REVIEW (Derleme)

SEMEN ANALYSIS FROM A POINT OF VIEW OF GYNECOLOGIST AND

RECENT DEVELOPMENTS

Omer Lutfi TAPISIZ1, Sadiman Kiykac ALTINBAS1, Faruk ABIKE2, Umit GOKTOLGA1

1 Department of Obstetrics and Gynecology, Etlik Zubeyde Hanim Training and Research Hospital, Ankara, Turkey
2 Department of Obstetrics and Gynecology, Ankara Medicana International Hospital, Ankara, Turkey

SUMMARY

Infertility is defined as the time of one year of unprotected intercourse without conception and affects approximately
15% of the couples. Data available over the years reveal that pathology is found among 55% of cases in women, 35%
of cases in men and the remaining 10% of cases are defined as unexplained infertility. Male infertility is commonly
due to deficiencies in the semen and semen quality is used as a surrogate measure of male fecundity. A semen analysis
evaluates certain characteristics of a male's semen and the sperm contained in the semen. It should be the first
laboratory investigation for men while evaluating a couple's infertility. Here, we tried to review the semen analysis
as a very important laboratory investigation in evaluating the infertile couples with all points in the light of last
published World Health Organization (WHO) criteria.

Key words: male infertility, semen analysis, spermiogram, World Health Organization (WHO)
Journal of Turkish Society of Obstetrics and Gynecology, (J Turk Soc Obstet Gynecol), 2012; Vol: 9 Issue: 1 Pages: 25- 31

J‹NEKOLOG GÖZÜ ‹LE SEMEN ANAL‹Z‹ VE SON GEL‹fiMELER

ÖZET

‹nfertilite, bir y›ld›r korunmas›z cinsel iliflkiye ra¤men konsepsiyonun oluflmamas› olarak tan›mlan›r ve yaklafl›k olarak
çiftlerin %15'ini etkiler. Y›llar içindeki mevcut data, patolojinin %55 olguda kad›nlarda, %35 olguda erkeklerde
oldu¤unu ve geri kalan %10 olgunun da aç›klanamayan infertilite olarak tan›mland›¤›n› göstermektedir. Erkek
infertilitesi s›kl›kla semendeki bir yetersizli¤e ba¤l›d›r ve semen kalitesi erkek fekunditesinin ölçümünde kullan›l›r.
Semen analizi erkek semeninin belirli özelliklerini ve semenin içerdi¤i spermi de¤erlendirir. ‹nfertil bir çiftin
de¤erlendirilmesinde erkek için yap›lacak ilk laboratuvar incelemesi semen analizi olmal›d›r. Biz de burada, infertil
çiftlerin de¤erlendirilmesinde çok önemli bir laboratuvar incelemesi olan semen analizini Dünya Sa¤l›k Örgütü'nün
(DSÖ) yay›nlad›¤› son kriterler ›fl›¤›nda bütün yönleri ile gözden geçirmeye çal›flt›k.

Anahtar kelimeler: Dünya Sa¤l›k Örgütü (DSÖ), erkek infertilitesi, semen analizi, spermiogram
Türk Jinekoloji ve Obstetrik Derne¤i Dergisi, (J Turk Soc Obstet Gynecol), 2012; Cilt: 9 Say›: 1 Sayfa: 25- 31

Address for Correspondence: Ömer Lütfi Tap›s›z. Turan Günefl Bulvar› Sedir Sitesi C2 Blok No: 6, Oran 06450, Ankara
Phone: (0505) 319 75 77
e-mail: omertapisiz@yahoo.com.tr
Received: 15 April 2011, revised: 27 July 2011, accepted: 11 August 2011, online publication: 08. December 2011

