Preparation and Selection of

Sperm for IVF and ICSI

LAB&&100
Lesson 7

AMERICAN SOCIETY FOR REPRODUCTIVE MEDICINE

REPRODUCTIVE SCIENCES CONTINUING EDUCATION

© 2014 American Society for Reproductive Medicine

Table of Contents

Course Overview ............................................................................................................................ 1

Exam Questions .............................................................................................................................. 4

Lesson Content ............................................................................................................................... 6

References ...................................................................................................................................... 26

These materials are copyrighted by the American Society for Reproductive Medicine. They are intended
for personal use and may not be reproduced or distributed without permission.
 Course Overview

ACTIVITY NUMBER: LABCC100 Lesson 17

ACTIVITY TITLE: Preparation and Selection of Sperm for IVF and ICSI

ACCREDITATION STATEMENT
The American Society for Reproductive Medicine is accredited by the Accreditation Council for
Continuing Medical Education to provide continuing medical education for physicians.

CREDIT DESIGNATION STATEMENT

The American Society for Reproductive Medicine designates this enduring material for a maximum of
0.5 AMA PRA Category 1 Credits™. Physicians should only claim credit commensurate with the extent
of their participation in the activity.

The American Society for Reproductive Medicine has been approved to provide Professional Enrichment
Education Renewal (PEER) credit through the American Board of Bioanalysis. Credits are pending for
this activity.

Release Date: October 15, 2014

Expiration Date: October 15, 2017

Estimated Time to Complete Activity: 30 minutes

NEEDS ASSESSMENT and IDENTIFICATION OF PRACTICE GAP

With developments in assisted reproductive technologies (ART) for the treatment of female infertility,
has come the development of and increasing use of ART for treatment of male factor infertility. Sperm
preparation techniques have also evolved to optimize conditions for sperm capacitation in preparation
for interaction with the oocyte, and presumably, successful fertilization. Several techniques for sperm
preparation have been developed that select for viable spermatozoa that can be used for standard
oocyte insemination or intracytoplasmic sperm injection (ICSI). Which procedure to use will depend not
only on the type of insemination (standard vs ICSI), but also on the quality of the fresh ejaculate,
including sperm concentration, motility and normal morphology.

This presentation provides an overview of the sperm preparation techniques, including conventional
preparation procedures, nutrient media, and supplements as they relate to the insemination technique.
Procedures for handling testicular biopsy specimens and epididymal aspirates, as well as special sperm
selection techniques will be discussed. This information is useful for clinicians, laboratory scientists,
embryologists and andrologists who will be involved directly or indirectly in the evaluation of the male
partner of the ART couple, and the preparation of sperm for use in ART cycles. The educational need for
this activity was based on the 2014 gap analysis review by the ASRM CME Committee.

EDUCATIONAL OBJECTIVES
At the conclusion of this educational activity, participants should be able to:
1. Discuss the goals of any sperm preparation technique to obtain sufficient viable sperm for
insemination of retrieved oocytes.
2. Discuss the laboratory methodology for sperm preparation for assisted reproduction, including

1
 media and supplements, swim-up and density gradients, testicular and epididymal sperm, and fresh
versus frozen sperm.
3. Discuss special techniques for sperm preparation including use of hypoosmotic buffer, hyaluronan
binding, calcium ionophore, and intracytoplasmic morphologically selected sperm injection
(IMSI)/motile sperm organelle morphology examination (MSOME) procedures.

TARGET AUDIENCE
This activity is designed to meet the educational needs of new and experienced laboratory scientists and
allied health professionals involved in assisted reproduction.

ACGME COMPETENCIES
Medical Knowledge
Patient Care

SUCCESSFUL COMPLETION REQUIREMENTS

Successful completion of this educational activity requires the learner to:
• View a course overview page, containing all CME and disclosure information, including
acknowledgement of commercial support and disclosure of unlabeled use, prior to the start of each
module.
• Complete a 10-question pre-exam prior to each module. Learners should note any pre-exam
questions answered incorrectly for clarification during module study.
• Be given the option of downloading a printed syllabus containing the presentation and narrative.
• Participate in the interactive activity: Audio narration is synchronized with PowerPoint presentation
that can be advanced, stopped or reversed as desired.
• Complete a 430-question post-exam, with feedback of correct/incorrect answers, scoring a
minimum of 70% in two attempts.
• Complete the evaluation survey.
• Print certificate of completion.

DISCLOSURES FOR PLANNERS AND FACULTY

Planners
Susan A. Gitlin, PhD: Nothing to Disclose
Michael Reed, PhD: Nothing to Disclose
Andrew R. La Barbera, PhD: Nothing to Disclose
Richard H. Reindollar, MD: Nothing to Disclose
Nancy Bowers, BSN, RN, MPH: Nothing to Disclose

Content Developers and Reviewers

 Morgan Amick, BS: Nothing to Disclose  Bruce Carr, MD: Grant/Research: AbbVie, Medicines
 Anthony Anderson, DSc: Nothing to Disclose 360; Speakers Bureau: Shionogi, Noven, Pfizer
 Marlane Angle, PhD: Consultant: Previvo  Marcelle Cedars, MD: Grant/Research: Nora
 C. Brent Barrett, PhD: Consultant: ReproSource Therapeutics; Ferring Pharmaceuticals
Fertility Diagnostics  Grace Centola, PhD: Consultant: New England
 Charles Bormann, PhD: Nothing to Disclose Cryogenic Center; Cryos International‐NY, Central
Park Fertility

2
 Carli Chapman, MS: Nothing to Disclose  Z. Peter Nagy, MD, PhD: Direct stockholder: My Egg
 Ri-Cheng Chian, PhD: Other: Cooper Surgical/SAGE Bank; Paid consultant: Origio, Fertilitech; Speakers
Company bureau: Merck MSD
 Susan Crockin, JD: Consultant: BMS, Merck,  Mike Neal, MSc: Nothing to Disclose
Prometheus; Advisory Board: GSK, CoStim, Aveo;  Fariba Nehchiri, MSc: Nothing to Disclose
Speakers Bureau: Pfizer Canada, BMS  Sergio Oehninger, PhD: Nothing to Disclose
 Judith Daar, JD: Nothing to Disclose  Kimball Pomeroy, PhD: Nothing to Disclose
 Erma Drobnis, PhD: Nothing to Disclose  Thomas (Rusty) Pool, PhD: Other: Auxogyn
 Kathryn Go, PhD: Nothing to Disclose  Marc Portmann, MHA, MT: Nothing to Disclose
 Kay Graff, MS: Consultant: Steptoe Therapeutics;  Catherine Racowsky, PhD: Consultant: Nora
Employee: Pelton & Crane Therapeutics
 Elizabeth Grill, PsyD: SouthEastern Fertility: Speakers  Lisa Rinehart, JD: Nothing to Disclose
Bureau  William Roudebush, PhD: Nothing to Disclose
 Shalini Gunawardena, RN, BSN: Walgreen's Nursing  Denny Sakkas, PhD: Consultant: Good Start Genetics;
Advisory Board Other: Fertilitech, Origio
 Lee Higdon, PhD: Nothing to Disclose  Mitchel Schiewe, PhD: Nothing to Disclose
 Kristen Ivani, PhD: Nothing to Disclose  Ira Sharlip, MD: Consultant: Pfizer, Lilly, Absorption,
 Sangita Jindal, PhD: Nothing to Disclose Vyrix
 Emily Jungheim, MD: Spouse is paid consultant:  Scott Smith, PhD: Product Review: Biocoat
Abbvie, Genentech, Spectrum, Celgene; Speakers  Amy Sparks, PhD: Nothing to Disclose
bureau: Genentech  Laurel Stadtmauer, MD: Nothing to Disclose
 Levent Keskintepe, DVM, PhD: Nothing to Disclose  Jason Swain, PhD: Consultant: Irvine Scientific
 Ann Kiessling, PhD: Nothing to Disclose  Tyl Taylor, MSc: Consultant; Spouse employed:
 Rebecca Krisher, PhD: Grant/Research: Serono Biodiseño
 Martin Langley, BS: Nothing to Disclose  Helen Tempest, PhD: Nothing to Disclose
 Michael Lee, MS: Consultant: Cook Medical  James Toner, MD, PhD: Speakers bureau: Merck
 Bruce Lessey, MD, PhD: Nothing to Disclose  Nathan Treff, PhD: Nothing to Disclose
 Dennis Matt, PhD: Nothing to Disclose  Tom Turner, MS: Nothing to Disclose
 David McCulloh, PhD: Consultant: Infertility and IVF  Matthew VerMilyea, PhD: Consultant: Auxogyn, Irvine
Medical Associates of Western NY; ReproART; Scientific, Genea Biomedx
Biogenetics Corporation; Employee: NYU Langone  Michael Vernon, PhD: Nothing to Disclose
Medical Center  Diane Wright, PhD: Nothing to Disclose
 Peter McGovern, MD: Nothing to Disclose  Hang Yin, PhD: Nothing to Disclose
 Yves Menezo, DSc, PhD: Consultant: Nurilia

