Sperm Processing and Selection Techniques in an IVF/ICSI 153

C HAPTER Sperm Processing and Selection Techniques 15 in an IVF/ICSI

Bhushan K Gangrade, Ashok Agarwal

n Introduction germinal/epithelial origin and (b) liquid fraction also called seminal plasma. The biochemical Fertility in the male depends on several sperm composition of human seminal plasma is complex parameters. Even though “number of spermatozoa and has been described in detail elsewhere.1 The per milliliter” of semen and the “total sperm count” main components of the seminal plasma are in the ejaculate are the main determinants of the enzymes such as acid phosphatase and alpha- fertility in men, other attributes such as the propor- glucosidase, sugar in the form of fructose and tion of morphologically normal and progressively several other substances including L-carnitine, motile sperm also contribute to the equation. In prostaglandins and zinc. addition, physiological, immunological and other During intercourse semen is deposited in the factors such as sperm DNA fragmentation may vaginal vault and only progressively motile further affect the fertility potential of male. The spermatozoa find their way into the cervix and normal values for sperm concentration and motile beyond. The nonmotile sperm and other cellular fraction in the ejaculate have been established by debris as well as the seminal plasma are left behind World Health Organization (WHO) and span over a in the vagina. Even though seminal plasma is the wide range. The fifth edition of the “WHO manual vehicle that carries sperm out of the male repro- for the examination of human semen and sperm- ductive tract, physiologically it is not the most cervical mucus interaction” notes the lower suitable environment for spermatozoa especially if reference limit for sperm concentration and total sperm are to stay in it for extended duration. sperm count at 15 and 39 million per milliliter Likewise for insemination or in vitro fertilization, respectively. In addition there is significant variation ejaculated sperm must be separated from the in sperm parameters (viz. ejaculate volume, seminal plasma as soon as feasible. concentration, total count, motility, vitality and In majority of the patients undergoing IVF, the progression) in repeat ejaculate samples over a sperm are isolated from the fresh ejaculate provided period in each individual. on the day of oocyte retrieval. In patients whose Spermatozoa produced in the seminiferous sperm parameters are within the reference range tubules in the testes are mixed with the secretions (normozoospermic) typically 50,000 to 100,000 of accessory glands namely seminal vesicles and normal motile sperm/ml are used to inseminate prostate which provide the bulk of the seminal oocytes. In patients with oligo- or asthenozoo- plasma. The ejaculate (semen) can be largely spermia, standard insemination of the oocytes may divided into two components: (a) cellular fraction result in complete failure or suboptimum consisting of live and dead sperm, immature sperm fertilization. Prior to the advent of ICSI,2 it was a cells, leukocytes, epithelial cells, round cells of challenge to obtain successful fertilization and 152 A Practical Guide to Setting Up an IVF Lab, Embryo Culture Systems and Running the Unit

pregnancies in couples with the diagnosis of severe sperm characteristics of the fresh ejaculate. The total male factor infertility. Development of ICSI made it sperm count and the proportion of motile sperma- possible to achieve fertilization and pregnancies in tozoa in the ejaculate are the main determining couples with severe male factor infertility. Interes- factors that are taken into consideration while tingly spermatozoa retrieved from any source deciding the optimum method for sperm (testes, epididymis, urine from retrograde ejaculate) preparation. It is equally important to take into can be used to fertilize oocytes by ICSI. In this account the method of insemination (IVF or ICSI). chapter we have described in detail the procedures For IVF it is preferable to have washed sperm for the isolation of spermatozoa from semen, urine preparation with high proportion of progressively (retrograde ejaculate) and testicular and epididymal motile sperm with minimal or no particulate debris. samples for insemination by IVF and ICSI. In contrast, for ICSI, the presence of cellular debris or reduced motility and progression in the sperm n Sperm-wash Media sample are not of critical importance. In fact such adverse sperm parameters do not appear to A variety of culture media are commercially avail- have any significant effect on the rate of fertilization able for sperm washing procedures. Some of these by ICSI. are Ham’s F-10, Human Tubal Fluid (HTF), Menezo’s B2 and B3 Medium, Bigger, Whitten and n Patient Preparation Whittingham Medium (BWW), and Earle’s Balanced Salt (EBS). Despite the differences in the compo- The patient should be instructed to abstain from sition between the media, the fact remains that all sexual intercourse for 2 to 3 days prior to providing these media are balanced salt solutions capable of semen sample. Shorter abstinence and frequent providing optimum environment and energy for ejaculations may result in lower volume and sperm survival and sustaining spermatozoa. For most count in the ejaculate. On the other hand, long term sperm washing techniques a HEPES-buffered abstinence of more than seven days may result in culture medium is appropriate. For standard increased proportion of nonviable and aged insemination of oocytes however, the final sperm spermatozoa in the ejaculate. Ideally, the sample suspension should be made in a bicarbonate-based should be produced by masturbation and collected culture medium recommended for IVF. The sperm- in a sterile specimen container. The specimen wash media should always be supplemented with container and other disposable plasticware should protein prior to use. The most common protein used be evaluated by either mouse embryo assay or to supplement sperm-wash media is the albumin sperm survival assay to ascertain the suitability prior fraction isolated from human serum (HSA) at a to its use. Patients should be encouraged to provide concentration of 5 mg/ml. It is important to note that the sample in a semen-collection room adjacent to only HSA preparations available commercially and the laboratory. The onsite sample collection avoids recommended for such application should be used. the possibility of exposure to extreme temperature The sperm wash medium should be stored that may be encountered if the sample is transported refrigerated at 4oC and warmed to 37oC prior to use. to the laboratory from a distance. Semen collection on the premise also enforces a chain of custody n Ejaculated Sperm starting at the point of origin. In some cases it may not be feasible for the patient to provide semen In majority of the couples seeking infertility sample on site. If the patient collects the ejaculate treatment through IVF, spermatozoa are isolated at an off-site location, he should be instructed to from fresh ejaculate provided on the day of the avoid lubricants that may have spermicidal effect. procedure. Several different procedures are avail- The patient should also be instructed to transport able for isolating sperm from seminal plasma for the sample container to the andrology laboratory insemination in assisted reproduction. The choice within 30 to 45 minutes of collection without of sperm preparation method largely depends on exposing it to extreme environmental temperature. Sperm Processing and Selection Techniques in an IVF/ICSI 153

