Sperm Preparation for IVF and ICSI
INTRODUCTION
Human spermatozoa at
ejaculation are incapable of in vivo fertilization and must undergo
maturational change during which they acquire the ability to fertilize oocytes.
This process, known as capacitation, was described more than 50 year ago by
both Austin and Chang . Capacitation is prevented in ejaculated
spermatozoa by at least one factor in seminal plasma. Additionally,
prolonged exposure to seminal plasma can inhibit the ability
of spermatozoa to
undergo the acrosome reaction in vitro and diminish their capacity to
fertilize. In the female genital tract, motile spermatozoa separate themselves
from seminal plasma, immotile spermatozoa, and debris
by actively migrating
through the cervical mucus. This active migration selects progressively
motile spermatozoa and allows them to undergo capacitation. Due to
the inhibitory effects of seminal plasma on sperm function,
it is critical that
spermatozoa used for clinical procedures such as in vitro
fertilization (IVF) or intracytoplasmic sperm injection (ICSI) be separated from the
seminal plasma as quickly as possible after ejaculation
and liquefaction.
Although IVF started
as a treatment for tubal infertility, the increasing number of men with
poor semen quality led to the development of a variety of sperm
preparation techniques. These techniques generally fallinto four categories:
(i) simple dilution
and washing.
(ii) sperm migration.
(iii) density
gradient centrifugation.
(iv) filtration or
adherence.
Regardless of
the technique, the objective of sperm preparation is to recover an enriched population
of motile and functionally competent spermatozoa while eliminating
dead spermatozoa and other cells, including bacteria
and leukocytes. The
technique should also minimize damage to the spermatozoa and eliminate
decapacitation factors and toxic substances such as reactive oxygen
species (ROS). Some of these techniques as well as theiradvantages and
disadvantages are presented here.
SPERM COLLECTION
Ejaculation
The semen specimen
should be collected by masturbation and the ejaculate produced into a
sterile glass or disposable plastic jar that has been checked for sperm toxicity.
As soon as the seminal plasma has liquefied, the specimen should be analyzed
according to the WHO guidelines and prepared for sperm isolation.
A second semen specimen may be requested if the semen specimen on the day
of IVF is of very poor quality . When liquefaction is delayed or the
specimen is especially viscous, drawing the sample through a 21 gage needle into a
syringe may help break up viscous globules. For men who are unable to
collect semen by masturbation, nontoxic condoms are commercially
available; guidelines for their proper use should be strictly abided by patient and
laboratory personnel. Ordinary contraceptive condoms must not be used
(even those without spermicide) because of their sperm toxicity.
Coitus inter-ruptus is also not recommended because of the risk of
incomplete recovery and potential iatrogenic contamination of the ejaculate. Semen may be
collected from men who are unable to achieve erection, emission, or
ejaculation because of neurological or psychogenic problems by electroejaculation
using direct vibratory stimulation of the penis or electrical stimulation of the
prostate. Ejaculates from spinal cord injured patients will frequently have high
sperm concentrations, decreased motility, and red blood cell
contamination. Sperm may also be recovered from the urine of patients whose
ejaculation is retrograde into the bladder. It is advisable that these patients be
prescribed stomach-acid buffering medications to make the urine pH more
hospitable for sperm.