It is the policy of the ASRM to ensure balance, independence, objectivity, and scientific rigor in all its
educational activities. All faculty/authors participating in this activity were required to disclose any
relationships they may have with commercial entities whose products or services are used to treat
patients so that participants may evaluate the objectivity of the presentations. The content and views
presented in this activity are those of the faculty/authors and do not necessarily reflect those of the
ASRM. Any discussion of off-label, experimental, or investigational use of drugs or devices will also be
disclosed. The disclosure statements were reviewed by the Subcommittee for Standards of Commercial
Support of the CME Committee of ASRM and any perceived conflicts of interest were resolved in
accordance with the policies of the ACCME.

STATEMENT OF SUPPORT
No commercial support has been provided for this activity.

3
 Exam
1. The pH of the culture medium used for sperm washing can be maintained using one or more buffer
systems. Which of the following buffer systems is most commonly used for sperm preparation?
a. MOPS
b. HEPES
c. Calcium-magnesium free buffer
d. Saline

2. Sperm capacitation and motility can be enhanced by

a. Incubation of washed sperm at 4°C for 1 hour
b. Incubation of washed sperm with pentoxyfylline
c. Incubation of washed sperm with denuded oocytes
d. Incubation of washed sperm with prostaglandin F

3. Which of the following sperm preparation methods guarantees removal of virus such as HIV or
hepatitis B from the washed sperm fraction?
a. Density-gradient centrifugation
b. Microfluidics
c. Glass-wool filtration
d. None of the above

4. The hypoosmotic swelling test can be used to

a. Choose viable, non-motile sperm for intracytoplasmic sperm injection (ICSI)
b. Enhance the fraction of motile normal sperm to be used for IVF/ICSI
c. Slow down sperm so that one can be picked up for ICSI
d. Remove the sperm tail prior to ICSI

5. A simple wash procedure appears to be best for

a. Specimen with large amounts of debris
b. Oligozoospermic specimen with count <20 million
c. Specimen with increased leukocytes
d. Epididymal sperm

4
6. Which of the following sperm-processing methods does not remove seminal leukocytes and reactive
oxygen species (ROS)?
a. Single-layer density-gradient centrifugation
b. Glass wool filtration
c. Hyaluronan-binding selection
d. Simple wash

7. Mature sperm will bind to which of the following substances used to coat a specially designed dish,
allowing easy pickup for ICSI?
a. Hyaluronidase
b. Platelet-activating factor (PAF)
c. Hyaluronan
d. Follicular fluid

8. Improved implantation and pregnancy rates have resulted from

a. Sperm cryopreservation
b. Use of intracytoplasmic morphologically selected sperm injection (IMSI)/motile sperm organelle
morphology examination (MSOME) procedures.
c. Incubation of sperm with pentoxyfylline
d. Use of glass-wool filtration to process sperm

9. Common supplements to culture media for sperm preparation include

a. Gelatin
b. Human serum albumin
c. Follicular fluid
d. Cholesterol

10. A male partner provides a semen specimen on the day of egg retrieval with the following
parameters: 28 million sperm/mL; 40% motility; 8% normal morphology; some debris and >4 white
blood cells/high power field. What method is best for preparation of this ejaculate for IVF?
a. Density-gradient centrifugation followed by swim-up
b. Dextran gel column filtration followed by swim-up
c. Microfluidic quartz chamber
d. Simple-wash centrifugation

5
 Welcome to the American Society for Reproductive
Preparation and Selection of Medicine’s eLearning modules. The subject of this
Sperm for IVF and ICSI presentation is Preparation and Selection of Sperm
for IVF and ICSI.
AMERICAN SOCIETY FOR
REPRODUCTIVE MEDICINE

Embryology Certificate Course

At the conclusion of this presentation, participants

Learning Objectives
should be able to:
● At the conclusion of this presentation, participants should be •Discuss the goals of any sperm preparation
able to: technique to obtain sufficient viable sperm for
1. Discuss the goals of any sperm preparation technique to
obtain sufficient viable sperm for insemination of retrieved
insemination of retrieved oocytes.
oocytes. •Discuss the laboratory methodology for sperm
2. Discuss the laboratory methodology for sperm preparation for
assisted reproduction, including media and supplements,
preparation for assisted reproduction, including
swim-up and density gradients, testicular and epididymal media and supplements, swim-up and density
sperm, and fresh versus frozen sperm.
gradients, testicular and epididymal sperm, and fresh
3. Discuss special techniques for sperm preparation including
use of hypoosmotic buffer, hyaluronan binding, calcium versus frozen sperm.
ionophore, and intracytoplasmic morphologically selected •Discuss special techniques for sperm preparation
sperm injection (IMSI)/motile sperm organelle morphology
examination (MSOME) procedures. including use of hypoosmotic buffer, hyaluronan
binding, calcium ionophore, and intracytoplasmic
morphologically selected sperm injection
(IMSI)/motile sperm organelle morphology
examination (MSOME) procedures.

6
 The goal of any sperm preparation method is first to
Goals of Sperm Preparation Procedures remove sperm from inhibitory factors present in the
● Remove sperm from inhibitory effects of the seminal plasma. seminal plasma. Factors in seminal plasma inhibit
● Obtain sufficient viable and morphologically normal sperm for spermatozoa from undergoing capacitation and the
insemination of retrieved oocytes.
acrosome reaction and reduce the ability to
successfully fertilize an oocyte. In addition, it is
obvious that the ultimate goal of any sperm
preparation method is to obtain sufficient viable and
morphologically normal sperm for use in the selected
assisted reproductive technology (ART).