25 DOI ID:10.5505/tjod.2012.60476
Ömer Lütfi Tap›s›z et al.

INTRODUCTION and making comparisons between individuals. Semen

sample should be collected following at least a 2-day
According to the definition by the World Health (2 to 7 days) sexual abstinence. To ensure
Organization (WHO) infertility is the absence of standardization, a 3-4 days period of sexual abstinence
conception despite one year of regular sexual intercourse will be appropriate. While short period of sexual
of a sexually active couple without contraceptive abstinence causes decreases in the volume and density
protection(1). Nearly 15% of couples fail to achieve of semen, that of long causes increases in its volume,
conception in a 1 year period and present to receive density and proportion of dead, immotile and
treatment for infertility(2). When reviewed, 55% of the morphologically abnormal sperms. Ideally, semen
reasons for infertility are found to be male-related and should be collected by masturbation in a sterile and
35% to be female-related, while 10% constitutes dry, wide-rimmed, glass of plastic container with no
infertility of unknown origin(3). In approaching to an previous spermicide toxicity. However, collection can
infertile couple, the first laboratory evaluation to be also be performed during intercourse using specially
performed will normally be a semen analysis in the produced plastic condoms (latex condoms affect the
process of investigating the presence or absence a vitality and motility of sperms) containing no
male-related infertility reason. We, in this review, spermicidal agents. Collection by withdrawal during
aimed to discuss semen analysis, which is of major intercourse is not recommended since this causes the
importance in evaluating an infertile couple, in all its loss of the first portion of sperm. The importance of
aspects and taking the latest criteria into consideration. collecting all the semen ejaculated should be kept in
mind, because, composed mostly of prostate fluid, the
first portion contains the highest sperm concentration,
SEMEN ANALYSIS AND INFERTILITY while the last portion is particularly vesicular fluid
forming the major portion of semen. Therefore, it is
Abnormal sperm analysis results are observed in the essential to avoid escapes and splits while collecting
major part of the infertility associated with male factor. semen. Germ, spit and lubricant contaminations should
First assessment should have at least a properly be avoided during semen collection. Previous to
performed semen analysis. If an abnormal result is collection hands and genitals should be washed with
encountered, other sampling should be performed after soap and rinsed thoroughly with water. Soap and other
at least 4 weeks(4). It should be noted that semen lubricants should not be used during masturbation.
parameters may differ very seriously in time and that Collecting semen in an isolated, special environment
may show temporal and seasonal differences even in within or near laboratory will be the most appropriate
fertile males. Therefore, performing more than one approach. It should be remembered that emotional
examination in the evaluation of semen analysis is stress and tension have negative effects on semen
important with respect to increase the degree of parameters particularly on volume, count and motility.
accuracy. Presence of a normal semen analysis will (5). Therefore, the importance of performing collection

often ensure an important male factor to be excluded in an environment in which patient will feel comfortable
if there is no complaint or suspicion concerning sexual and peaceful should always be kept in mind. If collection
dysfunction. In contrast, determination of abnormal is considered to be more proper to be done at home,
semen analysis will require additional endocrinological, semen sample should be ensured to be transferred
urological and genetic investigations. quickly while being kept at room or body temperature.
Semen sample taken in a way other than collecting
method should be evaluated within an hour(4).
COLLECTING SEMEN SAMPLE

It is important to have a certain standard method that MACROSCOPIC ANALYSIS of SEMEN

is explained in detail for collecting semen. Thus a
definite standardization will be ensured for evaluating Fresh ejaculate is white or grayish white opaque
the results of analysis, administering treatment protocols substance of a viscous nature. It has a typical odor. It