Surgical
The collection of
epididymal and/or testicular spermatozoa requires an office or outpatient
surgical procedure. Epididymal spermatozoa can beretrieved either by
microsurgery or by percutaneous needle puncture. As the typical
indication for epididymal aspiration is obstructive azoospermia rather than
testicular dysfunction, it is not uncommon for relatively largequantities of sperm
to be obtained and subsequently used for IVF, or even intrauterine
insemination (IUI), and any excess sperm may be frozen for future use. Depending
on operator skill, epididymal aspirates can be obtained with minimal
red blood cell and non-germ cell contamination, making the isolation
and selection of motile sperm quite easy. If large numbers of epididymal
spermatozoa are obtained, then density gradient centrifugation (see
below) is an effective method for preparing those spermatozoa for subsequent use. Testicular
spermatozoa can be retrieved by open biopsy (with or without microdissection) or
by percutaneous needle biopsy. Testicular specimens are contaminated
invariably with large amounts of red blood cells and testicular tissues; additional
steps are needed to isolate a clean preparation of spermatozoa. In order
to free the seminiferous tubule-bound spermatozoa, it is necessary to
use either enzymatic (collagenase) or mechanical methods. For the latter,
testicular tissues in supportive culture medium is macerated using glass cover
slips until a fine slurry of dissociated tissues is produced, and the resulting
suspension can then be processed for therapeutic use. Excess testicular
spermatozoa obtained in this manner can be frozen for future use in order to avoid
further surgeries. Testicular spermatozoa can also be obtained from a needle biopsy,
although only a small amount of tissue is usually retrieved and the
resulting sperm yield is proportionately low.
SPERM PREPARATION METHODS
Simple Washing and Dilution
The sperm preparation
method used for the first IVF cases involved dilution of the semen with
culture medium (usually at 2-10
times the volume) and separation of the
spermatozoa by centrifugation. After removal of the supernatant, the
pellet is resuspended in another aliquot of culture medium. Repeat
centrifugation, usually two or three times in total, is often used to ensure removal of
contaminating seminal plasma. The centrifugation is usually performed at 200-300
g and it should certainly be performed at centrifugal forces less than 800
g . Advantages of this method are that it is the simplest and the
least expensive to perform. One disadvantage of this technique is nonviable, and
immotile spermatozoa as well as any leukocytes, squamous epithelial
cells, or non-cellular debris that contaminated the original semen sample
will still be present in the washed sample. Another disadvantage is the concern about
potential damage caused by centrifugation. Aitken and Clarkson
reported that techniques involving the repeated
centrifugation of unselected populations of human spermatozoa generate cell
suspensions with significantly reduced motility. Moreover, these detrimental
effects of centrifugation were associated with a sudden burst of ROS produced
by a discrete subpopulation of cells characterized by significantly
diminished motility and fertilizing capacity. The ROSs were found to impair the
functional competence of normal spermatozoa in the same suspension,
reflected in impaired capacity for sperm-oocyte
fusion. It has also been shown
that ROS can cause DNA damage in human spermatozoa when exposed for time
periods consistent with clinical sperm preparation techniques for ICSI
or IVF . Thus, sperm preparation techniques that involve a washing
step in which semen is diluted with culture medium and centrifuged have
mostly been abandoned for alternative techniques such as direct swim-up from
semen or density gradient centrifugation.
Sperm Migration
Motile spermatozoa
separate themselves from seminal plasma in vivo by actively migrating
through cervical mucus in the female reproductive tract. There are a variety
of sperm preparation techniques that involve migration of spermatozoa, and
the element common and prerequisite to all these techniques is the self-propelled
movement of spermatozoa.
Swim-Up from Washed Pellet
The swim-up of
spermatozoa from a washed pellet technique was originally described by
Mahadevan and Baker and it is still a standard method for patients with
normozoospermia and female infertility . The procedure
involves dilution and
centrifugation (repeated two to three times) of a semen specimen to separate
spermatozoa from seminal plasma. The pellet of spermatozoa formed after the
final centrifugation can either be left intact or
gently resuspended in
the small residual volume of supernatant in the bottom of the centrifugal
tube. Swim-up from an intact sperm pellet requires that centrifugation
speeds be such that the final pellet is loosely compacted.
This can be verified
by gently and slowly tilting the test tube and observing whether the pellet
tilts as well. Each laboratory should determine the centrifugation time and speed that
will afford this attribute. If one chooses to
resuspend the sperm
pellet, then extreme care must be taken to ensure that no mixing occurs when
overlaying the non-compacted pellet with culture medium. If mixing
occurs, then the final aspirated supernatant (containing
sperm for subsequent
use) can be contaminated with immotile sperm, debris, and non-germ cells.
