage associated with comprehensive chro- tion failure and unexplained RPL.
• The initial optimism regarding the benefits of these by microarray CGH is used.
mosome screening (CCS) techniques.
models was overshadowed by the lack of ability to It should be clearly advised that before adopting PGS,
externally validate them. Microfluidic Technology
Comprehensive Chromosome Screening- the laboratory should be able to establish an extended
• According to Cochrane review,12 there is no difference embryo culture to day 5 or day 6. Improving culture conditions has been the central topic
in live birth, miscarriage, and stillbirth rates in trials based Preimplantation Genetic Screenings in assisted reproduction. It is well known that changes in
comparing TL systems to conventional incubation. • It can be performed through different methods. Clinical Studies on Preimplantation culture conditions like shifts in temperature, osmotic, and
• Further studies comparing the incubation environ- • The DNA is first amplified with multiple displace- pH fluctuations can be detrimental effect to the develop-
ment, the algorithm for embryo selection, or both, ment amplification (MDA), polymerase chain reaction
Genetic Screening ing embryo. Recently, attention is being focused on the
are required before adopting this system in routine (PCR), or targeted multiplex PCR. The amplified DNA • In a review published in 2015,15 Lee and colleagues platform on which gametes and embryos are cultured and
practice. is then analyzed using techniques like comparative demonstrated that CCS-based PGS resulted in a higher manipulated.
Invasive Assessment of Embryo genomic hybridization (CGH), single nucleotide poly- delivery rate per embryo transferred when compared The term “microfluidic” refers to the characteristic
morphism (SNP) arrays, next generation sequencing to the standard care, in both young and AMA patient streamlined fluid movement in microenvironment with
Preimplantation Genetic Screening (PGS) (NGS)-based CCS, or qPCR-based CCS. Among these populations. predictable flow patterns. These flow patterns allow a
• Preimplantation genetic diagnosis (PGD) is used to methods, array CGH was the first technology to be • Forman and colleagues16,17demonstrated no differ- laminar flow of media in parallel micro channels, with no
spot IVF embryos that are affected by specific inherited extensively used and validated. ence in live birth rates when single euploid blastocyst mixing, except by diffusion across the fluid-fluid interface.
458 The Infertility Manual Recent Advances in Infertility 459
These flow patterns of media can be used to introduce Secretomics and Metabolomics platforms have been shown in some studies to predict This procedure is still at its early trial stage and has the
sperm to egg or remove cumulus cells from eggs or to embryonic implantation potential.32 potential to benefit about 1.5 million women worldwide
change the medium surrounding an embryo as it grows. • Secretomics and metabolomics offer another strategy with absolute uterine factor infertility.
The volume of media needed with this technology would of determining which embryos are optimal to consider Noninvasive Sperm Selection
for uterine transfer.23,24
be greatly reduced thus decreasing the cost per IVF case.
• These are noninvasive technologies which attempt to Successful human reproduction relies partly on the inher- SUMMARY
The material used to produce microfluidic devices for
determine information about embryos from byprod- ent integrity of sperm DNA. Sperm DNA damage is asso- The field of ART is highly innovative. In future, with the
IVF is polydimethylsiloxane (PDMS) because it is non-
ucts that can be measured in the media culture fluid ciated with a significantly increased the risk of repeated advancement of technology we will be able to move closer
toxic, transparent, insulating, and permeable to gases,
surrounding the developing embryo.23 failures and increased pregnancy loss after IVF and ICSI. to nature and witness improved pregnancy rates with min-
hence ideal for embryo culture. This material is easily
• Secretomics is the evaluation of specific protein pro- Two new technologies have been developed as methods imal side effects at affordable costs.
moldable and can be adhered to glass chips as necessary
files found in the media culture fluid surrounding the capable of identifying a spermatozoon with low DNA
for some designs.
fragmentation.
developing embryo.23 While a variety of methods have PROBABLE QUESTIONS
Types of Microfluidic Devices Being been used in the past, mass spectrometry using sur- 1. Ultrastructural morphology sperm selection at high
face-enhanced laser desorption/ionization coupled to 1. Newer drugs in assisted reproductive technology.
magnification (IMSI)
Researched time-of-flight analysis is the most commonly utilized 2. Recent advances in noninvasive embryo selection.
2. Use of hyaluronic acid (HA) binding for sperm selec-
• Gravity driven passive flow devices modality currently to perform embryo secretomics.24-26 3. PGS: advantages and disadvantages.
tion for ICSI.
Specific protein profiles evaluated using this process 4. Describe microfluidics.
• Programmable infusion pump
has been shown by some investigators to be predictive The first technology has been discussed elsewhere. In
• Braille pin type computer controlled pumping system:
In this, tiny electric piezo actuators are used to move of implantation and pregnancy potential. Some of these this chapter, we will discuss use of HA for sperm selection. REFERENCES
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8. Park H, Bergh C, Selleskog U, Thurin-Kjellberg, Lundin K. No
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• Microfluidic culture systems have a great potential to however, significant advancements utilizing metabo- were performed.38 So far, four healthy babies have been rates? A randomized controlled trial. Fertil Steril.
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26. Katz-Jaffe MG, Schoolcraft WB, Gardner DK. Analysis of
protein expression (secretome) by human and mouse ART Guidelines
2013(b);27:140-6. preimplantation embryos. Fertil Steril. 2006;86:678-85.
11. Basile N, del Carmen Nogales M. Increasing the probability 27. Dominguez F, Gadea B, Esteban FJ, Horcajadas JA,
P Jyothishmathi Sharma, Manishi Mittal, Divyashree PS, Kamini A Rao
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12. Armstrong S, Arroll N, Cree LM, Jordan V, Farquhar C. Hum Reprod. 2008;23:1993-2000. Chapter Outline
Time-lapse systems for embryo incubation and assessment 28. Noci I, Fuzzi B, Rizzo R, Melchiorri L, Criscuoli L, • Various Guidelines over the Years • Semen Donors
in assisted reproduction. Cochrane Database Syst Rev. Dabizzi S, et al. Embryonic soluble HLA-G as a marker • Current ART (Regulation) Bill, 2014 • Cryopreservation
• Regulations with Respect to Surrogacy • Donors and Surrogates
2015;(2):CD011320. of developmental potential in embryos. Hum Reprod.
• Surrogacy (Regulation) Bill, 2016 • Recommendations
13. Fragouli E, Alfarawati S, Goodall NN, Sánchez-García JF, 2005;20:138-46.
Colls P. The cytogenetics of polar bodies: tnsights into 29. Navarrete Santos A, Tonack S, Kirstein M, Kietz S, Fischer B.
female meiosis and the diagnosis of aneuploidy. Mol Hum Two insulin responsive glucose transporter isoforms and
Reprod. 2011;17(5):286-95. the insulin receptor are developmentally expressed INTRODUCTION published the National Guidelines for Accreditation,
14. Kuliev A, Cieslak J, Verlinsky Y. Frequency and distribution in rabbit preimplantation embryos. Reproduction. Supervision and Regulation of ART Clinics in India as
of chromosome abnormalities in human oocytes. Cytogenet 2004;128:503-16. Infertility is emerging as a major health and social concern National Guidelines of Government of India in 2005.2
Genome Res. 2005;111(3-4):193-8. 30. Lipari CW, Garcia JE, Zhao Y, Thrift K, Vaidya D, in modern day India. According to the WHO, overall prev-
• Since these National Guidelines were not being appro-
15. Lee E, Illingworth P, Wilton L, Chambers GM. The clinical Rodriguez A. Nitric oxide metabolite production in the alence of primary infertility in India has been estimated to
priately followed, the ICMR formulated the draft ART
effectiveness of preimplantation genetic diagnosis for human preimplantation embryo and successful blastocyst be between 3.9 and 16.8%.1 Consequently, there has been
aneuploidy in all 24 chromosomes (PGD-A): systematic formation. Fertil Steril. 2009;91(4 Suppl):1316-8. (Regulation) Bill in 20083 which was again subjected to
a mushrooming of centers providing assisted reproduc-
review. Hum Reprod. 2015;30(2):473-83. 31. Nel-Themaat L, Nagy ZP. A review of the promises and extensive public debate.
tion throughout the country. Due to the great diversity in
16. Forman EJ, Hong KH, Ferry KM, Tao X, Taylor D, Levy B, pitfalls of oocyte and embryo metabolomics. Placenta. • Thereafter, the ART (Regulation) Bill was revised and
management protocols and absence of standard operating
et al. In vitro fertilization with single euploid blastocyst 2011;32(3):S257-63. finalized in 2010.4 The 2010 bill has now been revised
transfer: a randomized controlled trial. Fertil Steril. 32. Seli E, Sakkas D, Scott R, Kwok SC, Rosendahl SM, procedures, there is a necessity to develop country specific
guidelines for assisted reproduction. There is also a need by the Ministry of Law and Justice as Assisted Repro-
2013;100(1):100-7. e1. Burns DH. Noninvasive metabolomic profiling of
17. Forman EJ, Hong KH, Franasiak JM, Scott Jr RT. Obstetrical embryo culture media using Raman and near-infrared to curb unethical practices. An attempt has been made by ductive Technology (Regulation) Bill—2014.5
and neonatal outcomes from the BEST Trial: single embryo spectroscopy correlates with reproductive potential of Indian Council of Medical Research (ICMR) in this regard • Surrogacy (Regulation) Bill was passed in 2016.6
transfer with aneuploidy screening improves outcomes embryos in women undergoing in vitro fertilization. Fertil and a draft of assisted reproductive technology (ART) bill This bill prohibits all forms of commercial surrogacy
after in vitro fertilization without compromising delivery Steril. 2007;88:1350-7. was introduced for consideration in 2014. However, it is (Table 48.1).
rates. Am J Obstet Gynecol. 2014;210(2):157 e1-6. 33. Cayli S, Jakab A, Ovari L, Delpiano E, Celik-Ozenci C,
important that adequate care is taken so that both patients
18. Ubaldi FM, Capalbo A, Colamaria S, Ferrero S, Maggiulli R, Sakkas D, et al. Biochemical markers of sperm function:
Vajta G, et al. Reduction of multiple pregnancies in the male fertility and sperm selection for ICSI. Reprod Biomed and health workers mutually benefit from ART.