Bormann et al., 2010

Ideally, sperm preparation should be quick, easy, and

Ideally, Sperm Preparation Should...
nontoxic, isolating the highest concentration of
● Be quick and cost-effective motile, viable, and morphologically normal sperm.
● Isolate the highest number of motile/viable/normal sperm Procedures should also remove dead cells,
– Note, sperm do not need to be motile for IVF. leukocytes, bacteria, and toxic or bioactive
● Remove dead cells, leukocytes, bacteria, and contaminants
that would be toxic to oocytes.
substances—all of which would be harmful to
● Allow processing of high-volume ejaculates, and fresh or oocytes. Finally, the process should allow processing
frozen semen specimens. of high-volume ejaculates as well as fresh or frozen
specimens and tissue biopsies, such as testicular
biopsy tissue or epididymal aspirates. No one
technique exhibits all of these requirements.
Pagani et al., 2009;
Note that sperm do not need to be motile to be used
Mehta & Sigman, 2014
for in vitro fertilization (IVF), or intracytoplasmic
sperm injection (ICSI). The sperm must be viable and
morphologically normal. The technique of choice will
depend on both the quality of the sperm specimen
and the end use of the sperm for IVF, ICSI, or
intrauterine insemination (IUI).

7
 In summary, sperm isolation procedures should
Benefits of Sperm Isolation Techniques
remove or minimize viruses, bacteria, dead cells, and
● Removes or minimizes bacteria, viruses, antibodies, dead cells, leukocytes from the sperm, since exposure can be
leukocytes harmful to the sperm and to the oocytes. In doing so,
● Extends “shelf life” of sperm and promotes capacitation by
removal of seminal fluid components
such a procedure will extend the life of sperm, and
– Prostaglandins promote capacitation and the acrosome reaction.
– Oxygen radicals - reactive oxygen species (ROS) Finally, any sperm preparation procedure should
– Capacitation inhibitors
● Ability to resuspend sperm in a volume and concentration
result in a final volume of sperm fraction that is
appropriate for final use appropriate for the final use of the sperm: IVF, ICSI,
IUI.

Although sperm are typically thought of as originating

Semen Source
from ejaculated semen, many different sources exist.
● Ejaculated Sperm can either be ejaculated or surgically
– Fresh versus frozen retrieved. Ejaculated sperm include fresh semen,
– Electroejaculate
frozen-thawed semen, electroejaculated semen, and
– Vibration-induced ejaculation
– Retrograde retrograde semen, which is semen recovered from
● Surgically retrieved the urine. Special preparations are necessary to
– Fresh versus frozen obtain electroejaculated semen from spinal cord-
– Epididymal
– Testicular
injured men, as well as semen from the bladder in
men with retrograde ejaculation (due to diabetes,
result of pelvic surgery, etc.). Surgically retrieved
Image courtesy of Gianpiero Palermo, MD, PhD

sperm originate from epididymal aspiration or

testicular biopsy.

Raw semen must undergo processing in order to best

Spermatozoa Selection
prepare the sperm for fertilization. Several
• Simple wash: Centrifugation techniques have been developed to isolate sperm for
• Sperm migration: Swim-up use in assisted reproduction. These include 1) simple
• Density gradient wash-centrifugation; 2) migration techniques
Filtration
•
Semen
Seminal Live Sperm
in Fresh including swim-up from a washed pellet or directly
• New techniques: Microfluidics Plasma
Medium

from semen; 3) density-gradient centrifugation; 4)

Density
Debris &
Dead Sperm
filtration through glass-wool or dextran gel beads;
Gradient
and 5) new techniques including microfluidics. Each
of these procedures will be reviewed briefly.
Live
Sperm

Density Gradient

8
 The simplest technique for preparing sperm is a
Simple Wash Overview
simple wash procedure, where the fresh semen,
• Simplest of the IUI sperm preparations to perform diluted with nutrient media is centrifuged, usually
• Removes seminal fluids and concentrates the sperm into a twice, and the supernatant removed. The final pellet
workable volume
of sperm is resuspended in a volume appropriate for
• Not recommended with high concentrations of dead sperm,
cellular contamination, or debris the end use of the sperm. A simple wash procedure
• Good choice for samples with borderline concentration is not recommended for specimens with high
• <20 million/mL in initial sample
concentrations of dead sperm or debris, since dead
sperm, cells, and debris would not be removed, and
would be pelleted with the live sperm. This technique
is advantageous for specimens clean of debris and
cells, and those with lower concentrations. A single
wash-centrifugation method is commonly used for
frozen-thawed sperm.

A semen analysis should be conducted on each

Simple Wash Procedure
sample before and after the preparation. If a sample
1. Place semen in sterile centrifuge tube. is highly viscous, it may be necessary to either give
2. Add at least double the volume of sperm
wash media.
the sample more time to liquefy, draw it back and
3. Cap tightly, mix by inverting the tube 2–3 forth through a blunt needle and syringe or small
times, and centrifuge 10 min @ 300 g.
4. Decant supernatant, resuspend, and
pipette to help break up the sample, or wash it a
perform count and motility. second time if the sperm do not pellet well at the
– Repeat for second centrifugation.
5. Decant supernatant, resuspend in bottom of the tube after an initial wash. Generally
appropriate volume for concentration
needed for oocyte insemination.
speaking, it is best to minimize manipulation of the
sperm, such as with extra washes, etc. The basic
wash procedure is to place the semen in a sterile
centrifuge tube and add at least double the volume
of sperm wash media. After capping tightly, mix by
inverting the tube 2–3 times then centrifuge 10
minutes at 300 g. This step is generally repeated for a
second centrifugation/wash. Finally, decant the
supernatant, and resuspend pellet in 0.6-0.7 mL fresh
media for insemination. Once a preparation is
completed, the final sample should be drawn into a
syringe or packaged in some way for use, such as for
IUI, or placed into a sterile tube and provided to the
IVF lab.

9
 Advantages and disadvantages of the basic wash
Simple Wash
procedure are listed here. Advantages include that it
is easy and quick to perform, uses sterile plasticware
Advantages Disadvantages
● Simple and quick to perform ● Motile sperm concentrated in (centrifuge tubes, pipettes, media), and takes little
● High recovery of sperm presence of dead sperm, white
blood cells, and other seminal
time (30 minutes including all steps). This procedure
● Requires minimal processing of
sperm debris, which expose sperm to also results in a high recovery of sperm and requires
higher levels of reactive oxygen
species that may negatively affect minimal processing steps. However, with a simple
sperm function
● Usually not recommended alone wash, motile sperm are concentrated in the presence
for IVF (standard insemination)
of non-motile and dead sperm, white blood cells, and
other seminal debris. This exposes sperm to higher
levels of reactive oxygen species that may negatively
affect sperm function.
Image courtesy of Gianpiero Palermo, MD, PhD

A second, commonly used sperm preparation

Swim-up Procedure
procedure is a swim-up. This procedure allows sperm
● Allows motile sperm to migrate up (or migrate down) into a to migrate, or swim up into a fresh layer of nutrient
fresh layer of nutrient medium medium. Motile sperm are a prerequisite since
● Clean fraction of highly motile sperm obtained motile sperm can only migrate into the media. With
– Yields are low, but motility is high
o Not a problem for use in IVF/ICSI since required sperm motile swim-up, a clean fraction of highly motile sperm can
concentration is not necessarily a concern.
be obtained, but the concentration of motile sperm is
● Best for semen samples with high concentration and motility
low. The swim-up is best for specimens with high
concentration and motility. With IVF/ICSI procedures,
low yield is not usually a concern since fewer sperm
are needed for oocyte insemination.