J Turk Soc Obstet Gynecol 2012; 9: 25- 31 26

 Semen analysis from a point of view of gynecologist and recent developments

liquefies generally within 10-20 minutes and becomes d) Volume and pH of ejaculate
blurred. Normal ejaculate volume is expected to be between
1.5 and 5.0 ml(4). In determining the volume, it is
a) Color and odor recommended to place the sample in a wide-rimmed
Appearance of normal semen is homogenous, grayish- container and use gravimetric method. Measuring the
white in color and opaque. The lower the concentration volume by aspiration is not recommended (0.3-0.9 ml
of semen the less opaque it seems. Depending on the of mismeasurement). Normal pH of semen is >7.2.
prolongation of sexual abstinence, it may become Following liquefaction, preferably within 30 minutes,
yellowish in color. In case of presence of erythrocyte and 1 hour at the most, a pH assessment should be
in semen, the color will become reddish brown. Its done. Vesicula seminalis secretions contained in semen
typical color is thought to be resulting from sperm are alkaline while prostatic secretions have acidic
oxidation due to prostate secretions(5). The odor and properties. Both secretions should be in a certain
color of semen may differ according to duration of balance. It should be noted that pH of semen may
sexual abstinence and presence of infection. increase up to 8.0 in acute infections (infections of
prostate, vesicula seminalis, epididymis). No or little
b) Liquefaction time ejaculate volume should suggest an ejaculation failure,
Semen is coagulated during ejaculation by seminal short sexual abstinence, collection defects, congenital
vesicle secretion containing semenogelin I, and liquefies absence of vas deferens, ejaculatory duct obstruction,
at room temperature within approximately 20 minutes. hypogonadism or retrograde ejaculation. Evaluation
Liquefaction occurs by means of proteolytic enzymes of other semen parameters will help in differentiating
(fibronilisine, fibrinokinase, aminopeptidase) secreted the reason. The majority of semen is generated by the
by prostate. In evaluation of liquefaction, ejaculate is vesicula seminalis that develops from the same
placed in an incubator at 37oC and allowed to be embryological origin as the vas deferens. The secretion
liquefied. Liquefaction time more than 60 minutes or of the vesicula seminalis is alkaline and contains
no liquefaction longer is pathologic showing lack of fructose. In majority of the men without bilateral vas
prostatic enzyme or inadequate prostate function(6). In deferens, the vesicular seminalis is absent or
cases of no liquefaction or excess viscosity mechanical hypoplastic. In this case, semen will be acidic (pH<7.2),
stirring (e.g., withdraw and release by a pipette or an small in volume and of a quality containing no or little
injector) or dissolve in an enzyme (e.g., 1gr/l of fructose(8). Again in ejaculatory duct obstruction, the
bromelain or 0.5 cu/ml of plasmin) may be needed. It quality of semen will be similar and fructose
should be kept in mind that these practices may affect concentration will be reduced according to the level
seminal plasma biochemistry and sperm motility and of obstruction. If ejaculatory ducts are bilaterally
morphology(6). completely obstructed, semen will be acidic, and will
contain no fructose or sperm (for it contains only
c) Viscosity prostatic secretion). Also in hypogonadal men, since
Normal semen has a viscous texture. Increase in the testicular androgen production is low, vesicula
viscosity may occur due to hypofunction of vesicula seminalis and prostate, both of which are stimulated
seminalis. High viscosity may affect sperm motility by androgens, will not be able to produce adequately
and concentration. ‹ncrease in viscosity may reduce and ejaculate volume will be reduced. In the event that
the success of intrauterine insemination (IUI) and in ejaculate volume is less than 1 ml and that factors such
vitro fertilization (IVF). For evaluation, the sample is as hypogonadism, congenital absence of bilateral vas
withdrawn into pipette and observed leaving the pipette deferens, collection problems or short sexual abstinence
drop by drop under the effect of gravity. In case of period are excluded, a post-ejaculator urinary analysis
abnormal viscosity, the sample stretches like a thread. should be performed to determine retrograde ejaculation
If the elongation is more than 2 cm it is considered to Finding sperm in post-ejaculator urinary analysis
be pathological. Evaluation of elongation may be performed in men with no or little semen volume and
performed by using a glass bar(7). azoospermia should suggest retrograde ejaculation(4).

27 J Turk Soc Obstet Gynecol 2012; 9: 25- 31

Ömer Lütfi Tap›s›z et al.