Table 48.1. Salient features of guidelines and bills
advanced maternal age population after implementation Online. 2003;7:462-8. over the years
of an elective single embryo transfer policy coupled with 34. Huszar G, Celik-Ozenci C, Cayli S, Zavaczki Z, Hansch E, VARIOUS GUIDELINES OVER THE YEARS Year Salient features
enhanced embryo selection: pre- and post-intervention Vigue L. Hyaluronic acid binding by human sperm indicates
2005 •• First time guidelines were formulated
study. Hum Reprod. 2015;30(9):2097-106. cellular maturity, viability, and unreacted acrosomal status. Development of Guidelines •• Sperm donor 21–45 years; oocyte donor
19. Dahdouh EM, Balayla J, Garcia-Velasco JA. Comprehensive Fertil Steril. 2003;79(Suppl 3):1616-24.
chromosome screening improves embryo selection: a 35. Huszar G, Jakab A, Sakkas D, Celik-Ozenci C, Cayli S, • The ICMR proposed “National Guidelines for Accredi- 18–35 years
meta-analysis. Fertil Steril. 2015;104(6):1503-12. Delpiano E, et al. Fertility testing and ICSI sperm selection tation, Supervision & Regulation of ART Clinics in •• Allows both commercial and altruistic surrogacy
20. Chen M, Wei S, Hu J, Quan S. Can comprehensive chromosome by hyaluronic acid binding: clinical and genetic aspects. India” in 2002. This draft document then underwent 2008 •• Legally enforceable surrogacy agreement
screening technology improve IVF/ICSI outcomes? A meta- Reprod Biomed Online. 2007;14:650-63.
extensive public debate (seven cities were chosen— •• Oocyte donor—up to six times
analysis. PLoS One. 2015;10(10):e0140779. 36. Jakab A, Sakkas D, Delpiano E, Cayli S, Kovanci E, Ward D, •• Surrogacy—maximum thrice
New Delhi, Jodhpur, Mumbai, Bengaluru, Chennai,
21. Nir A. The fertilization dance: a mechanical view of the egg et al. Intracytoplasmic sperm injection: a novel selection •• Semen donor—maximum 75 times
rotation during the initial spermatozoa-ovum interaction. J. method for sperm with normal frequency of chromosomal Hyderabad, and Kolkata). The participants were given
2014 •• Age to avail ART services: women,
Theor. Biol. 2002;214:171-9. aneuploidies. Fertil Steril. 2005;84:1665-73. a prescribed proforma to enter their opinion (85% gen-
23–50 years; men, 23–55 years
22. Croxatto HB. Physiology of gamete and embryo transport 37. Parmegiani L, Cognigni GE, Bernardi S, Troilo E, eral public, 13% Indian doctors, and 2% international •• Only married infertile couples
through the fallopian tube. Reprod Biomed Online. Ciampaglia W, Filicori M. “Physiological ICSI”: doctors). •• Oocyte donor: only once in a lifetime.
2002;4:160-9. Hyaluronic Acid (HA) favours selection of spermatozoa • Based on this survey, along with comments and sug- Age 23–35 years
23. Fechner AJ, McGovern PG. The state of the art of in vitro without DNA fragmentation and with normal nucleus, gestions from the National Commission for Women •• Surrogate: only once in a lifetime
fertilization. Front Biosci (Elite Ed). 2011;3:264-78. resulting in improvement of embryo quality. Fertil Steril. •• Semen donor: maximum 25 times
and National Human Rights Commission, the National
24. Katz-Jaffe M, Gardner DK. Can proteomics help to shape 2010;93(2):598-604. •• Ban on commercial surrogacy for foreigners
the future of human assisted conception? Reprod Biomed 38. Brännström M, Johannesson L, Bokström H, Guidelines were finalized.
• The Ministry of Health and Family Welfare exam- 2016 •• Complete ban on commercial surrogacy
Online. 2008;17:497-501. Kvarnström N, Mölne J, Dahm-Kähler P, et al. Livebirth
ined these guidelines and after slight modifications •• Infertile couple married for at least 5 years
25. Katz-Jaffe MG, Gardner DK, Schoolcraft WB. Proteomic after uterus transplantation. The Lancet. 2014;385:
analysis of individual human embryos to identify novel 607-16.
462 The Infertility Manual Critical Analysis of the Current ART Guidelines 463
CURRENT ART (REGULATION) • To assist the State Boards in accreditation and regula-
tion of services, staff, and physical infrastructure of
BILL, 2014
ART Clinics and Banks
Chapter 1: Preliminary • To make regulations regarding permissible ART proce- Chairperson
dures and selection of patients
Definitions • Encouragement and promotion of training and
research in the field Representatives (Joint Secretary)
Definitions are given in Table 48.2.
• Regulation of third party reproduction including coun- 1. Department of Health Research
The ART Bill says no ART procedure shall be per-
seling of potential surrogate mother and oocyte donor 2. Ministry of Overseas Indian Affairs-Member, ex officio;
formed below the age of 23 years. This needs to be modi-
(possible long-term effects, psychological risks, and nominee of an Indian professional society-assisted
fied in case individuals are diagnosed to have conditions
vulnerabilities and possible effects on their existing reproduction-Member;
like azoospermia, cancers, etc.
relationship and children) a nominee of an National Commission for Woman-Member;
• Regulation of dissemination of information related to a nominee of National Commission for Protection of Child Rights-Member;
Chapter 2: Authorities to Regulate ART infertility and ART to the society a nominee of Medical Council of India–Member;
• Regulation of consents and records to be kept by the other expert members, not exceeding fifteen
National Board clinics and banks.
Members of National Board are shown in Figure 48.1.
• Functions of the National Board
State Board Maximum 23 members, At least 6 women, Term-3 years, <70 years of age
Board shall hold at least 3 meetings in a year
• To develop new policies in the area of ART Members of State Board are shown in Figure 48.2.
Fig. 48.1: Members of National Board.
Table 48.2. Important definitions and comments.
Term Definition Critical analysis
Infertility Inability to conceive after at least 1 year of unprotected Fixing a time limit of 1 year is not appropriate for
coitus or an anatomical or physiological condition that all cases. The American Society for Reproductive
would prevent a couple from having a child. Medicine (ASRM) recommends earlier evaluation
in older women7 (after 6 months if age >35 years Chairperson
or immediate if age >40 years).
Assisted All techniques that attempt to obtain a pregnancy by Intrauterine insemination (IUI) would also involve
Reproductive handling or manipulating the sperm or the oocyte out- manipulation of gametes outside human body. Representatives (Joint Secretary)
Technology side the human body and transferring the gamete or the Clinics performing IUI should also be registered as nominee from National registry;
embryo into the reproductive tract of a woman. ART clinics.
nominee of an Indian professional society-assisted
ART Bank Organization that is set up to supply sperm or semen, ART clinic cannot be ART bank. reproduction–Member;
oocytes or oocyte donors, and surrogate mothers to the Clinics previously recruiting semen donors have to
a nominee of an National Commission for Woman–Member;
assisted reproductive technology clinics or their patients depend on ART banks for donor semen.
a nominee of National Commission for Protection of Child Rights–Member;
ART Clinic Premises, other than the clinics of AYUSH System of No clear directions on minimum requirements, a nominee of Medical Council of India–Member;
medicine, equipped with the requisite facilities for carry- especially of laboratory differences between other expert members, not exceeding ten
ing out the procedures related to the assisted reproduc- centers carrying out IUI and centers with IVF
tive technology. facilities not mentioned.
Couple Relationship between a male person and female person No ART services for single parents, homosexu- Maximum 16 members, At least 4 women; Board shall hold at least 3 meetings in a year
who live together in a shared household through a rela- als single foreign individuals can adopt an Indian
tionship in the nature of marriage which is legal in India. child, but single Indian parent cannot avail ART.
National An Institution which shall be established under section National Registry should be set up immediately Fig. 48.2: Members of State Board.