There are several variations of how a swim-up can be

Swim-up Procedure
performed. The semen can be diluted with nutrient
1. Perform a simple wash on media, centrifuged into a pellet. Fresh media can be
the semen sample. layered over the washed pellet or a resuspended
– Resuspend to 0.6 mL
2. Gently layer 1 mL of fresh
sample. The top layer can be removed without
media on top of the disturbing the bottom and further concentrated or
resuspended pellet.
diluted depending on the insemination (IUI or
3. Incubate the sample at 37o
C for 1 hour. IVF/ISCSI). The protocol shown here uses a
4. Remove 0.6–0.7 mL of the resuspended sample rather than a pellet to reduce
top layer, without
disturbing the bottom, for the exposure of healthy sperm to reactive oxygen
the insemination. species that could negatively affect sperm function. It
is also helpful to incubate the swim-up at an angle, if
possible, to increase the surface area between the
layers and improve motile sperm recovery.

10
 The swim-up method is cost-effective and provides
Swim-up from Washed Sperm Pellet
for a very “clean,” highly motile final sample. It does,
however, take longer to perform than other
Advantages Disadvantages
● Cost-effective. ● Centrifugation of semen into a
preparations and the final concentrations may be
● Very “clean,” highly motile final pellet increases exposure of
motile healthy sperm to dead
low. A disadvantage of the swim-up from pellet
sample
cells and debris, ROS. technique is once again the problems with
● Not advantageous for specimens
with reduced motility and/or centrifuging raw semen into a pellet: motile, viable
concentration, or non-motile
sperm
sperm are centrifuged with dead sperm, cells and
● Takes longer time to perform debris, increasing the exposure of the “good” sperm
to oxygen radicals (ROS). The swim-up method also
takes longer to perform, and is not of benefit to
specimens with reduced motility and/or
ROS = reactive oxygen species
concentration.

With swim-up from semen, there is no initial

Swim-up from Semen
centrifugation of the semen into a pellet. Fresh media
1. Gently layer 1–2 mL of is layered onto the semen and incubated for
nutrient media over the fresh approximately 1 hour to allow motile sperm to
semen.
2. Incubate at 37o C for 1 hour.
migrate into the nutrient media. The top layer of
3. Remove most of the top layer, media, presumably containing the motile sperm, is
avoiding the semen layer. removed, and may be washed again to remove
4. May wash-centrifuge and
resuspend in fresh media
remnants of seminal fluid, resuspending the sperm
prior to use in IVF fraction in fresh media.
insemination.

The swim-up method is also cost-effective and

Swim-up from Semen
provides for a very “clean,” highly motile final
sample. It does take longer to perform than other
Advantages Disadvantages
● Cost effective ● Low yield of motile sperm
preparations and the final concentrations may be
● High concentration of motile and ● Exposure of motile sperm to low, although this might not necessarily be a concern
morphologically normal sperm semen for a longer period of time
● No contamination from non-germ ● Takes longer time to perform for IVF/ICSI. Most importantly, this method does not
cells, debris, and seminal
contaminants
require initial centrifugation of the semen, and thus
● Does not require initial avoids exposure of viable sperm to ROS from dead
centrifugation, thus no exposure
to ROS cells and leukocytes. It is important to set up the
tubes, and begin the incubation as soon as possible
after semen liquefaction to reduce exposure of the
sperm to the seminal fluid.

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 The density-gradient preparation works best for
Density Gradient Overview
samples with a normal or high initial concentration of
● Best for samples with normal or high initial concentration of motile sperm, because only a percentage of the
motile sperm. motile sperm are recovered. It should be
– Only a percentage of the motile sperm are recovered.
recommended in all cases where white blood cells in
● Sperm filtered through layers of silane-coated silica particles
suspended in nutrient media. the semen sample exceed the normal baseline. In
● Sperm with good motility pass through the layer interfaces density-gradient preparations, sperm are filtered
leaving seminal fluid, non-motile cells, contamination, and
debris behind. through layers of silane-coated silica particles
● Pellet of motile sperm at the bottom of the centrifuge tube suspended in nutrient media. Sperm with good
can be recovered, washed free from gradient media, and used
for insemination.
motility pass through the layer interfaces leaving
– May be followed by swim-up from the pellet to further select seminal fluids, non-motile cells, contamination, and
for highly motile sperm and reduce concentration for oocyte
insemination.
debris in the interface between the gradient layers
and semen. The pellet of motile sperm at the bottom
of the centrifuge tube can be recovered, washed free
from the gradient media, and used for insemination.
For IVF, density-gradient centrifugation can be
followed by a swim-up to further select for a highly
motile and clean preparation.

The density gradient preparation can be performed

Density Gradient: Discontinuous 2-Layer Protocol
with several variations. First, prepare a gradient
● Prepare gradient column
1. Pre-centrifugation column by placing 1.5 mL of 90% gradient media in a
– 1.5 mL of 90% gradient media in a
centrifuge tube John Doe
centrifuge tube and gently layer an additional 1.5 mL
of 40% gradient media on top of the 90% media.
MRN: 585
– Gently layer an additional 1.5 mL DOB 1/1/8
2. Post-centrifugation
of 40% gradient media on top
● Layer up to 3 mL of semen on top Next, layer up to 3 mL of semen on top of the 40%
of the 40% layer
John Doe

● Centrifuge for 15-20 minutes at

MRN: 585
DOB 1/1/8
layer and centrifuge for 15 minutes at 300 x g. Each
280-300x g Semen centrifuge has to be set for speed based on
● Remove and discard the
revolutions per minute, to equal the g force (300 x g)
40%
supernatant and gradient media to
90%
just above the sperm pellet
● Wash the sperm pellet with 3-5 mL
that is needed. Remove and discard the supernatant
and gradient media to just above the sperm pellet.
Remove
of sperm wash media. Centrifuge at
300x g for 10 minutes.
● Decant and resuspend to final Sperm pellet
Wash the sperm pellet with 3-5 mL of sperm wash
volume OR perform swim-up.
media, then decant, and resuspend to final volume.
The concentration of the gradient media layers can
be altered, or a single layer of concentrated gradient
media can be used, if preferred. As a general rule,
the total volume of semen processed should not
exceed the volume of the media in the gradient
column. Recovery of motile sperm can also be
decreased if the gradient layers are too large. If it is
desired to process more semen, extra tubes with
gradient columns can be prepared and used.

12
 The density-gradient preparation can be performed
Density Gradient: 1-layer Protocol
using one layer of gradient solution, usually 80% or
● Prepare gradient column 1. Pre-centrifugation 90%. The procedure is the same for all subsequent
– 1.5 mL of 90% gradient
media in a centrifuge tube John Doe
steps.
MRN: 585

● Layer up to 1.5 mL of semen on DOB 1/1/8

2. Post-centrifugation
top of the 90% layer
● Centrifuge for 15–20 minutes John Doe
MRN: 585
● Remove and discard the DOB 1/1/8

supernatant and gradient media

to just above the sperm pellet
● Wash the sperm pellet with 3-5 Semen

mL of sperm wash media 90%

● Decant, and resuspend to final
volume OR perform swim-up Remove

Sperm pellet

The density gradient preparation filters out

Density Gradient Centrifugation
nonmotile sperm, amorphous cells, white blood cells,
and debris; the final sample has an increased
Advantages Disadvantages
● Filters out nonmotile sperm, ● More involved, takes longer to
percentage of motile sperm, and exposure of the
amorphous cells, white blood
cells, debris
perform, requires special media sperm to reactive oxygen species is reduced.
● Only a fraction of the motile
● Final sample has increased sperm is recovered. May not be a Compared with other methods, the procedure takes
percentage of motile sperm good preparation when sperm
● Reduced exposure of sperm to concentration or motility is longer to perform and requires special media. Only a
ROS reduced.
fraction of the motile sperm is recovered, thus this
may not be a good preparation when sperm
concentration or motility is reduced. Motile sperm
concentration in the final sample often exceeds 90%.
While the density-gradient and swim-up methods do
a good job of providing a “clean” final sample, no
preparation can guarantee a final sample completely
free from infectious agents or cellular debris.