MICROSCOPIC ANALYSIS of SEMEN Concentration (C): Total counted sperms (N) / number
of rows evaluated x 1 / volume of one row x dilution
In this examination sperm count and motility rate
characteristics, morphological structure, presence or C = (N/n) x (1/20) x dilution factor (n: number of
absence of agglutination, leukocyte and round cell square where count is performed)
staining and count, and, if necessary, vitality
investigations are performed(5). Furthermore, according Normal values according to the WHO criteria defined
to criteria defined by the WHO in 2010, sperm in 2010 are stated as spermatozoa/ml: 15x106, total
aggregation is determined by evaluation of binding of sperm count: 39x106(9).
immotile sperms to motile or immotile sperms, mucus If no sperm is seen in microscopic examination, a new
fibers, non-sperm cells and cellular wastes(9). Once fresh preparation is prepared. If again no sperm is seen,
the semen is liquefied, examination of unstained wet then azoospermia may be suspected. Semen sample is
preparations using phase-contrast optical system will centrifuged at high speed ( (3000g/15 minutes), and
be more advantageous. Through this initial examination, concluded as criptozoospermia and azoospermia if and
important information about the concentration and if not, respectively, sperm cells are detected. The
motility of sperm is obtained(5). incidence of azoospermia among all men is 1%, while
it is 10-15 % in infertile men(11). In new recommendations,
a) Agglutination use of fixed, non-centrifuged samples for evaluation
This is a status in which motile sperms clump and stick and indicating the sensitivity of counting method are
together. Types of agglutinations may be in head-to- underlined. On the other hand, it has been emphasized
head- tail-to-tail or head-to-tail fashion. Presence of that centrifuge methods are needed in sufficient cell
plurality of tail-to tail motile dimers may be suggestive collection for therapeutic methods and in post-
of the presence of antisperm antibodies(5). Who has vasectomy semen evaluation(9).
defined four groups for the purpose of grading
agglutination: Grade I, isolated, <10 spermatozoa per Sperm motility
agglutination, most of the sperms are free; Grade II- Spermatozoon need to be actively motile to pass cervical
Moderate, 10-50 spermatozoa per agglutination, free mucus and fertilize the ovum in tuba uterine. Evaluation
sperms are present; Grade III, Extended >50 of sperm motility should be performed within 60
spermatozoa per agglutination, some spermatozoa are minutes, preferably 30 minutes, following semen
free; Grade IV-Whole, all spermatozoon are agglutinated liquefaction. Evaluation should be performed by
(9). counting 200 sperm cells in total at room temperature
or 37oC, at the depth of 20 µm and in 200X or 400X
b) Sperm count and concentration magnifications. Evaluating motility subjectively causes
Neubauer hemocytometer, Makler chamber or single different laboratory results at present. To prevent this
use counting devices (Mikro-Cell) are used to determine and ensure objectivity, the WHO has determined sperm
sperm cell counts in ejaculate(5). For sperm count, motility evaluation criteria and 3 groups have been
hemocytometer counting chambers of 100 µm depth defined(9):
are recommended. Semen should be diluted to correctly - Progressively motile; actively motile whatever its
count the sperms. The degree of dilution is determined speed (both linear and wide circular,
according to sperm cell count obtained per image - Non-progressively motile; all motility patterns
square for 200X or 400X magnification in fresh without progression (swimming in tight circle),
preparation and the number of sperms per milliliter is - Immotile
calculated(10). For determining sperm concentration,
at least 200 cells are counted following a suitable According to WHO, progressively motile sperm rate
dilution. Calculation is made according to following of minimum 32% and total motile sperm rate of
formulas using developed Neubauer hemocytometer; minimum 40% has been defined as normal (9) .
According to previous criteria published by the WHO
in 1999 gathering progressive motility in single group