Registry 18 at Indian Council of Medical Research, New Delhi to curb the malpractices with respect to multiple
and shall act as central data base of all the Assisted oocyte and semen donations at various centers.
Reproductive Technology Clinics and Banks in India Why should the National Registry be under ICMR
Chapter 3: Procedure for Registration application for registration as an assisted reproductive
and helping the State Boards and National Board technology clinic or assisted reproductive technology
in accreditation, supervision, and regulation of the
as ART procedures are no longer research? and Complaints bank under this Act
assisted reproductive technology clinics and banks in • Within a period of 90 days from the date of constitution • Apply to the state board
country and help in policy making, respectively. of the Registration Authority under this Act, make an • Registration can be issued or rejected within 90 days
464 The Infertility Manual Critical Analysis of the Current ART Guidelines 465
• Valid for 3 years Critical Analysis preimplantation genetic diagnosis (PGD), shall be Critical Analysis
• Need to submit the copies of certificates of all the per- done only when the embryo suffers from preexist-
sons employed While uploading the results would help us know the out- ing, heritable, life-threatening, or genetic diseases. Aadhar card has been made voluntary. Hence other proof
comes at each individual clinics and also the number of of identification is to be used
positive outcomes from a particular donor, especially
Critical Analysis semen donors, it is unclear as to where should the details
Critical Analysis • In case of death or disability of the oocyte donor, it
shall be presumed to be caused by the negligence of
No specific mention of qualifications of treating doctors be uploaded. Research should be allowed on any embryo donated for the ART clinic unless proven otherwise.
and embryologists. such purposes and not only on embryos with known
• All records, charts, forms, reports, consent letters, and
all other documents required to be maintained under
diseases. Critical Analysis
Chapter 4: Duties of ART Clinic and Bank this Act and the rules made under shall be preserved Chances of critical ovarian hyperstimulation syndrome
• Should not disclose the identity of oocyte donor to for a period of 10 years and after which the records Chapter 5: Sourcing, Storage, Handling, (OHSS) are low as there is no conception [low human
recipient couple or anyone else except in case of medi- shall be transferred to the National Registry of Assisted and Record Keeping for Gametes, chorionic gonadotropin (hCG) levels] and gonadotropin-
cal emergency or order of a competent court. Reproductive Technology Clinics and Banks in India of Embryos, and Surrogates releasing hormone (GnRH) agonist trigger can be used
• Ensure that patients, donors, and surrogates are free the Indian Council of Medical Research. which is well known to prevent OHSS.
from viral infections. • If the ART Bank closes before 10 years, the records shall Oocyte Donor • The risk of other serious acute complications like infec-
be immediately transferred to the National Registry.
Criteria critical analysis (Box 48.1) tion, hemorrhage, and torsion is less than 0.5%.11
• The number of oocytes or embryos to be placed in a
Critical Analysis • Also the responsibility of the ART Bank that recruits
woman in single treatment cycle would be specified by 1. Ever married the donors must be clarified upon.
However, the ART bill does not mention anything about the National Board or the concerned State Board. 2. 23–35 years of age • Oocyte donated by a relative or known friend of either
human immunodeficiency virus (HIV) discordant couples 3. One live child of 3 years of age of the couple should not be used.
who want to become parents. This is an important aspect Critical Analysis 4. Once in lifetime
of legislation as patients with HIV or hepatitis B cannot
• Till such a board is constituted, there is no limit to 5. Requires consent of spouse (Box 48.1) Critical Analysis
be denied the benefit of assisted reproduction, which will • Not more than seven eggs should be retrieved
number of embryos transferred leading to more high
help prevention of transmission of the virus from husband from one donor Lot of couples requiring oocyte donation request to obtain
order multiple pregnancies and related complications.
to wife or vice versa while providing the joy of parenthood. • Oocytes from one donor can be shared between oocytes from their relatives or friends. The present guide-
There is an urgent need to curb this problem.
European Society of Human Reproduction and Embry- two recipients only, but each recipient should get lines prevent this but it should be given a second look as
• On the other hand, strict limitation on the number, as
ology (ESHRE) has specific guidelines for embryology labs minimum of seven oocytes. social complications arising from such a situation are less
required by law in few countries, prevents individuali-
to prevent viral transmission.8 because ultimately birth mother is the recipient.
zation of cases.
American Society for Reproductive Medicine (ASRM)
• It is recommended that each center must monitor its Critical Analysis On one hand the ART bill does not allow altruistic
has specific guidelines for serodiscordant couples.9 Such oocyte donation, on the other hand the Surrogacy Bill, 2016
own data and develop protocols depending on patients’ The provision of obtaining just seven oocytes from the
guidelines are lacking in the current ART bill. recommends only altruistic surrogacy.
clinical features, to decrease adverse events like multi- donor is controversial as on the one hand Act allows for
• All assisted reproductive technology banks shall cryo- ple pregnancy, and maintain good success rate.10 sharing of oocytes with seven eggs to be given to each REGULATIONS WITH RESPECT
preserve semen sample for a quarantine period of at • Furthermore, since only embryos and not oocytes are party, on the other hand how that is possible if only seven
least 6 months before being used. artificially transferred into a woman’s uterus, the term TO SURROGACY
eggs can be obtained from one donor.
“oocyte” should be removed from the statement in the
• Aadhar card is to be used as proof of identity Issues with Commercial Surrogacy
Critical Analysis bill.
Cannot mix semen from husband and donor This is shown in Figure 48.3.
No mention of quarantine for oocyte donors. Ideally, even
No transfer of gametes of more than one individual Box 48.1. Criteria of critical analysis.
oocyte donors should be tested twice to avoid seroconver-
at a time Ever married: Even legal experts are unable to explain the
sion of the recipient in case the donor is in window period. Exploitative: Rural background, poor, illiterate women
No self and surrogate embryo transfer simultane- meaning of the term ever married •• Agents and brokers get the bulk of money
• Specific instructions and written consent with regard ously Lower age limit for semen donor is 21 years, but for oocyte
Health Issues: Forced to deliver by C-section
to death or incapacity of any of the parties is manda- In case the spouse has imminent death—his/her donor it is 23 years. Women can get married at 18 years of
age, but cannot be an oocyte donor until 23 years. •• Repeated pregnancies can affect cardiovascular health
tory before freezing human gametes and embryos. gametes can be procured for use by surviving spouse.
•• Psychological stress
• All consents and agreements should be in local language. Ova from fetus cannot be used under any circum- Previous ART guidelines and present ASRM have suggested
• All information regarding biochemical and clinical stances maximum six cycles of oocyte donation in a woman’s Unbalanced Act: Leave home for the duration of the pregnancy
pregnancy should be uploaded online within 7 days of The destruction or donation, with the approval lifetime,11 but present bill restricts this to only once •• Rights of their own children compromised.
receiving the information, withholding identity of the of the patient, to an approved research labora- Consent of spouse cannot be obtained if the woman is
tory for research purposes, of an embryo after separated or is a widow. Fig. 48.3: Issues with commercial surrogacy.
patient.
466 The Infertility Manual Critical Analysis of the Current ART Guidelines 467
Issues with Altruistic Surrogacy consanguinity.12 However, instead of keeping a limit on the Table 48.3. Rights and duties of donors and surrogates.
number of semen samples from a donor, it is more impor-
• Family members may be forced to become surrogates. Appropriate formula and mechanism needs to be developed under Rules Specific guidelines regarding monetary compen-
tant to document the number of pregnancies. for payment of compensation to the gamete donor and to transfer the sation for gamete donors are required.
• Develop bonding with the unborn child
funds to the bank account of the gamete donor.
• No form of monetary compensation
• Women in need may not find an appropriate surrogate CRYOPRESERVATION If there are any complications that have arisen during pregnancy (i.e. Insurance companies may not come forward to
gestational diabetes, chronic hypertension, etc.) which are likely to con- provide insurance for a lifetime.
at all!! • A human embryo may, for such appropriate fee as tinue for the rest of her life then it shall be covered appropriately under
may be prescribed, be stored for a maximum period of insurance.
SURROGACY (REGULATION) BILL, 2016 5 years and at the end of such period such embryo shall A surrogate shall relinquish all parental rights over the child or children. Appropriate adoption guidelines are required.
be allowed to perish or donated to an research organi-
• Complete ban on commercial surrogacy
zation.
• For the intending couple: Age of female partner should
• No donor gamete shall be stored for a period of more Offences and Penalties 3. Nongovernmental organizations (NGOs) or gov-
be between 23 and 50 years and male partner between ernment organization made in charge of third party
than 5 years. • No ART Clinic shall offer a couple to provide a child of
26 and 55 years. banks to curb malpractices
• The intending couple should not have a living child predetermined sex. 4. Promote oocyte banking similar to semen banking
born biologically or through adoption or through sur- Critical Analysis • Offenders shall be punishable with imprisonment for with proper quarantine
rogacy earlier. a term which may extend to 5 years or with fine which 5. More debate on altruistic surrogacy.