This latter technical problem is less of an issue when the sperm pellet is left
intact. Regardless of whether an intact or disrupted sperm pellet is used,
culture medium is layered over the pellet and the tube is incubated at 37_C
for 30-60
minutes to allow the spermatozoa to swim up from the pellet. As with all
techniques involving the mixing of spermatozoa with medium, it is
important to choose a culture medium that is buffered appropriately for the atmosphere in
which the technique takes place. Therefore, if the incubator
atmosphere is the same as the laboratory and the temperature is 37_C,
then the medium should be buffered with HEPES or a similar buffer, and the caps of the
swim-up tubes should be tightly closed. If the incubator atmosphere is 5-6%
CO2 and the temperature is 37_C,
then the medium is best buffered with
sodium bicarbonate or a similar buffer, and the caps of the test tubes should
be loose. Adherence to the aforementioned will ensureculture pH that is
compatible with sperm survival. To facilitate the
release of motile spermatozoa from the sperm pellet, the test tube may be
placed at 45_,
thereby increasing the surface area interface between the
sperm pellet and the culture medium. Alternatively, aliquots of the
resuspended pellet may be placed in 4-well dishes before culture medium is layered
over each aliquot. The use of 4-well dishes will also increase the
interface between the pellet and the culture medium. Evidence that sperm have
successfully swim-up into the overlaying culture medium is reflected
by an increase in turbidity. If the culture medium appears clear, then
more time may be needed to allow spermatozoa the opportunity to swim
out of the pellet. After the incubation, the upper layer of culture medium
containing spermatozoa is carefully aspirated without disrupting the
interface and transferred to a clean test tube from which concentration, motility, and
morphology can be assessed. Advantages of the
swim-up from washed pellet method include the recovery of a high
percentage of motile sperm and the absence of other cells and debris. Another
advantage of this technique is that it consistently produces suspensions of
spermatozoa with increased swimming velocity and more normal sperm
morphology . The swim-up method also results in significant
improvement in the rates of acrosome reaction, hypo-osmotic swelling (HOS), and
nuclear maturity . A disadvantage of the swimup
from washed pellet is
the low overall recovery of motile spermatozoa; motile spermatozoa
trapped at the bottom of the pellet may never be able to reach the
interface with the culture medium. Thus, the efficiency of the
technique is based
not only on the initial sperm motility in the ejaculate, but also on the size,
level of compaction, and exposed surface area of the final pellet. Another
disadvantage is the previously discussed concern about
potential damage
caused by centrifugation of unselected populations of human spermatozoa.
Direct Swim-Up from Semen
A swim-up technique
that avoids centrifugation of unselected populations of spermatozoa is the
direct swim-up from semen, in which aliquots of liquefied semen are placed
underneath a layer of culture medium in either 4-well
dishes or a series of
test tubes. The interface between the semen layer and the culture medium is
increased by placing the tubes at 45_ in the
incubator. Depending on the
initial ejaculate volume, sperm concentration, sperm
motility, multiple
test tubes, or 4-well dishes may be used to increase the recovery of motile
spermatozoa. The interface can often be cleaner when the liquefied semen
is layered under the culture medium with a syringe and
needle rather than
layering the culture medium over the semen. The test tubes are
incubated at 37_C
for 30-60
minutes to allow the spermatozoa to swim
up from the liquefied semen. Evidence that sperm have successfully swum up
into the overlaying culture medium is reflected by an increase in
turbidity. If the culture medium appears clear, then more time may be needed to
allow spermatozoa the opportunity to swim out of the pellet. After
incubation, the upper layer of culture medium in each tube is carefully aspirated
and removed to a clean centrifugal tube. The suspension is then centrifuged at
300-600
g for 4-10
minutes after which the supernatant is removed and the
pellet resuspended in fresh culture medium to achieve the desired
concentration of motile spermatozoa. Advantages of the
direct swim-up method include the recovery of a high percentage of
motile sperm and the absence of contaminating dead or immotile
spermatozoa, non-germ cells, and debris. In a comparison of
four methods for
sperm preparation, Ren et al. found that the direct swim-up method
provided the best sperm motility. Another advantage is the elimination of the
centrifugation step prior to the swim-up, which reduces
ROS production by
white blood cells and dying spermatozoa.Adisadvantage of the direct swim-up
from semen is the low recovery of motile spermatozoa.