J Turk Soc Obstet Gynecol 2012; 9: 25- 31 28

 Semen analysis from a point of view of gynecologist and recent developments

and change in determined proportions (in 1999, fast Then sperm morphology is evaluated microscopically
progressive motility ≥ 25%, fast progressive + slow at 1000X magnification. In normal sperm morphology,
progressive motility ≥ 50%) forms the main differences the head should be oval in shape and acrosome should
(12) constitute main differences. Total motile sperm constitute the 40-70% of the front part of the head.
count (ejaculate volume x Concentration x proportion Vacuole number in head section should < 2 and vacuole
of progressively motile sperm) is an important should not be larger than 20% of the area occupied by
parameter. In circumstances of total motile sperm count the head. As for the tail section: the tail should not be
of < 5x106, treatment using techniques supporting broken or curved and should not rotate 360o(9,12,15).
reproduction will be a more appropriate approach(13). Normal reference values for sperm morphology are ≥
4% in 2010 WHO criteria(9), while are >3% in that of
c) Sperm vitality 1992(16). As for the 1999 WHO declaration, IVF
Vitality of sperms is defined by membrane integrity success has been stated to be markedly reduced when
of cells. Proportion of vital sperms is determined by normal sperm morphology is < 15%(12). The reference
counting total of 200 cells. According to the WHO value for normal sperm morphology determined by
criteria this is recommended to be performed routinely Kruger is > 14%(15). 1999 vs. 2010 the WHO reference
on all semen samples. If particularly the proportion values for normal sperm morphology are given in
progressively motile sperms is ≤ 40%, vitality test Table I.
should definitely be performed(9). In evaluation of
sperm vitality, staining tests (Eosin Y, Eosin-nigrosin) Table I: Normal sperm morphology criteria determined WHO in
and hypoosmotic swelling (HOS) test are performed. 1999 an 2010(9,12).
According to the latest WHO criteria vitality of more PARAMETERS WHO 1999 (12) WHO 2010 (9)
than 58 in staining tests and HOS test has been Head
determined as normal reference value(9). Width 2.5-3.5 µm 2.8 µm
Length 4.0-5.0 µm 4.1 µm
Length/witdh ratio 1.5-1.75 1.5
d) Differentiation of round cells and leukocytes Acrosomal region should cover 40-70% of the head
In semen, other than sperms, there are round cells such Neck and middle piece
as epithelium and prostate cells, spermatogenic cells Width <1.0 µm 0.6 µm
Length 1.5x of the length 4.0 µm
(immature germ cells) and leukocytes. In classical
of the head
semen analysis, immature germ cells and leukocytes Cytoplasmic residues normal head area < 1/3
can easily be confused with each other. Leukocytes in Tail
ejaculate are specifically formed of neutrophills. In Length ~45 µm
Width < middle piece
circumstances such as infection, reduction of ejaculate
volume and sperm dysfunction due to oxidative stress
there may be excess of leukocyte in semen. In normal
semen leukocyte count must be < 1x 106/ml. When NORMAL REFERENCE VALUES IN SEMEN
the number of round cell in semen is > 1x 106/ml, ANALYSIS
leukocyte diagnosis test may be performed. In
determining leukocyte count, test for the presence of Limits which we consider as normal reference values
intracellular peroxidase and specific antigen tests are in semen analysis are values obtained based on the
used. In the test based on intracellular peroxidase, comparison of semen parameters in fertile and infertile
leukocytes are stained in brown using benzidine and couples (17-19) . On the other hand, it should be
hydrogen peroxide added to the medium. Leukocytes recognized that normal reference values do not always
are thus differentiated from other cells(14). reflect the absolute minimum values necessary for
pregnancy to possibly occur. There are many men who
e) Morphological evaluation are fertile despite being out of normal limits and there
Two smears are prepared from semen sample and are those who are infertile despite being within normal
allowed to dry in the air. After that, these smear samples limits. However, in approaching a normal couple,
are fixed and stained (Papanicolaou, Shorr, Diff-Quick). abnormal semen analysis results suggest male factor