There cannot be an arbitrary limit of 5 years for cryo-
• Couple should be married for 5 years. may extend to rupees 10 lakhs or with both.
preservation. If the couple is willing for birth spacing then
• The transfer of a human embryo into a male person or
Women suffering from disorders like Müllerian agen- this time period can be extended. Also it is important to CONCLUSION
into an animal that is not of the human species shall be
esis need not wait for 5 years to avail surrogacy. increase this time limit in cases of cancer survivors.
an offence. The ART is a technology that has opened new frontiers.
• Certificate of proven infertility Along with bringing new hope to infertile couples across
Regulation of Research on Human the globe, it has brought in its wake a slew of unethical
Rather than a certificate of proven infertility, more Finance, Accounts, Audits, and
appropriate would be a certificate to declare inability Embryos practices promoted by unscrupulous businessmen seek-
Reports ing to exploit the emotions of such couples. Hence, there
to carry the pregnancy to viability. • The transfer of any gametes and embryos to any coun-
• This chapter in the bill deals with the regulations is a need to bring this technological revolution under the
• All records shall be preserved for a period of 25 years. try outside India for research is prohibited. rule of law. However, the law has to keep pace with the fast
regarding salaries of members of national and state
• Any offence shall be cognizable, nonbailable, and • Research only on gametes and embryos donated for developing science. The Indian bill is already 15 years in
boards.
noncompoundable. such purpose the making with still no established laws or accreditation
• Regulations regarding the maintenance of accounts
• For research, permission of the Department of Health bodies. At this rate, the law will already be obsolete by the
and audits by the national and state boards.
Critical Analysis Research to be obtained time it is passed.
• No human embryo created in vitro is maintained for a Society viewpoint about newer technologies will
• No specific numbers of abortions or implantation fail- period exceeding 14 days or such other period as rec- Chapter 10: Miscellaneous
always differ depending on moral, religious, and scientific
ures to qualify for surrogacy ommended by the National Board. Miscellaneous chapter deals with the dispute and their opinions. Hence, it is difficult to please all. The responsi-
• Adoption of unborn child—no legal procedure
settlement between national and state boards and/or bility of ethical practice lies with the ART practitioners.
described
• Legal implications to the surrogate, if she terminates
Rights and Duties of Patients, Donors, government Simultaneously, the law must ensure that physicians are
the pregnancy Surrogates, and Children not harried and unnecessarily persecuted in the name of
Critical Analysis patient rights as this will lead to fearful practice which in
Child Rights turn will hamper patient management.
SEMEN DONORS There are large areas where specific guidelines are yet to
• A child born to a woman artificially inseminated with come. PROBABLE QUESTIONS
Criteria the stored sperm of her dead husband shall be consid- Examples—Medical tests for gamete donors, amount
ered as the legitimate child of the couple. of monetary compensation, manner of harvesting oocytes, 1. Critically analyze the current ART bill.
• Age: 21–45 years
• A child or children may, upon reaching the age of 18, manner of storage of embryos, etc. 2. What are the legal aspects in relation to third-party
• Screened for infectious diseases
ask for any information, excluding personal identifica- reproduction?
• Consent of spouse, if married
tion, relating to the donor or surrogate. 3. What are the regulatory authorities for artificial repro-
• Maximum of 25 times. RECOMMENDATIONS
ductive technology in India?
Research has shown that in a population of 80,0000, 1. Immediate set up of national registry 4. Critical analysis of surrogacy (Regulation) Bill, 2016
there should not be more than 25 pregnancies from
DONORS AND SURROGATES 2. Finger prints can be used as proof of identity instead 5. Guidelines for regulation of ART over the years. Write
single semen donor in order to prevent inadvertent Rights and duties are mentioned in Table 48.3. of Aadhar card a short note.
468 The Infertility Manual Index 469
REFERENCES 8. ESHRE Guideline Group on Good Practice in IVF
Labs, De Los Santos MJ, Apter S, Coticchio G, Debrock S,
1. World Health Organization. Infecundity, infertility, and
childlessness in developing countries. DHS Comparative
Lundin K, et al. Revised guidelines for good practice in IVF
laboratories (2015). Hum Reprod. 2016;31(4):685-6. Index
Reports No 9. Calverton, Maryland, USA: ORC Macro and 9. Practice Committee of American Society for Reproductive
the World Health Organization; 2004. Medicine. Recommendations for reducing the risk of viral
2. Government of India. Ministry of Health and Family Welfare transmission during fertility treatment with the use of
(Department of Health Research). National Guidelines for autologous gametes: a committee opinion. Fertil Steril.
Accreditation, Supervision and Regulation of ART Clinics 2013;99(2):340-6. Page numbers followed by f refer to figure, fc refer to flowchart, and t refer to table.
in India. 2005. 10. Practice Committee of American Society for Reproductive
3. Government of India. Ministry of Health and Family Medicine; Practice Committee of Society for Assisted
Welfare (Department of Health Research). The Assisted Reproductive Technology. Criteria for number of A Amino acid 284, 396, 397 Authorized foreign adoption agency 347
Reproductive Technologies (Regulation) Rules, 2008. embryos to transfer: a committee opinion. Fertil Steril. Ampicillin 214 Azoospermia 127, 140, 141fc, 298
4. Government of India. Ministry of Health and Family Welfare 2013;99(1):44-6. Abdominal distension 277 Anastrazole 253 causes of 141
(Department of Health Research). The Assisted Reproductive 11. Practice Committee of the American Society for Abdominal myomectomy 206 Androgen 145, 245, 262 ductal obstruction 141
Technologies (Regulations) Bill. New Delhi; 2010. Reproductive Medicine; Practice Committee of the Abortion and tubal subfertility, induced 176 excess 143 management of 140
5. Government of India. Ministry of Health and Family Society for Assisted Reproductive Technology. Repetitive Acridine orange test 134, 135 role of 187 physical examination 140
Welfare (Department of Health Research). The Assisted oocyte donation: a committee opinion. Fertil Steril. Acrosomal cap 11 supplementation 187
Reproductive Technology (Regulations) Bill. New Delhi; Acrosome reaction 11 Androgenic drugs 245
2014.
2014;102(4):964-6.
12. Practice Committee of American Society for Reproductive
Adenomyomectomy, challenges of 200 Andrology instruments 451 B
Adenomyosis 45, 195, 198, 199 Anejaculation 149
6. Government of India. The Surrogacy (Regulation) Bill. 2016. Medicine; Practice Committee of Society for Assisted Bacterial vaginosis 212
diagnosis of 199 Aneuploidy prediction models, time
7. Practice Committee of the American Society for Reproductive Reproductive Technology. Recommendations for gamete Bariatric surgery besides 118
pathogenesis of 198 lapse and 456
Medicine. Diagnostic evaluation of the infertile female: and embryo donation: a committee opinion. Fertil Steril. Barker’s hypothesis 81
ultrasound 199 Aniline blue staining 134
a committee opinion. Fertil Steril. 2015;103(6):e44-50. 2013;99(1):47-62. Basal body temperature 268, 268f
Adenomyosis-associated infertility Antibiotics 398
Basement membrane 18f
management of 200 Antiestrogens 145
Bench top incubator 440, 441f
pathophysiology of 200 Antimetabolite 178
advantages of 440
Adnexal lesion Antimüllerian hormone 259, 260, 260t,
Bicornuate uterus 46, 46t, 47f, 171
classification of 48f 340, 364
Bicorporeal uterus 168f
ultrasound of 45 role of 5
Biological clock and fertility
Adoptee 349 Antioxidants 90, 143
preservation 159
Adoption Antiphospholipid antibody 331
Biological safety cabinet 367f, 450
age criteria for 348t Antral follicle 51f, 66, 67f
Biopsy
coordinating agency 347 count 27, 259, 340
guide 310
disruption of 349 Aplastic uterus 168f
technique 154
legislative history of 348 Arcuate uterus 46, 46t, 47f, 172
Biosafety cabinet 451f
procedure of 349f Aromatase inhibitors 60, 87, 143, 232,
Bladder injury 316