Column filtration methods are based on the fact that

Column Filtration Method
dead or dying sperm are sticky and will attach to
● Glass wool filtration glass-wool or dextran gel beads even in the presence
● Cross-linked dextran gel column filtration of high protein concentrations. The preparation of
– Dead or dying sperm are extremely sticky and will adhere to the glass
fibers or dextran gel beads.
the column can be somewhat difficult, and with glass-
wool columns, it is important to rinse the column
thoroughly to remove loose glass-wool fibers. These
methods also require an initial wash centrifugation of
the semen, with layering of the washed and diluted
pellet onto the column for a second centrifugation
step.
Mortimer and Mortimer, 1992;
Bormann et al., 2010.

13
 Glass wool filtration results in a similar recovery of
Glass-Wool Filtration
motile sperm as in the density-gradient
centrifugation method, yielding an average of 50%-
Advantages Disadvantages
● Advantageous for ● Final product not as clean 70% progressively motile sperm. It is most successful
asthenozoospermic specimens or
those with sperm demonstrating
● Is not a commonly used for specimens with a high percentage of sluggishly
procedure
an abnormal hypoosmotic swelling ● Slightly more expensive and can motile sperm, or low total motility
(HOS) test 1,2
● Uses entire ejaculate
be time-consuming and difficult
to make the glass-wool column (asthenozoospermia), or where viability is low by the
● Separates sperm from urine in
cases of retrograde ejaculation3
● Initial centrifugation may result in hypoosmotic swelling (HOS) test. Glass-wool
increased ROS
● Eliminates leukocytes, and thus filtration also uses the entire ejaculate, thus
reduces ROS
● Yields significantly more increasing the probability of recovering more sperm,
chromatin condensed sperm than
swim-up and gradient
removes leukocytes, and effectively separates sperm
centrifugation 4 1
3
Remrev et al., 1989; 2 Borman et al., 2010;
Henkel et al., 2003; 4 Henkel et al., 1994
from urine following retrograde ejaculation.
However, glass-wool filtration results in a sperm
fraction that may not be as clean as other methods,
particularly with its risk of contamination with glass-
wool fibers. The set-up is more costly and time-
consuming to prepare the columns. All in all, glass-
wool filtration column filtration is not a commonly
used procedure for sperm preparation for ART.

Cross-linked dextran gel-column filtration is based

Cross-Linked Dextran Gel-Column Filtration
upon the same principles as the glass-wool filtration
columns. This type of filtration results in a high yield
Advantages Disadvantages
● High yield of motile sperm. ● Added expense
of motile sperm, and is more advantageous for
● Best for specimens with normal ● Not as clean as other methods. specimens with normal motility and morphology but
motility and morphology, but ● Initial centrifugation may result in
reduced concentration. increased ROS. reduced concentration. A disadvantage is the added
expense, time, and technique for preparation of the
columns, and the resulting fraction again may not be
as clean as other methods, particularly the density-
gradient centrifugation and swim-up methods.

14
 The most common sperm preparation techniques are
Microfluidics
suboptimal for patients with severe oligozoospermia,
● Alternative to traditional sperm separation since many of these specimens exhibit not only low
procedures, especially for severe
oligozoospermic specimens1
sperm count but large amounts of cells and debris.
● First developed by Kricka and colleagues in Microfluidics has been proposed as an alternative to
19932
– Silicon and glass microfluidic devices traditional sperm separation methods, especially in
– Gravity-driven pumping system,
microscale integrated sperm sorter
Image courtesy of Charles
Bormann, Ph.D. suboptimal, oligozoospermic semen specimens.
(MISS)3 Kricka and colleagues in 1993 first developed
● Significant decrease in DNA fragmentation
as compared to initial semen, as well as microfluidic devices using silicon and glass devices for
following wash centrifugation, density-
gradient centrifugation, and swim-up4
sperm isolation. Such devices use a network of
● Quartz microfluidic system; highly motile branching microchannels that separate or
sperm within 5 minutes, used for ICSI5
1 Bormann et al., 2010; 2 Kricka et al., 1993; 3 Cho et al., 2003;
characterize sperm based upon motility and forward
progression. More recently, Cho and colleagues
4 Schulte et al., 2007; 5 Shibata et al., 2007

developed a gravity-driven pump system, termed a

microscale integrated sperm sorter, or MISS. These
systems contain inlet/outlet ports, fluid reservoirs,
gravity-driven power sources, and converging
microchannels with laminar flow. All components
then facilitate sperm sorting. Nonmotile sperm, cells,
and debris are moved into the waste reservoir.
A more recent study compared microfluidic sperm
sorting with wash centrifugation and density-gradient
centrifugation. This group demonstrated a significant
decrease in sperm with D-N-A fragmentation. In a
quartz microfluidic system, a high percentage of
highly motile sperm were separated within 5 minutes
and used for ICSI, and a 50% fertilization rate was
achieved.

Microfluidic separation systems are simple and safe,

Microfluidics
and result in motile sperm with normal morphology
and reduced DNA fragmentation as compared with
Advantages Disadvantages
● Simple and safe ● Not capable of processing
traditional methods. However, microfluidics devices
● Reduces percentage of entire ejaculate are not capable of processing an entire ejaculate, and
sperm with DNA ● Does not isolate all motile thus do not isolate all motile sperm. However,
fragmentation sperm
adequate numbers of sperm for use in ICSI can be
easily obtained in a short period of time from
severely oligozoospermic specimens.
Microfluidic devices for sperm separation continue to
be developed and tested, and are not yet in the
Bormann et al., 2010; mainstream of standard sperm separation
Schuster et al., 2003
procedures.

15
 Patients may choose to use cryopreserved semen
Preparation of Cryopreserved Sperm for ART
samples for ART for many reasons, including use of
● Reasons for using cryopreserved sperm may include: donor sperm (which must be cryopreserved to
– Use of donor sperm comply with US Food and Drug Administration [FDA]
– Client depositors:
o Military duty or extended travel
regulations) or for those with military duty or
o Samples frozen prior to fertility-damaging treatments extended travel. Some have samples frozen prior to
 Chemotherapy
 Radiation
fertility-damaging treatments such as chemotherapy,
 Vasectomy radiation and vasectomy. When working with
o Collection and storage of multiple samples to be combined for
increased concentration of available motile sperm cryopreserved samples, it is important to realize that
● Cryopreserved samples have a shorter lifespan and should be samples will inevitably have a lower number of motile
subjected to minimal handling during preparation
– Simple wash/centrifugation sperm than they do prior to freezing. As a result,
– Swim-up from thawed semen patients should be counseled on how many vials or
units of sperm to use based on post-thaw analysis.
Most often, patients will purchase at least 2 vials of
donor sperm, or request 2 vials of client depositor
semen for an ART cycle so that adequate numbers of
motile sperm can be recovered for insemination.
When preparing cryopreserved samples for ART,
minimal handling is best, as cryopreserved samples
are less robust than fresh samples. A single
centrifugation basic wash is typically adequate to
remove seminal fluids and cryoprotectants. It is also
important to dilute the thawed semen
cryopreservative slowly, so as to avoid osmotic shock
to the spermatozoa.