Migration Sedimentation
The
migration-sedimentation method was developed by Tea et al. and it combines the swim-up
technique with a sedimentation step in special glass or plastic tubes
containing an inner cone. Spermatozoa swim up directly
from liquefied semen
into the overlying culture medium and subsequently settle
gravitationally in the inner cone of the tube. Incubation is usually 60 min at 37_C,
after which the medium in the cone is removed and centrifuged at 300g for
5-10
minutes. Sperm count and motility are then determined on the
resuspended pellet. The dvantages of the
migration-sedimentation method are similar to those of the direct
swim-up technique: the migration-sedimentation method is a very gentle
separation method and it yields a clean fraction of highly motile spermatozoa.
In addition, ROSs are reduced because of the lack of centrifugation prior
to sperm igration. The disadvantages of the technique include a very low
yield of motile spermatozoa and the equirement for special glass or plastic
tubes. A comparative study
by Gabriel and Vawda demonstrated that specimens from
fertile males processed using the migration-sedimentation method had the
greatest increase in motility and the only increase in morphology versus
specimens processed either by filtration (SpermPrep1) or swim-up from
washed pellet. Specimens from subfertile males also showed significantly
increased sperm motility and morphology when the migration-sedimentation method was used. Gabriel concluded that migration sedimentation should
be the method of choice unless the original sperm count is low. Sanchez et al.
modified the migration-sedimentation method to include an initial
centrifugation of the neat semen at 400 g for 10 minutes with the resulting
pellet diluted in 500
mL of seminal fluid before being placed under culture
medium in special glass tubes and incubated for 2-3 hours at 37_C.
After the incubation, the medium in the cone is removed and centrifuged at 300 g
for 5-10
minutes. Sperm count and motility are then determined on
the resuspended pellet. The extra centrifugation step and the lengthened
incubation allowed them to recover a sufficient number of motile spermatozoa
even in cases with severe oligozoospermia and/or asthenozoospermia. Using this modified
method, Sanchez et al. demonstrated significantly better
results in progressive motility, normal morphology, chromatin
condensation, and reduction in the ercentage of dead spermatozoa when compared with
density gradient centrifugation. In spite of the findings of Sanchez
et al., one must bear in mind the same cautions when subjecting unselected
sperm populations to centrifugation
Density Gradient Centrifugation
Density gradients may
be either continuous or discontinuous although the discontinuous
gradients have been used almost exclusively since the late 1980s . Discontinuous
gradients are usually prepared with two or three
layers. Colloidal
silica with covalently bound silane molecules is probably the most common
density gradient material currently used for clinical IVF and andrology.
PureSperm1 (NidaCOn
International AB, Go"teborg, Sweden), Isolate1 (Irvine
Scientific, Santa Ana, California, U.S.A.), IxaPrep (MediCult, Copenhagen,
Denmark), and Enhance1 (Conception Technologies, San Diego,
California, U.S.A.) are examples of silane-coated silica
particle solutions
that can be used for discontinuous gradients. These products are made isosmotic by
the inclusion of polysucrose; they have very low toxicity, are
nonirritating, and are approved for human in vivo use. As with any product, it is
important to follow the manufacturer's
recommendation for proper use and
application.