cost of 349t 233f, 252
reasons for 347 Blastocyst
dosage 233
review and current status 347 cell types of 17f
indications 233
triad 347, 347f cryopreservation 406
mechanism of action 232
Adoptive parents 350 Blastomere aspiration pipette 437, 437f
side effects 234
Adrenal hypothesis 81 Blood flow
uses 233
Adrenarche 71 Aspiration tubing 312 detection studies 147
Advanced hysterosalpingography 175t Aspirin 190, 291 in mature follicle 51t
Agency adoption 348 role of 291 Blunt cannula 424, 424f
Age-related fertility decline 159 Assisted hatching 190, 414 Bocyte retrieval 313t
Air quality analyzer 450 indications for 414 Body mass index 113t
Air-jacketed incubators 439 methods of 415 Bologna consensus 181
Albumin 278 Assisted reproduction 289 Bologna criteria, limitations of 182
Allis forceps/tenaculum 418 Assisted reproductive technology 16, 89, Breast development, tanner staging 72f
Altruistic surrogacy 341 107, 176, 289, 297, 308, 309, 330t, 335, Breathing disorder 277
Aluminum clips 445, 446f 335t, 379, 383, 396, 403, 458, 462 Bulls-eye pronucleus, single 392f
American College of Obstetricians bank 462 Buserelin 241
and Gynecologists 208 clinic and bank, duties of 464
American Society for Reproductive
Medicine for Tubal Factor
clinics, supervision and regulation
of 461
C
Evaluation 176 Asthenozoospermia 298 Cadherins 18
American Society of Clinical Oncology 163 Asynchrony 256 Calcium infusion 279
470 The Infertility Manual Index 471
Cancer 403 Comparative genomic hybridization, Cumulus oocyte complex grading 369t Egg future directions 23 laparoscopy 27
therapy advantages of 457 Current ART (regulation) Bill, 2014 462 banking 343 morphological markers of 21 parts of 173, 173f
and fertility 159 Compound microscope 437, 437f Current ART guidelines, critical analysis donation 340 Endometrioma 312 pathophysiology 173
outcome of 160fc Compromised respiratory function 280 of 461 Ejaculation 11 Endometriomics, potential uses of 23 Falloposcopy 175
potential outcomes of 159 Congenital uterine Cusco’s pelvic speculum 418 premature 149 Endometriosis 195, 298, 330 Female
Candidiasis 211 abnormalities, management of 170 Cutting needle biopsy 154 Ejaculatory additional 197 age and fertility preservation 159
Capinsert for cryovial 446f anomalies 46t, 166t, 168 technique 154 disturbances 149 associated infertility 198 antisperm antibodies 298
Carbohydrates 396 malformations CVM ring fiber plug 444, 445f duct, transurethral resection of 142 classification of 197t factor infertility 157
Carbon activated gas filtration 449 classification 166 Cyst wall, ablation of 198 failure 298 development of 195 fertility 37
Carrier genetic match 344 diagnosis 169 Cytoplasmic maturation 368 Electroejaculation 150 management 198 infertility 99f
Catheter etiology 166 Cytoplasmic morphology assessment Electroejaculator 452, 452f medical management 198 reproductive system 177f
components of 426 reproduction 166 393, 393t, 394t Electronic monitoring functions 362 mild 255 Fertility 36, 39
types of 301, 321 Conical BD falcon tube 419, 419f Elevated triglycerides 83 surgical management 198 management of cancer patients 159, 160
Ceftriaxone 214 Continuous gonadotropin Embryo 114, 405f, 442f, 465 Endometriosis-associated infertility, preservation 159, 160, 162fc
Cell gel electrophoresis, single 134, 135 administration 272
D assessment, conventional management of 198 established methods of 161
Cellular adhesion molecule 17f, 18, 18f Contraceptive history 26 Defective sperm condensation 132 methods of 454 Endometriotic females, indications of 159
types of 19f Controlled ovarian Delayed puberty 76, 77fc cryopreservation 161 cyst 50f options post treatment 163fc
Center well dish 434, 434f hyperstimulation 371, 383 Denudation 368, 380 development 17, 17f implants 196f post-treatment 163
Central adoption resource authority stimulation 249, 258 Dexamethasone 244 donation 34 Endometritis strategies 161
347, 348 Conventional insemination 432 mechanism of action 245 implantation of 20, 65 acute 48 sparing
Central obesity 83 Conventional open biopsy 154 intrafallopian transfer 319 chronic 48 interventions 161fc
Diacyl glycerol 387
Central pathway 115 technique 154 advantages 319 Endometrium 22, 44, 52, 114 surgeries 162
Didelphys uterus 171
Central precocious puberty 72 Conventional step-up dose protocol 88f disadvantages 319 during menstrual cycle 44 Fertilization 12, 12f, 14
Diet-induced obese 114
Centrally acting anorexiant loading techniques 429 thin 256
Conventional straw vitrification 405f Dimethyl polysiloxane 430 abnormal 390
medications 118 preimplantation genetic screening 456 triple line 270f
Cook aspiration unit 424, 424f Direct cover vitrification 408 assessment of 382, 386
Cervical mucus 268, 298 selection 333, 334t Endoplasmic reticulum 373, 387
Cook catheter 427, 427f Dish change 431f check 388
removal of 322 parameters 413 Endoscopic methods 175
Corifollitropin alfa 453, 454f Dish preparation 431 key players of 9
Cesarean section 176 technologies evaluating 454 Enzyme concentration 370
benefits of 454 Donor media 431
Chemical zona drilling 415 transfer 53, 319, 320, 322, 333, 334t Epididymal maturation 6
Corpus luteum 106 and surrogates 466 Fertiloscopy 175
Chemoattractant cytokines 18 and laboratory phase 332
Cortical granulation 386 rights and duties of 467t Epididymal sperm aspiration, Fertoprotective adjuvant therapy 163
Chemokines 18 catheter 323, 426
Cortical strips, cryopreservation of 407 central database of 343 microsurgical 142, 152, 153t, 403 Fetal origin 81
Chlamydia 210 catheter quality control tests 426
Corticosteroids 90, 144, 279 commercial 339 Epididymis 10, 152 of adult disease 81
trachomatis 215 clinical touch method of 321
Counselling, types of 33 gametes 341 Eppendorf micromanipulator 438, 439f Fibroid 45, 48t
Chorionic gonadotropin 284 day of 190
Craft suction unit 425, 425f insemination 339 Erectile dysfunction 103f, 148fc, 148t in infertility
preparations 239 management, difficult 320
Critical analysis, criteria of 465 sperm 299 classification 148t expectant management 205
Chromomycin 135 medium 322
Cryo storage 447f thalassemia screening of 340 disorders 148t investigation 204
Chromopertubation 175 optimisation of 53
Cryobiology 402 types of 339 problems 148t management 205
Classic and sure wallace catheters 426, 427f site of 323
Cryogloves 446, 447f Dopamine agonists 245, 279 Estradiol 85, 271 medical therapies 205
Clinical hypothyroidism 37 techniques 333, 334t, 429f
Cryoleaf 443, 444f Double lumen addition 263 pathophysiology 203
Clinical ovarian stimulation monitoring 269 timings of trial 320
Cryolock 443, 443f oocyte retrieval needle, parts of 422f levels 271 surgical treatment 206
Clomiphene 230f, 230t types of 319
Cryoloop 443, 443f principle of 424 Estrogen 244, 289, 290 role of 203
citrate 58, 86, 87, 87t, 108, 252, 229, 302 Embryological development 166
Cryopreservation 161, 402, 466 Doxycycline 214 receptor 21f treatment of 205fc, 207, 207fc
contraindications 232 Embryology instruments 418
basic principles of 404, 404f Dual trigger 286 Ethylene glycol 405 Flat tape sensor pt1000 447
dosage 230 Embryoscope 413, 441, 441t
Cryoprotectants 403 Dysmorphic uterus 167f Enclomiphene 230 Ethylenediaminetetraacetic acid 397 Flexi pipettes 432, 433f
failure 232
Cryoprotective agents 405 Endocrine control 7 Euploid blastocyst 188f Fluorescence in situ hybridization 456
indications 231
Cryostraws 444, 444f European Society for Human Reproduction analysis, limitations of 456
mechanism of action 230
Cryotop 443f, 444
E of spermatogenesis 7fc
Endocrinological and Embryology Classification 167 Flushing media 431
pharmacology 229
risks and complications 232 Cryovial 445f Early pregnancy disorders of infertility 63 European Society for Human Reproduction Focal adenomyosis 48t
side effects 231 workstation rack 445, 446f assessment of 53 mechanism 116 and Embryology Guidelines 176 Follicle
feedback mechanism of 231f Cullen’s sign 318 services 177 Endometrial European Thyroid Association 38 atresia 4
structure of 230f Culture media 396, 429, 430f scan 53 biopsy 22 curretting 314
development of 66, 66t, 186t
CO2
changer 448, 449f
components of 396
types of 429
Ectopic gestation 54
Ectopic pregnancy 173, 174, 177f, 178, 179
timing of 23f
changes during menstrual cycle 68
F flushing 314
incubators 357, 360 Culture systems 396, 398 classification 177 hyperplasia 48 Fallopian tube formation 4
regulator 448, 449f sequential culture media 398 diagnosis of 176 pathology 48 assessment of 175, 175t and maturation 4t