With improvements in antiviral treatments and

Preparation of Virus-Positive Specimens
longer survival rates, there are more patients who are
● Goal to completely remove all virus particles from infected positive for HIV or hepatitis who are seeking assisted
semen specimens reproductive treatments. Andrology and embryology
– Is that possible?
– HIV virus binds to leukocytes and macrophages.
laboratories should be well prepared to process both
● Treat such semen specimens using same procedures as sperm and oocytes from virus-positive patients. The
determined by quality of specimen. methodology for sperm preparation for the virus-
● All semen specimens handled using universal precautions
– Separate work bench? Separate laboratory?
positive male would be determined based on the
– Separate storage tank for frozen specimens known to be viral quality of the initial fresh or frozen semen specimen.
positive? Vapor storage?
Ideally, the goal of sperm preparation for ART would
be removal of all virus particles from infected semen
specimens. However, this is not really possible with
current methodology. Virus-positive specimens
should be handled with universal precautions in the
same manner that any biological specimen would be
handled. Suggestions include using a separate work
bench in the laboratory, separate incubator, and
separate centrifuge, with decontamination after each
use. At a minimum, known virus-positive semen
specimens that are frozen and stored for future use

16
 must be housed in impenetrable containers such as
cryogenic storage straws and/or stored in the vapor
phase of liquid nitrogen.

With certain medical conditions there may be no

Retrograde Ejaculation
antegrade ejaculation, with semen passing in a
● Recovery of sperm following retrograde ejaculation retrograde fashion into the bladder. The acidic and
● Neutralize or alkalinize the urine hypoosmotic nature of the urine is detrimental to
– Oral bicarbonate sperm. Sperm can be recovered from the urine
– Direct instillation of buffer into the bladder
● Following ejaculation, examine a urine/buffer sample
following retrograde ejaculation and used for assisted
– Simple wash with nutrient medium reproduction. In order to recover viable sperm, the
– Concentrate for sperm urine must be neutralized by oral bicarbonate
– Further processing, such as swim-up, gradient centrifugation,
glass-wool filtration, etc. can be done based on the quality of treatment or by instilling appropriate buffer into the
the initial specimen.
bladder using a sterile catheter. Following
masturbation, any antegrade ejaculate should be
recovered and processed. A urine sample should be
produced within 5 minutes of ejaculation or orgasm,
and should be analyzed. Based on the quality of the
retrograde specimen (sperm concentration and
motility), the urine will be washed free from the
sperm using a simple wash technique. Other
processing techniques such as swim-up, density
gradient centrifugation, or glass-wool filtration can be
done based on the quality of the specimen and the
end use of the sperm in ART.

17
 Sperm can be surgically retrieved from either the
Surgically Retrieved Sperm
epididymis—termed microepididymal sperm
● Epididymal sperm aspiration (MESA)—or retrieved directly from the
testicle, known as testicular sperm extraction (TESE).
It has been more than 20 years since reports of the
first ICSI procedure using sperm retrieved directly
Photo courtesy of Larry Lipshultz, MD
from the testicle. Not only has ICSI become a
standard procedure, but TESE has been routinely
● Testicular Sperm
used for males with obstructive and nonobstructive
azoospermia.

Photo courtesy of Kirk Lo, MD

Epididymal aspirates are usually very low in volume

Preparation of Testicular and Epididymal Sperm
and must be carefully manipulated. Sperm from the
● Epididymal sperm epididymis may show twitching or slow movement.
– Usually shows some movement Depending on the initial analysis, epididymal sperm
– Depending on concentration, use simple wash, swim-up,
motility enhancement methods
can be washed by simple centrifugation or even
● Testicular sperm swim-up. Often the concentration is low, and thus
– Usually not motile these specimens would be used for IVF/ICSI rather
– Simple wash preferred
– Incubate overnight under oil. Some sperm may show movement
than intrauterine insemination. Epididymal sperm
and can be picked up for ICSI 1 may also be good candidates for motility
enhancement and selection by hypoosmotic swelling.
Sperm retrieved from testicular tissue generally are
Schmidt et al., 2007 not motile and are few in number. The sperm may be
teased from the tubules and washed by simple
centrifugation. If frozen-thawed, sperm can be found
in the fluid, or teased from the tubules in the tissue.
It has been shown to be advantageous to incubate
testicular sperm overnight under oil, which can result
in a twitching movement, thus facilitating pickup of
“viable,” normal sperm for ICSI.

18
 Often the biopsy tissue is examined in the operating
Surgically Retrieved Sperm for ART
room to determine the presence of sperm. The
● Examine for presence of sperm in surgeon will continue to biopsy different areas of the
the operating room testicle until sperm are found. However, sperm are
● Meticulous laboratory search of
mechanically processed testicular
not always identified in the operating room. A
tissue, possibly to include meticulous laboratory-based search of mechanically
enzymatic treatment
– Chance of finding sperm after
processed tissue, including enzymatic treatment, can
chemical digestion when sperm be performed to increase the chances of finding
were not initially found is 25%–30%1
– Other studies report a success rate sperm. Some studies have reported a 25%–30%
of 7% using mechanical preparation
followed by multi-hour, many-
chance of finding sperm following enzymatic
technician search in the laboratory. 1
Photo courtesy of Grace Centola, PhD
digestion, when sperm were not initially found after
1 Dabaja & Schlegel, 2013
mechanical manipulation. Additional studies have
reported a success rate of 7% when mechanical
preparation is followed by multi-hour, multi-
technician search for sperm.

Both epididymal aspirates and testicular tissue can

Cryopreservation of Surgically Retrieved Sperm
be cryopreserved so that sperm might be available
● Epididymal aspiration: microsurgical (MESA), percutaneous (PESA) at a later date and for multiple IVF cycles. Surgical
– Must be kept in isotonic media capable of supporting the sperm until
processed
retrieval with cryopreservation eliminates the need
– Synthetic HEPES-buffered human tubal fluid (HTF) supplemented with for surgery on the day of oocyte retrieval.
0.5% human serum albumin (HSA) and antibiotic
– HEPES-buffered Ham’s F-10 with HSA
Epididymal aspirates and testis tissue
● Surgical retrieval: Testis biopsy (TESE), microdissection TESE, testis fine- cryopreservation pose unique requirements for
needle aspiration (FNA)
– Tissue is minced or seminiferous tubules teased apart using needles. cryopreservation. Aspirates must be kept in
Tubule contents squeezed into surrounding buffered medium such as
HEPES-buffered HTF media isotonic medium until the specimen can be
– Sperm removed, used immediately or stored for future use transported from the surgical suite to the laboratory
– Sperm will not be progressively motile, but may be twitching
– Freeze tissue pieces separately from cell suspension for processing. Such media include HEPES-buffered
– Standard TEST-yolk-glycerol cryopreservative synthetic human tubal fluid supplemented with
human serum albumin as a protein source, or other
media such as Ham’s F-10 medium. Likewise, testis
tissue is placed into buffer at the time of retrieval
for transport to the laboratory. The tissue is then
minced, and seminiferous tubules can be teased
apart from the larger tissue pieces. The tubules can
then be compressed and squeezed to release sperm
into the surrounding medium. Testicular sperm are
generally not motile per se. A few twitching sperm
may be found, but this is variable. Both the
spermatozoa and testis tissue pieces are frozen
using standard TEST-yolk-glycerol cryopreservative
in separate containers.