29 J Turk Soc Obstet Gynecol 2012; 9: 25- 31

Ömer Lütfi Tap›s›z et al.

and require additional clinical and laboratory evaluations TERMINOLOGY IN SPERM ANALTSIS
(4). The importance of evaluating all parameters as a ACCORDING TO WHO (2010) CRITERIA(9)
whole should always be remembered. For example, if
semen volume, sperm motility and abnormal sperm Normozoospermia: Sperm sample complying with
ratios are at or within normal limits, slightly low sperm (fertile) the WHO parameters.
density would not be a very important issue. Among Oligozoospermia: Spermatozoa concentration
semen parameters, those that have the greatest effect lower than 15 million/ml.
on fertility are concentration, motility and morphology. Asthenozoospermia: Proportion of progressively motile
The risk in terms of fertility increases 2-3 fold if there spermatozoa less than 32%.
is a problem related to one of these parameters, 5-7 Teratozoospermia: Proportion of spermatozoa with
fold if there is a problem in two and 16-fold if in all normal morphology less than < 4%.
three(20). Oligoasthenoteratozoospermia: Co-presence of above
How to perform semen analysis has been determined described three defects
in detail by the WHO through new regulations made in terms of number,
over the years (9,12,16,21) . Presently, however, motility and morphology
methodology and reliability of semen analyses indicates severe male
performed in many centers show marked differences. infertility.
What is recommended is to perform semen analysis Azospermia: Absence of spermatozoa in ejaculate
in laboratories with certain standards confirmed by Criptozoospermia: Presence of spermatozoa after
quality control programs (eg., Clinical Laboratory high speed centrifuge, which is
Improvement Amendments CLIA; absent in fresh sample.
www.hcfa.gov/medicaid/clia/cliahome.htm)(22). The Necrozoospermia: Less vital, more immotile
WHO has published new reference values for semen spermatozoon in ejaculate.
parameters in 2010(9). In Table II, the most recent Leukospermia: Presence of leukocyte in ejaculate
normal reference values for semen analysis published in an amount greater than reference
by the WHO are given in comparison with those values.
published in(9,12). Aspermia: Absence of ejaculate.

Table II: Lower reference values accepted for semen parameters

published by the WHO in 1999 and 2010. CONCLUSION
PARAMETERS WHO 1999 (12) WHO 2010 (9)
Semen volume (ml) ≥2 1.5 (1.4-1.7) In approaching to an infertile couple, semen analysis
Total sperm count (106/ejaculate) 40 39 (33-46) appears to be the most essential first-to-be-done
Sperm concentration (106/ml) 20 15 (12-16) laboratory test for investigating the presence or absence
Total motility (PR+NP, %) (a+b+c) >50 40 (38-42)
of a male-related factor. To provide a more correct
Progressive motility (PR, %) a+b >50, a >25 32 (31-34)
Vitality (vital spermatozoa, %) 75 58 (55-63) result, semen analysis should include the evaluation
Sperm morphology of at least two samples taken at four weeks interval.
(Normal forms, %) 30 (WHO), ≥4 In terms of standardization and obtaining proper results,
14 (Kruger)
the importance of complying with the necessary sexual
pH > 7.2 > 7.2
Peroxidase positive leukocyte (106/ml)< 1.0 < 1.0
abstinence time and collection conditions should be
MAR test (particle-bound, appreciated. It should be noted that evaluation should
motile spermatozoa, %) < 50 < 50 be performed by specially trained relevant people in
Immunobead test (test positive centers that meet certain conditions. An analysis
motile spermatozoaa, %) < 50 < 50
performed in compliance with standards and concluded
PR: Progressive, NP: Non-progressive, MAR: Mixed antiglobulin reaction. objectively using criteria set forth by the WHO shall
play an important role in the process in which clinicians
determine the route they will follow in terms of
treatment. For reaching this result, this review provides

J Turk Soc Obstet Gynecol 2012; 9: 25- 31 30

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