Coda tower 449f single step media 398 management of 176, 177fc polyp 48 evaluation of 27 recruitment of 66
Coda unit 450f Cumulus complex 367 signs of 177 receptivity 17, 20, 21 functions of 173 stimulating hormone 231f, 233f, 236,
Colloidal silica density gradient 300 Cumulus corona oocyte complex symptoms of 177 and implantation 16 hycosy 27 249, 271, 340, 454f
Commissioning couple 341 grading 368 uncommon ectopics 177 array 22 hysterosalpingography 27 structure of 236f
472 The Infertility Manual Index 473
Follicular receptor polymorphism 183, 183t Hot plate 448, 449f I treatment, financial aspects of 31 K
antrum 364 side effects of 240 Human chorionic gonadotropin 143, 178, tubal factors, unexplained 57
aspirate 318 structure 235 179fc, 245, 283, 284, 289, 335, 383 Iatrogenic luteal phase deficiency 289 ultrasound in 43 Karyopyknotic index 268f
aspiration set 311 therapy 253 Human embryo 388f, 390f, 396, 466 Immature oocyte retrieval for in vitro unexplained 56, 57, 231, 255, 298 Kinetic TL parameters 455
dynamics of 309 Gonadotropin preparations 239 cryopreservation 406 maturation 315 Inflammatory disorder 195 Kisspeptins 283, 286
fluid 318fc development of 237 Human endometrium, dynamics of 16f Immature ovum aspiration needle 422, 423f Injection and stimulation test,
contaminated with blood 365 Gonadotropin releasing hormone 107, 145, Human fertilisation and embryology Immunoglobulins 18 combined 147
production of 364 231f, 289, 332, 335, 453 authority 329, 344 Immunotolerance of pregnancy 20 Injection pipette 436f L
protein 364 agonist 279, 283, 285, 291, 335 Human growth hormone 78 Implantation Inositol 90 Labotect catheters 428, 428f
screening 364, 365 preparations 241 Human immunodeficiency virus 313, 340 contributors of 16f deficiency 82f Lacker’s model 252
growth, stages of 66f analogs 163, 240, 302 Human menopausal gonadotropin 237 steps of 17, 18f deficient state 82, 82t Lactoferrin 364
monitoring 50 antagonist 187, 242 Human oocyte In vitro fertilization 16, 59, 107f, 113, 258, triphosphate 387 Laminar flow cabinet 359, 366
output ratio 184, 184f dosage 243 cryopreservation 405 267, 297, 313, 328, 329, 335, 379, Insemination 297, 301 Laminar flow workstation 358
phase factors 107 mechanism of action 242 fine structure 10f 380, 387, 413 artificial 297 Laparoscopic
rupture 52, 283 regimen, advantages of 243 Human ovary 3 advantages of 380 mode of 301 assisted myomectomy 207
screening 364 side effects 243 Human reproduction, successful 459 cycle 107, 109 procedure 301 myomectomy 207
stimulation 237 structure 242 Human serum albumin 398 ultrasound monitoring in 269t timing of 301 ovarian drilling 88, 254
Folliculogenesis 185, 185t dependent 73, 76 Human sperm laboratory Instruments Laparoscopy 57, 175
Form of third-party reproduction 344t independent 73 and egg 9 advancements 454 laminar air flow 366 L-arginine 190
Fort index 184, 184f secretion 115, 116 survival assay 419 cleanliness 360 vacuum pump 366 Laser assisted hatching 415
Free androgen index 85 Gonococcal infections 215 Human spermatozoa 402 disinfectants 446 Insulin Late follicular phase endometrium 52f
Fresh oocyte donation 343 Gonorrhea 210 Hyalase 432 time lapse technologies in 413 hypothesis 82, 82t Leptin
Frydman rigid catheter 428, 428f Goserelin 241 Hyaluronan 364 In vitro follicle maturation and culture 164 like growth factor 364 in obesity 115
Functional corpus luteum 50f Granular cytoplasm 371 Hyaluron-bound sperm 459 In vitro maturation 89, 278, 383, 384, 454 resistance 115f resistance 82
Granulosa cells 367 Hyaluronic acid 398 of oocytes 162 sensitizing agents 90t Letrozole 188, 252
Ground glass appearance 50f Hyaluronidase and mechanical Incubation time 368 syringe 429, 430f advantages of 234
G Growth factor beta 1, transforming 364 methods 368 Incubator 439, 439t International Ovarian Tumor Leuprolide 241
Galactorrhea 100 Growth hormone 189, 190t, 244, 263 Hydrosalpinx 49f, 174 box type 440f Analysis Group 45 Light field LC-polscope 412f
Gas in line filter 449, 450f mechanism of action 244 impact 176 types 439 Intracervical insemination 297 Limulus amebocyte lysate test 421
Gene 116 Guidelines and bills over years 461t Hypergonadotropic hypogonadism 255 Indian Council of Medical Research 461 Intracytoplasmic morphologically Live birth rate 329, 330t, 331t
product 116 Gynetics catheters 428, 428f Hyperprolactinemia 95, 255 Infertile couple selected sperm injection 379, 382 L-methylfolate 90
Genetic causes of 98f female history 26 Intracytoplasmic sperm injection 14, 297, Low-dose
Hyperthyroidism 37, 39, 39fc initial investigations 26 313, 379-382, 459
couple 341 H and female infertility 36 male history 26 indications of 379
aspirin 245
step-up protocol, chronic 88f
factors 183
mechanism 117 Halo sperm assay 135 and male infertility 39 Infertile female population 38t procedure 381, 382f Lumen needles
screening 340 Hatching 17, 414 signs of 37t Infertility 30, 31, 36, 40, 95, 195, 203, 462 Intratubal insemination 297 double 311
testing 29 Hemiuterus 168f symptoms of 37t career compromise 31 Intrauterine insemination 297, 301-303 single 311
Genital development Hemorrhagic cyst 50f Hypogonadism 100 cause of 330, 330t advantages of 297 Lumen oocyte retrieval needle
phase of 166 Heparin, role of 291 Hypogonadotropic hypogonadism 140 cervical factors, unexplained 58 catheter 418, 418f, 419t double 422, 422fc
timetable of 166t Hepatitis Hypo-osmotic swelling 128 chances of conception, unexplained 58 cycles 109, 301, 302 parts of single 422f
Genomic activation 17 B surface antigen 340 Hypoplastic uterus 46 counseling 32, 34 endpoints 303 single 421, 422f
Genomic hybridization, comparative 456 C virus 313, 340 Hypothalamic dysfunction 231 counselor 32 indications of 297, 298 Luminal endometrial epithelium 16
Germ cell number 5t, 65f Hera cell incubator 439, 440f Hypothalamic gonadotropin embryological factors, unexplained 58 instruments 418 Luteal cells 68
Germinal vesicle 371, 380, 380f Herpes genitalis 213 releasing hormone 96 endometrial factors, unexplained 57 role of 89 Luteal follicular transition 68
oocyte 372f High security straw 443, 444f Hypothalamus 99 expectant therapy and lifestyle steps of 299 Luteal phase 161
Gestational surrogacy 341 Hindu Adoptions and Maintenance Act 348 gonadotropin releasing hormone modifications, unexplained 58 trolley 418 defect 106, 107, 107f
Ghrelin 116 Hirsutism, management of 86 pulses induce secretory pulses factors generating stress 30f Intravaginal transmyometrial embryo deficiency 231, 289, 289f
Glycodelin 19 Histrelin 241 of LH 96f male factors, unexplained 58 transfer 319 theories regarding pathogenesis
Glycoprotein 236 Hoffman modulation contrast Hypothyroidism 37 management of 86 Inverted microscope 438f of 107t
hormones 235 microscopy 438 in male infertility 39 unexplained 58, 60fc Issues with altruistic surrogacy 466 diagnosis, normal 107
Glycosaminoglycan hyaluronate 398 Holding pipette 436, 436f signs of 37t ovarian and endocrine factors, Issues with commercial surrogacy 465 factors 107
Gonad 3 Homogenous granular cytoplasm 372f symptoms of 37t unexplained 57 Istanbul consensus 390, 391t incidence, normal 106
development of 3f Hormonal Hysterosalpingo-contrast sonography 175 peritoneal factors, unexplained 57 manipulations 186
Gonadarche 71
Gonadotropin 59, 87, 99, 146, 235,
changes 37, 39
oral contraceptive pill, combined 86
Hysterosalpingogram 57
Hysterosalpingography 175
pharmaceutical advancements 453
physics of ultrasound 43
J normal 106, 108t
support 289-291, 302, 333, 335t
237, 240t, 253 profile in azoospermia 140t Hysteroscopic subendometrial embryo prognosis, unexplained 60 Juvenile Justice (Care and Protection treatment, normal 108
dose 261 Hormone delivery 319 sharing and confinement in social of Children) Act 348 Luteinising hormone 115f, 231f, 271, 283
increasing dosage of 185 assay 272 Hysteroscopy 57 circle 31 Juvenile Justice (Care and Protection of structure of 236f
products 237f replacement therapy 23f Hyteroscopic myomectomy 206 support counseling 33 Children) Amendment Act, 2006 348 testing 268
474 The Infertility Manual Index 475
M Microtools, distal end of 436f Nuclear maturation 12, 383 agonist cycle 312 Ovarian lesions, sonographical Pelvic inflammatory disease 213
Microtubule organizing center 387 Nuclear precursor bodies 389, 390f and embryo transfer 308 classification of 49t alternative parenteral regimen 214