In the discontinuous
density gradient method, the ejaculate is placed on top of the density
gradient medium and is centrifuged at 300-400
g for 15-30
minutes. As the density gradient medium is a colloid rather than a solution, it has low
viscosity and it does not retard the sedimentation of spermatozoa due to
centrifugation . Highly motile spermatozoa move actively in the
direction of the sedimentation gradient and can penetrate the boundary faster
than poorly motile or immotile spermatozoa . Thus, the soft pellet at
the bottom is enriched for highly motile spermatozoa. The pellet is washed with
culture medium and centrifuged at 200 g for 4-10 minutes. The wash and
centrifugation is then repeated to ensure removal of contaminating
density gradient medium. The final pellet is resuspended in culture medium so
that concentration and motility can be determined. Density gradient centrifugation usually results in a clean fraction of highly motile
spermatozoa. As the whole volume of the ejaculate is used in density gradient
centrifugation (as it is in the swim-up techniques), it yields a
significantly higher total number of motile spermatozoa and it can be used for patients
with varying degrees of suboptimal semen parameters (e.g.,
oligozoospermia and asthenozoospermia). Other advantages of density gradient
centrifugation include the elimination of leukocytes and the significant reduction
of ROS . Additionally, Nicholson et al. demonstrated that
centrifugation through one brand of silane-coated silica particles (PureSperm)
efficiently reduces bacterial contamination. Hammadeh et al. reported that
another advantage of the density gradient method is the
recovery of a higher percentage of morphologically normal spermatozoa than
found in conventional swim-up or glass wool filtration. The technique has
also been shown to yield sperm populations with better DNA quality and
chromatin packaging . Further, preliminary reports suggest that
specimens known to be contaminated with sexually transmissible viruses
can effectively be "cleaned
up" using density gradient centrifugation and
the isolated spermatozoa can be used for therapy with exceptionally low
risk for horizontal disease transmission . One disadvantage of density gradient
centrifugation is that the density gradient medium is a bit more
expensive than either of the swim-up techniques.
Adherence Filtration
These methods are based on the phenomenon that dead and moribund spermatozoa are extremely sticky and will attach to glass surfaces even in the presence of relatively high concentrations of protein.
Glass Wool Filtration
In this method,
motile spermatozoa are separated from immotile spermatozoa by means of densely
packed glass wool fibers. The principle of this technique involves
both the self-propelled movement of the spermatozoa and the filtration
effect of the glass wool. The method initially employed vertical Pasteur pipettes
filled with glass wool fibers on to which the ejaculate was placed and
allowed to filter by gravity (27). The method has evolved such that in a
current variation (28), the filter is created by placing 30 mg of pre-cleaned glass
wool microfibers in the barrel of a 3mL disposable syringe and gently packing it
down using the syringe plunger (minus its rubber tip). The syringe is
suspended vertically in a 15mL centrifuge tube and rinsed several times
with culture medium to remove any loose wool fibers prior to filtration.
Meanwhile, the ejaculate is washed with an equal volume of culture medium,
pipetted into 15mL centrifuge tubes (no more than
3 mL/tube), and
centrifuged at 300 g for 3 minutes. Each resulting pellet is resuspended in 1mL of
culture medium, and centrifuged again at 300 g for 3 minutes. The pellet in
one tube is resuspended with 300
mL of culture medium, and this
single supernatant is sequentially added to resuspend the sperm pellet in
any remaining tubes (the total volume should not exceed 400
mL).
The washed sperm suspension is gently pipetted over the pre-wet glass wool column and
then allowed to filter by gravity into a clean 15mL centrifugal tube.
When the dripping stops, 100
mL of culture medium is added to the
filter and allowed to drip through. The filter is removed and the filtrate can be
assessed for sperm oncentration and motility.
The success of this method is related to the kind of glass wool used the chemical ature
of the glass, the surface structure and charge of the glass wool, and the
thickness of the glass wool fibers. Glass wool from Manville Fiber Glass Corporation
(Denver, CO) or SpermFertil1
columns
from Mello Holzhausen,
Germany) has been tested extensively in clinical practice . Glass wool
filtration and two-layer, iscontinuous density gradient centrifugation resulted
in an average recovery of 50-70%
of the progressively motile and about 50%
of the HOS-positive spermatozoa . Additionally, glass
wool filtration tended to be more successful than density gradient
centrifugation when the ejaculates were asthenozoospermic or had an abnormal HOS test.