19
 Is fresh or frozen epididymal or testicular sperm
Fresh vs. Frozen Surgically Retrieved Sperm
better for ICSI?
● Motile, frozen-thawed epididymal sperm have ICSI outcomes Reports in the literature have generally concluded
comparable to fresh epididymal sperm. that there is no difference between fresh and frozen-
● No differences in fertilization rates or clinical and ongoing
pregnancy rates between fresh and frozen-thawed testicular
thawed sperm. However, with cryopreservation and
sperm. thawing, any motility is lost, and most centers prefer
– Significant decrease in implantation rates seen to work with motile sperm, since motility points to
– Sperm viability decreases following cryopreservation
viability and some sense of normalcy. Nonmotile
fresh testis sperm appear preferable for ICSI, since
viability rates approach 90% in fresh testis sperm. A
meta-analysis showed that fertilization rates, clinical
Shin & Turek, 2013
pregnancy rates, and ongoing clinical pregnancy rates
do not differ between the fresh and frozen groups.
However, a significant decrease in implantation rates
was observed with frozen-thawed testis sperm,
presumably due to the decreased viability following
thaw.

Once sperm are made available for IVF/ICSI, the

Sperm Selection
choice of next steps are dependent on the quality of
● Motility stimulating agents the sperm fraction. Certainly in cases where a good
– Pentoxyfylline sperm concentration and motility are apparent,
– Caffeine
conventional techniques for oocyte insemination are
● Hypoosmotic Swelling (HOS)
– Selection of viable non-motile sperm chosen. Even if the concentration is low, but the
● Hyaluronan (HA)-binding selection morphology and motility are adequate, it is relatively
– Selection of mature sperm based on binding to HA easy to choose sperm for oocyte injection.
● MSOME
– Motile sperm organelle morphology examination
However, in the severely suboptimal specimen, such
– Evaluation of nuclear morphology at high magnification in real as severely oligozoospermic, asthenozoospermic,
time
● IMSI
immotile, and teratozoospermic specimens,
– Intracytoplasmic morphologically selected sperm injection advanced sperm selection techniques may be
warranted to assist in selection of an optimal sperm
for ICSI. Advanced strategies include motility
stimulation procedures and selection of sperm based
on sperm maturity and ultramorphology as listed
here.

20
 The standard method for determining sperm viability
Motility-stimulating Agents
has always been sperm motility. However, in cases
● Pentoxifylline where sperm are retrieved from the testis, sperm are
– Methylxanthine derivative most often immotile. Frequently, cryopreserved
– Most commonly used method for motility enhancement in sperm show decreased or no motility. Several
both fresh and frozen-thawed sperm
– 3–4 mM for 30 minutes
chemicals are known to enhance sperm motility,
● Caffeine most notably, pentoxyfylline and caffeine.
● Platelet-activating factor (PAF) Pentoxyfylline is a methylxanthine derivative that is
an inhibitor of phosphodiesterase, which then
increases intracellular levels of cyclic AMP (cAMP).
cAMP stimulates motility, velocity, and
hyperactivation of sperm, and has been shown to
enhance the acrosome reaction. Interestingly,
pentoxyfylline is approved by the FDA for treatment
of vascular disease. The stimulant effects of caffeine
can be used to improve or stimulate sperm motility.
Platelet-activating factor also improves motility, and
enhances sperm capacitation, the acrosome reaction
and oocyte penetration. The mechanism of action of
PAF is not completely understood and its use requires
further study.

Viable cells are able to maintain an osmotic gradient

Hypoosmotic Swelling Test
across the cell membrane. When sperm are placed
● Viable sperm swell in hypoosmotic solution into a hypoosmotic buffer solution, water will flow
● Selection of nonmotile but viable sperm for ICSI into the cell to maintain the intra- and extracellular
osmotic balance. When water flows into the cell, it
will expand in volume. Nonviable sperm will not
exhibit swelling in a hypoosmotic buffer.
The sperm tail will swell or coil in such a buffer, and
the viable sperm will thus be visible under phase
contrast optics. The viable sperm can be picked up,
washed from the hypoosmotic buffer, and used for
ICSI.

21
 The Sperm-Hyaluronan Binding Assay® (HBA) is a
Sperm-Hyaluronan Binding Assay® - HBA
commercially available sperm function test that
● Ability of mature, normal sperm with intact acrosome to bind determines the ability of the sperm to bind to a slide
to hyaluronic acid (HA), the main mucopolysaccharide of the coated with hyaluronic acid. Live, mature sperm will
cumulus
– Hyaluronic acid does not bind to immature sperm bind to the hyaluronic acid coating the plate. These
● Commercially available kit for testing and use sperm also have been shown to be normal and with
– PICSITM plate (Biocoat, Inc)
intact acrosome. Studies have shown that the HBA
– Hyaluron Binding Assay (HBA)
o FDA 510K approved test correlates with the level of sperm maturity, strict
● Rapid, simple test morphology, and chromatin integrity, as well as
● Correlation with sperm maturity, strict morphology,
chromosomal integrity, progressive motility, hemizona assay
results of the HZA, SPA, and fertilization in vitro.
(HZA), sperm penetration assay (SPA), and fertilization in vitro

This photomicrograph shows sperm binding to

Sperm Hyaluronan Binding
hyaluronic acid that coats a commercially available
● Advantages dish. Bound sperm can then be picked up by the
● Highly specific embryologist and used for oocyte injection. One
● Minimal safety
concerns
advantage of using this system for sperm selection is
● Improved fertilization, that the hyaluronan binding is highly specific, as only
less embryo
fragmentation, higher
mature sperm bind. There are minimal safety
pregnancy rates using concerns since hyaluronan is a naturally occurring
ICSI-selected sperm 1,2
● No adverse events component of cervical mucus, follicular fluid, and
Photo courtesy of Mike Reed, Ph.D., Albuquerque, NM
reported cumulus matrix. A study by Worrilow and colleagues
● Disadvantages3
● Time-consuming
reported a significant improvement in fertilization
1 Bormann et al., 2010; 2 Worrilow et al., 2007
rates, less embryo fragmentation rates, and higher
beta-hCG levels and pregnancy rates with
3 Mehta and Sigman, 2014

hyaluronan-bound selected sperm. The miscarriage

rate was also decreased when this system was used
to select mature sperm for ICSI. A single
disadvantage is the time commitment, particularly if
multiple oocytes have been retrieved and need to be
injected.

22
 Selection of Sperm Based on High-Magnification
A new approach for selection of morphologically
Morphology normal sperm using high magnification observation
● Motile sperm organelle morphology examination (MSOME) of unstained sperm was first reported in 2001 by
– Real-time, high magnification observation of sperm Bartoov, termed motile sperm organelle morphology
o >6000x magnification examination, or MSOME. Sperm can be visualized in
– Assesses 6 organelles: acrosome, post-acrosomal lamina,
neck, tail, mitochondria, and nucleus
real-time at magnifications of at least 6000x, which
– Selection of sperm free of nuclear vacuoles enables visualization of the sperm nucleus. MSOME
allows selection of sperm free of nuclear vacuoles,
● MSOME + ICSI = IMSI which are related to impairment of embryo
– Intracytoplasmic morphologically selected sperm injection
development. Together with micromanipulation
systems, MSOME has allowed a modified ICSI
Souza Setti et al., 2013; Bartoov et al., 2001
procedure termed IMSI, or intracytoplasmic
morphologically selected sperm injection.