The semen as it comes out of the urethra is in procedure in IVF since the presence of nonviable the form of dense liquid, however soon after the sperm, leukocytes and round cells in the close ejaculation, semen transforms into a jell-like proximity of oocytes may adversely affect fertili- coagulum. When allowed to stand at room tempe- zation and subsequent embryonic development. rature or at 37oC for approximately 30 to 45 minutes the coagulated semen liquefies completely. An Method initial semen analysis on the ejaculate should be • Measure the volume of the ejaculate. performed at this time (usually within one hour of • Add 4 × volume of sperm wash medium to the providing the sample) to determine basic para- ejaculate and mix well. meters such as the concentration, volume, total • Centrifuge at 300 × g for 10 minutes. sperm count, percent normal morphology, percent • Discard the supernatant. motility and progression. • Re-suspend the pellet in 1.0 ml sperm wash In the following section we describe the medium methods that are widely used in reproductive • Centrifuge at 300 × g for 10 minutes. laboratories for the preparation of spermatozoa • Discard the supernatant from the ejaculate: • Resuspend the washed pellet in 0.1 to 0.5 ml of A. Simple sperm-wash the culture medium. B. Swim-up from washed pellet • Mix gently. Perform postwash analysis and C. Swim-up from semen record the sperm concentration, motility, D. Gradient separation. morphology and progression as per standard procedure. SIMPLE SPERM WASH Note: Viscous semen samples should be treated (Recommended for oligospermic and with a-chymotrypsin (Bovine pancreas; CHY5S; asthenozoospermic semen samples; Sigma-Aldrich) before wash to obtain a good Recommended method of insemination-ICSI) yield of sperm. The procedure for enzymatic digestion of viscous sample is described below: Simple washing of sperm essentially removes the Aseptically dissolve the vial (5 mg) of a-chymo- seminal plasma without any significant decrease in trypsin in 2.5 ml of warm (37oC) sperm wash total sperm count. Sperm parameters such as the medium. Aspirate 0.5 ml of solution (a-chymo- percent of morphologically normal and motile trypsin, 1.0 mg) using a tuberculin syringe and add sperm in postwash samples usually remain to the viscous ejaculate. Mix by swirling the speci- unchanged however the proportion of sperm men container. Allow to stand for approximately 15 exhibiting rapid forward progression and minutes. Proceed to step 2 as described above. hypermotility may sometimes increase primarily due to the removal of seminal plasma. The simple sperm wash method has the inherent disadvantage SWIM-UP FROM WASHED PELLET of retaining and packing all the dense components (Recommended for normozoospermic semen of the semen in a tight pellet following centrifu- samples; Recommended method of insemination- gation. The motile sperm form a pellet along with IVF or ICSI) cellular and other debris present in the ejaculate. Some of the components such as white blood cells, Simple sperm wash procedure as described above round cells and nonviable sperm are known to be essentially separates cellular and particulate the source of reactive oxygen species that may be fraction from the seminal plasma. In order to harmful to live sperm.3 The simple sperm wash enhance the motile fraction of spermatozoa, a technique should therefore only be limited to isolate procedure that utilizes the upward migration of sperm from severe oligo- and asthenozoospermic progressively motile sperm is employed. The semen samples. The use of simply washed sperm is preparation at the end usually results in a high not recommended for standard insemination percentage of motile and activated sperm and is 154 A Practical Guide to Setting Up an IVF Lab, Embryo Culture Systems and Running the Unit