After processing, the activity of the zona lysing enzyme acrosin
increased approximately two- to hreefold, but no significant improvement in the
percentage of normal sperm forms occurred. Glass wool filtration was also
more effective in removing non-motile and HOS-negative spermatozoa than
density gradient entrifugation when the percentage of these types of
spermatozoa in the ejaculate is high. This method can use
the whole volume of the ejaculate and thus yield asignificantly higher
total number of motile spermatozoa, which means it can be used for patients
with oligozoospermia and/or sthenozoospermic. It is also possible to
prepare motile spermatozoa from patients with retrograde ejaculation . Another
advantage of glass wool filtration is the elimination of up to 90% of the
leukocytes present in the ejaculate . As leukocytes are a major producer of
ROS, elimination of a majority of leukocytes should significantly reduce
ROS. Finally, glass wool filtration was also found to yield a significantly
higher percentage of chromatin-condensed spermatozoa than swim-up or
density gradient centrifugation . Disadvantages of the glass wool filtration
method include the added expense of the glass wool and a filtrate that
is not as clean as it is with other sperm preparation methods because remnants of
debris may still be present. Sephadex Columns Sperm separation
using Sephadex beads is another filtration method, and a kit based on
this principle (SpermPrep) is commercially available (Fertility Technologies,
Inc.). Basically, liquefied semen is diluted with
culture medium and
centrifuged at 400 g for 6 minutes. The supernatant is iscarded and the
sperm pellet resuspended in culture medium to a concentration of 100_106
sperm/mL. One milliliter of the washed semen is placed in the filter column
containing hydrated filtration beads and mixed gently. The bottom cap is
removed from the filter column and fluid is allowed to filter for 15
minutes. The filtrate is entrifuged at 400 g for 6 minutes and resuspended in 1mL of
culture medium before being assessed for oncentration, motility, and
morphology.
In a comparative
study, the yield of spermatozoa post-processing was highest with
SpermPrep than with swim-up or migration sedimentation in both fertile and
subfertile men , and for that reason the authors recommended that specimens with a
lower than normal sperm count but normal motility and
morphology should be processed with SpermPrep. Disadvantages of Sephadex bead
filtration include the added expense of the kit and a filtrate that is
not as clean as it is with other sperm preparation methods because remnants of
debris may still be present. In addition, the prefiltration centrifugation step
might generate ROS.
POST-SEPARATION
TREATMENT OF SPERMATOZOA
Improvement of Motility
and Sperm Function
Pentoxifylline
The use of
methylxanthine derivatives such as pentoxifylline for the stimulation of sperm functions,
especially motility, is well known. Pentoxifylline is a nonspecific
inhibitor of phosphodiesterase that has stimulatory effects on sperm motility and
motion characteristics like sperm velocity or hyperactivity.
The stimulatory
effect is attributed to increased intracellular levels of cAMP via inhibition
of its breakdown by cAMP phosphodiesterase. Pentoxifylline is also reported to
enhance the acrosome reaction presumably
due to the increasing
levels of cAMP. The results of pentoxifylline treatment in assisted
reproduction are equivocal. Depending on the conditions, especially the time
of stimulation relative to the capacitative state of the
spermatozoa and the
concentration of pentoxifylline in the medium, overstimulation can
result in a premature acrosome reaction . Thus, pentoxifylline tends
to be used on a limited basis in IVF programs and some
programs choose to
use pentoxifylline only in the preparation of epididymal and testicular sperm
for assisted IVF. Spermatozoa retrieved
from the testis have not experienced the maturation-inducing
influence(s) afforded during epididymal transport, and therefore, are in
a different physiologic state than epididymal or ejaculated
spermatozoa.