Macromolecules 397 Mitochondrial activation, application Nucleic acid precursors 396 anesthesia for 309 Ovarian rejuvenation 191 causative agent 213
Magnetic cell sorting technique 403 of 191, 191f Nucleotide polymorphism, single 456 antagonist cycle 312 Ovarian reserve 27, 160f clinical features 213
Magnetic resonance-guided focused Monitoring endometrium 270 Number of insemination 301 complications of 315, 315t assessment of 57, 340 diagnostic considerations 214
ultrasound surgery 207 Monitoring follicular dynamics, needle 421 markers of 259t etiology of 215
Makler chamber 420, 420f methodology of 269 preparation of 312 test 183, 331 oral treatment 214
Male Monitoring liquid nitrogen levels 362 O procedure 312 Ovarian sensitivity, concept of 184 parenteral treatment 214
antisperm antibodies 298 Monopolar electrocautery 254 rate 308 Ovarian stimulation 21, 161, 227, 229, 244, treatment of 214, 215fc
Obesity 112, 118t, 329
factor infertility 123 Motility, activation of 11 transmyometrial 314 258, 260, 262f, 267, 299, 453 Pelvic structures 43, 312
and male sexual dysfunction 117
infertility 39fc Mouse embryo assay 419, 421 spindle positive and negative 412 cycles 250 evaluation of 43
and polycystic ovarian syndrome 83
evaluation of 28 Mucus causes retention 426 structure of 9 goal of 181 Pelvic ultrasound test 84
and reproductive health 113
reproductive system 116 Müllerian agenesis 171 Oogenesis 3 monitoring of 267 Pentoxifylline 145
categories of 113t
sterility 31 Multilocular lesions 49 Oogonial mitosis 4 protocols 229, 331, 332t Percutaneous epididymal sperm
combined procedures 119
Maternal spindle transfer 345f Multilocular solid cyst 50 Oophoropexy 162 types of 268t aspiration 142, 153, 153t, 403
effect of 113, 116
Maturation 10 Multiple displacement amplification 456 limitations of 162 Ovarian tissue cryopreservation 162fc, technique 153
first-line treatment of 86
and activation of spermatozoa 10 Multiple follicles 49f Open and closed cooling 443f 163, 407 Percutaneous testicular sperm
genes 116t
Mature follicle 51 Open fine needle aspiration 152 indications 163 aspiration 142
Multiple sperm defect indices 129 goal of therapy 117
Mature oocyte 284 advantages 153t limitations 163 Peripheral precocious puberty 72
Multiple vacuoles 373f lifestyle modification 117
cryopreservation 161 disadvantages 153t Ovarian torsion 315, 317 Peritoneal hemorrhage 315, 316
Myomas 204, 330 management 117
Mayer-Rokitansky-Küster-Hauser technique 152 differential diagnosis 318 Periumbilical hematoma 318
and infertility 203 mechanism of 116
syndrome 341 Optimizing in vitro fertilization investigations 317
and pregnancy 204 medical treatment 117 Persistent anuria 279
Mean spindle retardance 412 outcome 328 symptoms 317
Myometrial bulkiness 199f pathogenesis of 115f Persistent oliguria 279
Mechanical partial zona dissection 415 Optimizing ovarian stimulation treatment 318
Myometrial cyst 199f pathophysiology 115 Petri dish 434
Meiosis, resumption of 283, 284f protocol 331 Ovarian transposition 162
Myometrium 44 restrictive procedures 119 pH meter 448, 448f
Meiotic division Oral Ovaries
select treatment 117 Phospholipase C zeta 386, 387
completion of first 4 contraceptive 244
surgical artificial 164 Pituitary dysfunction 231
completion of second 4 N options in 119t
pill 86t evaluation of 27, 44 Placental protein 19
Meiotic spindle ovulogens 229
treatment 118 ovulation 27 Plasma membrane, depolarization of 386
angle of deviation of 412 N-acetyl cysteine 90 progesterone 108
types 113 Ovulation Plastic wares in in vitro fertilization
role of 411 Nafarelin 241 Orchidometer 451, 451f
Obstructive azoospermia 140, 152, 155, 403 assessment of 56 laboratory 432
Members of national board 463f Narishige micromanipulator 438, 438f Organelle redistribution 12
treatment of 141 induction 251, 252 Polar body 374
Members of state board 463f National Guidelines for Accreditation 461 Orgasmic disorders 150
Offences and penalties 467 agents 87 biopsy pipette 436, 437f
Menstrual Natural cycle of Osmolality 399
in vitro fertilization 263 Oligoasthenospermia and superovulation 249, 250 Polscope 411
cycle 65, 161, 249 Ovarian cycle 249, 250
intrauterine insemination 301 evaluation of 144 in anovulation 251 additional applications of 412
abnormalities 113 Ovarian cyst theory 195
Natural fertility 113 management of 140, 144 timing of 68 application of 412
different phases of 69f Ovarian cystectomy 198
Nausea 12 mild 255 Ovulatory dysfunction 113 embryoscope and assisted hatching 411
follicular phase 65 Ovarian endometrioma 196f, 198
Needle aspiration biopsy 153 moderate 255 Ovum pick up 313, 359, 425 Polycystic ovarian syndrome 80, 80f, 82f,
neuroendocrine control of 70 ultrasound images of 196f
advantages 154t Oligoasthenoteratozoospermia 298 ultrasound guided procedures 52 83, 84t, 86, 278
overview of 250t Ovarian enlargement 277
disadvantages 154t Oligospermia 298 Ovum quality 51 and menopause 92
phases of 45t Ovarian failure post chemotherapy, risk
Needle immersion vitrification 408 Oocyte 113, 361, 412 Oxandrolone 78 and pregnancy 91
disturbances 37 of 160f
Needle tip markings 422, 423f abnormality, classification of 372f Oxidative stress diagnosis and evaluation 80
dysfunction, management of 86, 86t Ovarian follicle
hemorrhage 200f Neisseria gonorrhoeae 214, 215 activation of 386, 387f and low-grade inflammation 83 etiopathophysiology 80
dysfunction 182
history 26 Neuroendocrine dysfunction 81 and embryo cryopreservation 404, treatment of 135 in adolescents 91
pool, depletion of 182
phase 161 Neuroendocrinology of puberty 72fc 405, 441 Oxygen species, reactive 133 lifestyle modifications 86
Ovarian folliculogenesis, life cycle of 249f
Metabolic syndrome 83, 83t Neuropeptide 115 aspiration in in vitro maturation 384 Ovarian fragmentation 191, 191f management of 86
chromosomal of 313 metabolic complications 83
Metabolomics 458
Metaphase of second meiosis 411
Nitric oxide 364
Nocturnal penile tumescence 147 cumulus complex 364, 365f, 367
Ovarian hyperstimulation 267 P phenotypes 80
Ovarian hyperstimulation syndrome 161,
Metformin 90, 234, 263 Nonconsummation of marriage 147 donation 34, 341t 259, 275, 276t, 279, 283, 332, 335 Pain 315, 316 classification of 81t
Methotrexate 178 Noninvasive embryo selection 454 donor, screening of 340, 340t classification 276 acute 277 Polycystic ovary 47, 51f
Metronidazole 214 Noninvasive sperm selection 459 dysmorphisms 371 clinical symptoms and signs 276 Papillary structures, absence of 49 syndrome 330, 335
Microfluidic Nonobstructive azoospermia 140, 143, formation 4 early and late forms 275 Particulate absolute filtration, Polymerase chain reaction 456
devices being researched, types of 458 152, 155, 403 increase number of 187, 189 high-risk factors 277 high-efficiency 358 Polyvinyl alcohol 398
potential application of 458 gonadotropins 143 maturation 12, 383 incidence 277 Pasteur pipette 433, 433f Poor cervical mucus 298
technology 457 surgical management of 144 and trigger protocol 332, 333t pathophysiology 275 Pelvic infection 315 Poor ovarian
Micromanipulation 435, 437 treatment 143 physiology of 283 prevention of 272, 277, 278 management 316 reserve 182
Micromanipulator equipments 438 Non-permeating cryoprotective agents 405 near 11 risk of 314 prevention 316 response 181, 182
Micropipette holder 437, 437f Non-sperm cells 129 nuclear maturity evaluation 370, 371 Ovarian hypothesis 81 source of 315 acquired factors 183
Microscope eyepiece magnification 367f Nontoxic syringe 429, 430f retrieval 308t, 312, 332, 333t, 360, 421 Ovarian implants 196 symptoms 315 lifestyle-related factors 183
476 The Infertility Manual Index 477
pathophysiology 182 Prolactinomas, treatment of 102t Remi centrifuge 420, 420f Sexual dysfunction 99 retrieval 143 Test tube
risk factors 182 Pronuclear Reproduction, third-party 339 in male infertility 147 retrieval techniques 152 heater 425, 425f
Portiovaginalis of cervix 323 abnormalities 390 Reproductive tract infections 210, 210t classification 147 advantages of different 142t warmer 312
Poseidon strategy 182, 182t, 184t, 186t, morphology 393, 393f agents of 210t treatment 149 classification of 152t Testicular atrophy, bilateral 141
187t, 189t scoring 389 causes of 210t Sexual history 26 disadvantages of 142t Testicular failure, primary 140
Postacrosomal region 11 size 393 compilations of 210t Sexually transmitted infections 210 selection 332 Testicular retrieval techniques 154
Postpartum hemorrhage 341 transfer 345f treatment of 210t Shanghai protocol 189, 189f techniques 333t Testicular sperm aspiration 153