There are advantages and disadvantages of IMSI. It

IMSI
has been proposed as an alternative to standard ICSI,
especially in couples with repeated ICSI failures as
Advantages Disadvantages
● Significantly improves the ● Prolonged sperm
well as those with increased DNA fragmentation.
percentage of quality manipulation Some reports have shown improved implantation and
embryos ● Special instrumentation pregnancy rates and decreased miscarriages rates
● Improves implantation and ● Significant cost
pregnancy rates ● High level of technical
after IMSI. Disadvantages include the need for
● Reduces miscarriage rate expertise and prolonged sperm manipulation, special high-cost
reproducibility
instrumentation, and the need for a high level of
technical expertise. However, reports have been
confusing and conflicting. Some studies have not
shown significant differences between ICSI and IMSI.
Clearly, more studies are needed to determine the
utility of MSOME and IMSI in assisted reproduction.

Media used for ART are usually classified according to

Media For Sperm Preparation
the buffer system used to maintain the pH of the
● Broadly classified according to the pH buffering system used medium. Two buffering systems are commonly used
● Bicarbonate-buffered media with human gamete preparation. Bicarbonate-
– Requires CO2 for pH control buffered medium relies on carbonic acid/bicarbonate
– Incubate sperm in media with higher pH (7.6–7.8) since a more
acidic pH can inhibit capacitation. equilibrium for maintaining pH. Bicarbonate-buffered
– Adjust CO2 accordingly to maintain an alkaline pH. medium requires carbon dioxide to maintain the pH
● Non-bicarbonate buffer, specifically HEPES or phosphate- with the weak acid, dissolved carbon dioxide. This
buffered media
– CO2 not required; designed for use in ambient air medium is designed to maintain close to, or slightly
– Most commonly used buffer system below, neutral pH of 7.0 using the carbon dioxide
environment.
HEPES = hydroxyethyl piperazineethanesulfonic acid
Non-bicarbonate media, specifically hydroxyethyl
piperazineethanesulfonic acid (HEPES), are the most
commonly used media for sperm preparation. Carbon
dioxide is not required with this type of buffer to

23
 maintain the media pH. HEPES buffer has a working
pH range of 6.8–8.2. The second most common
buffer is phosphate, such as that used in phosphate-
buffered saline where the pH is approximately 7.2–
7.4. The greatest advantage to these buffering
systems is that a carbon dioxide environment is not
needed. The processing and incubation of washed
sperm can be done in tightly closed tubes in ambient
air.

Protein supplementation of culture media for sperm

Media for Sperm Preparation
preparation is essential for sperm preparation.
● Protein supplementation - mimics in vivo environment Human serum albumin, purchased commercially, is
– Human serum albumin the most common protein supplementation.
o 3-5 mg/mL
o Commercially available; pathogen and endotoxin tested
Synthetic serum supplementation has been popular
– Protein promotes removal or efflux of cholesterol from the sperm as well, especially for oocyte and embryo culture.
membrane, facilitating capacitation; also promotes sperm longevity.
● Addition of antibiotics Addition of protein helps to mimic the in vivo
– Gentamicin most common; replaced penicillin/streptomycin to avoid environment, and promote longevity of the
patient penicillin sensitivity
● Types of media for sperm preparation
spermatozoa. The protein is also known to promote
– Synthetic human tubal fluid (HTF) efflux of cholesterol from the sperm membrane, thus
– Ham’s F-10 or F-12 media
– Media for sequential IVF steps
facilitating sperm capacitation and ultimately the
acrosome reaction.
Antibiotics are also commonly added to culture
media for sperm preparation. The ejaculate
commonly contains bacteria that are usually sensitive
to antibiotics. The process of washing sperm in media
with antibiotics should take care of any bacteria
present in the semen or bound to sperm, prevent
effects of bacteria on the sperm and oocytes, and
prevent intrauterine infection after insemination or
transfer.

24
 There are several methods of choice for sperm
Summary and Conclusions
preparation for assisted reproduction. These include
● There are multiple techniques for preparing sperm for assisted simple washing, density-gradient centrifugation,
reproduction, each of which has advantages and filtration, and swim-up. The method chosen should
disadvantages.
● The method for sperm processing should be chosen based on
be based on the quality of the sperm, specifically the
the sperm concentration and motility, whether it is fresh or sperm concentration and motility, whether the sperm
frozen, or ejaculated or surgically retrieved sperm.
are fresh or frozen, or whether ejaculated or
● Selecting the optimum sperm for ICSI can be done using
hypoosmotic swelling or MSOME/IMSI. Motility-enhancing surgically retrieved. Additionally, methods for sperm
chemicals can also be used to enhance sperm motility and selection include use of hypoosmotic swelling buffer
thus selection of viable sperm.
to choose viable sperm and high magnification
morphological assessment termed MSOME/IMSI.
Furthermore, motility-enhancing chemicals such as
pentoxyfylline, platelet-activating factor, or caffeine
can be used to improve or stimulate sperm motility
and allow for selection of motile and thus viable
sperm for assisted reproduction.

Thank you for participating in this educational

Thank you! activity.

We hope you enjoyed the course!

25
 References
1. Bartoov B, Berkovitz A, Eltes F. Selection of spermatozoa with normal nuclei to improve the
pregnancy rate with intracytoplasmic sperm injection. N Engl J Med 345: 1067-1068, 2001.
2. Boitrelle F, Guthauser B, Alter L, Bailly M, Bergere M, Wainer R, Vialard F, Albert M, Selva J. High
magnification selection of spermatozoa prior to oocyte injection: confirmed and potential
indications. Reprod Biomed Online 28:6-13, 2014.
3. Bormann CL, Alagretti JR, Da Motta ELA, Serafini P, Smith GD. Preparation and selection of sperm for
IVF and ICSI. IN Reproductive Endocrinology and Infertility. Integrating Modern Clinical and
Laboratory Practice, DT Carrell and CM Peterson, eds., Springer, New York, 2010, pp. 579-590.
4. Cho BS, Schuster TG, Zhu X, Chang D, Smith GD, Takayama S. Passively driven integrated microfluidic
system for separation of motile sperm. Anal Chem 75:1671-1675.
5. Dabaja AA, Schlegel PN. Microdissection testicular sperm extraction: An update. Asian J. Androl
15:35-39, 2013.
6. Ebner T, Fillicori M, Tews G, Parmegiani L. A plea for a more physiological ICSI. Andrologia xx: 1-18,
2011.
7. Henkel RR, Franken DR, Lombard CJ, Schill W-B. Selective capacity of glass wool filtration for
chromatin condensed human spermatozoa: a possible therapeutic modality for male factor cases? J
Assis Reprod Genet 11: 395-400, 1994.
8. Huszar G, Sbracia M, Vigue L, Miller DJ, Shur BD. Sperm plasma membrane remodeling during
spermiogenic maturation in men: relationship among plasma membrane beta 1,4 galactosyl-
transferase, cytoplasmic creatine phosphokinase, and cratine phosphokinase isoform ratios. Biol
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