Treatment of immotile or very poorly motile fresh or cryopreserved testicular
spermatozoa with pentoxifylline very frequently simulates some form
of motion, whether it is twitching, nonprogressive motility, or
progressive motility. The goal in any ICSI procedure is to use spermatozoa that are
viable, and motion is the best indicator ensuring both a functional
(protective) plasma membrane and patent metabolic processes. The combination of
these two attributes lends greater assurance that the DNA has not been made
more vulnerable to the deleterious effects of ROS. Platelet-Activating Factor Platelet-activating
factor (PAF) is a biologically active phospholipid thought to be a
cellular mediator in reproduction that has been found in spermatozoa of many
different species, including human (33). PAF has been reported to have
positive effects on motility, capacitation, acrosome reaction, and oocyte
penetration , and these stimulatory actions on spermfunction can be
inhibited by PAF antagonists . Although the molecular mechanism of action
has yet to be fully elucidated, the positive effect of PAF on sperm function has
led to its use in assisted reproduction. Roudebush et al. reported that
pregnancy rates in IUI cycles were significantly increased after the
spermatozoa from normozoospermic males were prepared with a medium
containing PAF. Detection of Viability Sperm motility is an
important indicator of viability, especially when performing ICSI. In the absence
of native or stimulated sperm motility, the assessment of
viability becomes critical. There is a simple vitality test based on the
semi-ermeability of the intact and physiologically functional plasma membrane which causes
spermatozoa to swell under hypo-osmotic conditions, when an influx of
water results in an expansion of cell volume . This vitality test is
known as the HOS test. The HOS test can be
used for specimens where the spermatozoa are all immotile. When setting
up a dish for the ICSI procedure, a small (5mL)
drop of HOS solution is
placed near the PVP drop and two extra drops of culture medium are placed
nearby. A small volume of sperm suspension is placed in one of the extra
drops. When spermatozoa are located, they are picked up in the ICSI
micropipette and placed in the HOS solution. Immediately after contact with the
hypo-osmotic medium, the tails of some spermatozoa will begin to coil or
swell. Tail swelling or curling indicates that the spermatozoon is undergoing
hypo-osmotic stress, the plasma membrane is functional, and the cell is still
viable. The spermatozoon is then picked up in the ICSI micropipette and
placed in the other extra drop of medium in order to wash off excess
hypo-osmotic medium from both the micropipette and the spermatozoon.The spermatozoon is
then placed in the PVP drop in order to proceed with ICSI.
SUMMARY
The choice and
application of the appropriate sperm preparation technique can be a major
contributor in influencing whether a patient will become pregnant. Ejaculates
from subfertile males very frequently contain or have the potential for
producing ROS, which are known to compromise sperm function and damage
DNA. Therefore, it is imperative that the laboratory technologist selects
a technique that will directly separate functional and highly motile
spermatozoa from all that remains. Although it might be argued that density
gradient centrifugation is the most effective method, not all laboratories
may have access to the equipment and/or resources needed to perform the
technique. Thus, depending on the initial sperm parameters, the
direct swim-up from semen can safely and effectively be used and the
post-swim-up centrifugation step may be omitted.Although every
specimen is considered valuable, oftentimes the specimens being handled are
precious and/or expected to serve as a resource for many attempts at
paternity. Thus, one must be even more selective whenchoosing a sperm-preparation technique. For example, a male undergoing medical therapy may
be producing his last sperm-containing ejaculate, so optimization in the total number
of motile sperm recovered is necessary. Further, due care is advised
when processing an operatively obtained specimen, as repeat surgery
exposes the patient to additional (and unnecessary) risk. After reading this
piece, one may ask: What kind of techniques might be on the horizon?
Current technologies have afforded new knowledge about sperm biology.
Expression of membrane proteins is not only a reflection of properly
functioning spermatogenesis and spermiogenesis, but also sperm maturation.
This characteristic may be exploited by using techniques that isolate cells
based on the ioniccharge of expressed membrane proteins (i.e.,
electrophoretically) or by using reversible binding techniques that involve cell
function.
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