Preantral follicle 66, 67f Pronuclei Retrograde ejaculation 149 Sildenafil, role of 291 transport 7 advantages 153t
Precocious puberty 71, 74fc axis of 388f, 389f management of 149 Simplex optimized media 396 ultrastructure 10f disadvantages 153t
etiology of 73 breakdown, timing of 413 Right tubal ectopic pregnancy 178f Skeletal abnormality 168 vitality 128 technique 153
evaluation of 73 formation of 13 Rocket pump 425f Slow freezing 404 Spermatogenesis 3, 5 Testicular sperm extraction 142, 144, 403
Pregnancy observation of 387 Room air 367 Smooth endoplasmic reticulum development 5 microsurgical 154
after cancer treatment 163 Proopiomelanocortin 115 Round bottomed BD falcon tube 419f cluster 373, 373f differentiation 6 Testis 99, 152
occurrence 277 Round well dish 434, 434f Soft core 212 meiosis 6 Testolactone 145
Propanesulfonic acid 432
rates 16 Round-bottomed BD falcon tube 418 Soy protein 203 process of 6f Testosterone
Prophase of meiosis, diplotene stage of 4
Preimplantation 17, 17f Specialized adoption agency 347 proliferation 6 gel 188
Prophylactic antibiotic 309
genetic Sperm 13f Spermatozoa 361 test 85
diagnosis 456
Prostaglandins 19 S acrosome reaction 11f total number of 125 Thawing process 403
Protein
screening 457 and pronuclear formation, fusion of 386 Sperm-soluble factor, release of 386 Therapeutic counseling 33
kinase C 387 Salivary ferning kit 268
Preovulatory follicle 67, 68f cell, structure of 9 Spindle imaging 411 Thermal mechanism 117
synthesis 12 Salpingoscopy 175
luteal phase 68 chromatin limitations of 413 Thiazolidinediones 90
Pseudosac 54t Salpingotomy, steps of 179
ovulation 67 dispersion test 134, 135 Spindle length 412 Third-party reproduction, future of 343
Psychological aspects of infertility 31 Screening genetic couple 342t
Preovulatory phase 161 packaging defects, tests for 134 Spindle orientation 412 Three-parent babies 344
Puberty and surrogate 342 Spontaneous ovulatory cycles 109
Primordial follicle 66, 67f structure 132 Thyroid
abnormal 71 Secretomics 458 Standard and sure view wallace 427f
Primordial germ cells 66 assay 134, 135 antibody 40
classification of abnormal 72fc Secretory endometrium, stages of 17f Statins 91
Probe covers 421f collection of 149 autoimmunity 38, 38t
etiology of delayed 76 Secretory phase, stages of 17 Steiner pistol device 319
Probe movements, basic 44t count 127 disorders 36
evaluation of delayed 77 Selective salpingography 175 Stereo zoom microscope 367
Problematic cervix 320 cryopreservation 402, 403 function 39
normal 71 Semen 125 Stereomicroscope 437, 438f
Progesterone 85, 108, 271, 289 damage 133t stimulating hormone 85, 235
treatment analysis 28, 40, 57, 125, 132 Sterile container with lid 418, 419f
optimal dose of 290 abortive apoptosis 133 Thyrotoxicosis, causes of 36
of delayed 78 initial microscopic examination 127 Sterile culture conditions 360
routes and doses of 108t antibiotics 136 Time lapse imaging 441f
of precocious 73 liquefaction 126 Stimulation
safety of 109 antioxidants 136 Time lapse monitoring
Progestins 78 Pubic hair development, tanner staging 72f method of collection 125 double 189, 189f
cause-specific approach 136 features of 413
Prolactin 85, 95 Pyridostigmine 190 parameter 56, 129 individualization of 258
dietary antioxidants 136 system 413
actions of 96 procedure 128 Stress, treatment induced 30
effects of DNA damage 133 safety of 414
and infertility 98 steps 125 Subclinical hypothyroidism 37, 38, 38t Tissue culture media, complex 396
and ovarian stimulation 103 Q testing for immunoassays 129
evaluation of 132
indications 133 Subseptate uterus 46, 46t Toluidine blue staining 134
biosynthesis 95 collection 299 Sulbactum 214 Total asthenozoospermia 155
Quality control 355 mechanisms of 132
causes of 97, 98t donors 466 Surgical sperm retrieval 152 Total leukocyte count 317
in ART laboratory 355 oxidative stress 133
high levels of 98t macroscopic examination of 126 selective isolation 136 contraindications 152 Towako transmyometrial
of culture medium and disposables 360
diagnosis of high levels of 100 pH 126 supplementary antioxidants 136 indications 152 ET catheter system 319
of humidity 362 operative techniques 152
functions of 97, 97f preparation techniques 300t tests 134 needle 429, 429f
of laboratory equipment 357 Surrogacy 34, 341
high levels of 97, 98, 99t, 100, 100f, processing 299 treatment 135 Trachomatis 210
of lasers 362 viscosity 126 (regulation) Bill, 2016 343, 466
102f, 103f of pH 362 varicocelectomy 136 Transdermal estrogen therapy 78
in females 96 volume 126 deoxyribonucleic acid 132 commercial 341 Transition electron microscopy 14
of procedures 362 majority of 125 terminologies 341
in males 97 fragmentation 29 Transrectal ultrasound 196
program, components of 356 Seminiferous tubule traditional 341
laboratory evaluation, high levels of 101 donation 339 Transvaginal
Quality guidelines, references of 355 cross-section of 6f Surrogate 341, 342, 465
management of high levels of 100 freezing guided oocyte retrieval 308
Quality management, total 355 single 154 screening 342t
medical treatment, high levels of 101 techniques of 403 hydrolaparoscopy 175
physical examination, high levels of 100 Septa, absence of 49 thawing process 404f Synchronize follicle development 186 scan 269, 340
Synechiae 48
radiotherapy, high levels of 101 R Septate uterus 46, 46t, 47f, 167f, 171 function tests, additional 29
Synthetic kisspeptin analogues 286
transcervical intrauterine ET 319
receptors 95 Sequential medium 399t head swells 14 ultrasound 196
role of 99 Raloxifene 234, 253 Serial serum hormone levels 271 incorporation 13f Syphilis spread by sexual contact 212 disadvantages of 43
secretion Randomized controlled trial 185 Serodiscordant couples 298 morphology 128 methodology of 43
RCOG recommendations for tubal
neuroendocrine regulation of 96, 97f
patterns 96 assessment 176
Serosa 44
Serum
motility 127
grading of 128
T Trichomoniasis 211
Triptorelin 241
source of 97 Recombinant gonadotropins 238 albumin 398 oocyte Tamoxifen 87, 234, 253 True sac 54t
structure of 95, 95f Recombinant luteinizing hormone 239, 285 human chorionic gonadotropin 318 fusion 12 Teflon tubing 424, 424f Tubal
surgery, high levels of 101 Refractile bodies 374, 374f Sex hormone therapy 78 interaction 386 Temperature measurement 448 assessment categories 175t
treatment of high levels of 101 Relaxin 19 Sex partners, management of 215 preparation 380 Teratozoospermia 298 blockage 174
478 The Infertility Manual
cannulation 175, 175t Uterine Venereal disease research laboratory
damage 173 anomalies 46, 169t 313, 340
causes of 174f, 174fc aplasia 46 Vertebral osteomyelitis 315, 317
incidence of 173 artery embolization 207 Vibrect vibrator 452, 452f
risk factors of 174fc body, pathologies of 45 Vitamin 396
factor cycle 249, 250 A 78
evaluation of 175 factor infertility, absolute 459 D 90
infertility 173 fibroid tumors 205t deficiency 83
infertility 176 transplantation 459 Vitrification 404
Uterus carrier devices for 405
patency 57
assessment of 57 kit 441, 442f
pathology 330
diadelphous 46 laboratory procedure for 406
management of 176
evaluation of 28, 44 protocol 442f
surgery 176
hysterosalpingography 28 techniques 408
Tuberculosis fallopian tube 174 Volunteer donors 339
Tubes hysteroscopy 28
normal 167f Vomiting 277
assessment of 57
three-dimensional ultrasound 28
evaluation of 45
Turner syndrome 78
T-shaped 46 W
ultrasonography 28
Waist to hip ratio 112
U V
Wallace catheter 426, 426f
Water bath 446, 447f
Ultrarapid vitrification 405f Water-jacketed incubators 439
Vacuole 371
Ultrasound 46 Well dish 434, 435f
single large 373f
guided aspiration 198 WHO semen analysis parameters 28
Vacuum erection devices 452, 452f
guided procedures 52 Vaginal
complications 53
technique 52
cleaning 309 Y
disinfectants 316
machine 421 hemorrhage 315 Y chromosome microdeletion 29
Unicornuate uterus 46, 171 probe 310
Unilocular lesions 49
Ureteric
thrush 211 Z
ultrasonography-guided embryo
fistula 316 transfer 322 Z score 391t
injuries 315, 316 Varicocele Zona pellucida 17, 376, 412
stricture 316 grading of 28 function of 414
Urinary repair 144 thick 376f
alkalization 149 Varicocelectomy 144 Zuclomiphene 230
human menopausal gonadotropin 251t Vascular control systems 207 Zygote
tract, dilation of 317 Vascular endothelial growth factor 106, 364 abnormal 392f
Urine pregnancy test 318 Vaso-epididymostomy 142 formation 14, 14f
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