essentially free of the cellular debris. Sperm swim-up from sperm pellet as described above. The preparation by this method may be used for IVF. only difference is that instead of washing the sperm before the swim-up, the whole semen is used. Method Method • Prepare the sperm pellet by simple sperm wash as described earlier. • Take 0.75 to 1.0 ml of sperm wash medium in • Add 20 to 30 microliter sperm wash medium to sterile round bottom tube (Falcon 2003). Prepare the conical centrifuge tube containing the pellet 4 to 8 tubes for each patient. Incubate the tubes o and mix gently. Be careful not to introduce air at 37 C. bubbles to the thick sperm suspension. • Allow the ejaculate to liquefy and perform basic • Take 1.0 ml of pre-warmed (37oC) sperm wash semen analysis. medium in a sterile round bottom tube (Falcon • Using a sterile glass pipette, very gently underlay 2003). the liquefied semen (0.1–0.2 ml) in the bottom • Using a long (9.5") sterile glass pipette, very of the sterile tubes without disturbing the gently underlay the thick sperm suspension at semen-medium interface. Even slight the bottom of the Falcon 2003 tube containing disturbance of the interface may result in the prewarmed medium. Care should be taken not contamination of nonmotile/nonviable sperm in to disturb the suspension-medium interface. the media column. • Gently place the round bottom tubes in a • Place the tubes gently at 37oC for one hour. humidified incubator at 37oC for 60 minutes. • Motile spermatozoa migrate into the column of The motile spermatozoa migrate upwards into the culture medium. Gently aspirate the culture the column of the sperm wash medium. medium column, starting at the top. The • Gently aspirate the upper column of the uppermost layer, farthest from the interface medium taking care not to disturb the bottom typically contains the highest proportion of semen layer. The swim- up column from all the motile sperm. Take care not to disturb or aspirate tubes should be combined in a conical the residual suspension at the bottom of the centrifuge tube. tube. • Centrifuge the conical tube containing the • Transfer the aspirated swim-up fraction to a pooled swim-up fraction (sperm suspension) at fresh conical tube. 300 × g for 10 minutes • Centrifuge at 300 × g for 10 minutes and • Discard the supernatant. resuspend the pellet in 0.5 to 1.0 ml of culture • Resuspend the pellet in a small volume (0.1–0.25 medium. ml) of sperm wash medium. • Perform postwash semen analysis on the • Perform postwash analysis on the sample. sample. Note: As described earlier, for use in IVF sperm Note: If post wash sperm are to be used for IVF, wash medium may be substituted with it is recommended to use a bicarbonate-based bicarbonate-based IVF medium. IVF culture medium for swim-up (Step 3). In this case, the swim-up tubes are incubated at 37oC GRADIENT SEPARATION in a 5 to 6% CO2 environment. Gradient separation of motile spermatozoa is based SWIM-UP OF SPERMATOZOA on the premise that during centrifugation on a FROM WHOLE SEMEN density-gradient column, cells accumulate at a point (Recommended for normozoospermic semen of equilibrium that is identical to their own density samples; Recommended method of (isopycnic point). Motile/live sperm are denser than dead sperm or other cells present in the semen. insemination-IVF or ICSI) Following centrifugation, the live sperm accumulate This is a simple technique based on the separation as a pellet at the bottom of the centrifuge tube of motile sperm by upward migration similar to the whereas dead spermatozoa form a ring in the upper Sperm Processing and Selection Techniques in an IVF/ICSI 155

portion of the gradient column. The density gradient glass Pasteur pipette. Viscous semen samples method provides high proportion of motile sperm should be treated with chymotrypsin as with good yield and is one of the most popular and described earlier to reduce viscosity and widely used methods for sperm wash in reproduc- enhance the recovery of spermatozoa. tive/andrology laboratories. • Centrifuge at 400 × g for 20 minutes. Several variations of the density gradient • Using a glass pipette, gently aspirate the seminal method have been described.4 Some of these are: fluid and the upper PureCeption layer taking a. One step density gradient care not to disturb the sperm pellet in the b. Two layer discontinuous density gradient bottom. c. Three layer discontinuous density gradient or • Resuspend the sperm pellet in 3.0 ml sperm mini-density gradient. washing medium. Mix gently. In mid to late 1980s Percoll, a colloidal suspen- • Centrifuge at 400 × g for 5 to 10 minutes. sion of silica particles coated with polyvinyl- • Discard the supernatant and resuspend the pyrolidone (PVP) gained widespread acceptance as washed sperm pellet in 0.1 to 0.5 ml medium. the product of choice for use in density gradient • Perform postwash analysis on the sample. separation of spermatozoa. However, some batches of Percoll were reported to have high levels of TWO STEP DISCONTINUOUS endotoxins. In addition, this product was not FDA- DENSITY GRADIENT PROCEDURE approved for use in human clinical laboratory. In (Recommended for normozoospermic semen 1996, Pharmacia AB (Uppsula, Sweden) the samples; Recommended method of manufacturer of Percoll, withdrew it from use in density gradient separation of spermatozoa in insemination-IVF or ICSI) reproductive laboratories. This forced the • Warm the reagents to 37oC prior to use. development of other products (such as Nycodenz, • Prepare the following two gradients as Isolate, Iodixanol and PureCeption) for use in sperm described: preparation. Of these PureCeption, a Silane-coated – 80% PureCeption= 8.0 ml isotonic silica particle solution has gained popularity. Comparison of several density gradient media for PureCeption + 2.0 ml sperm wash medium selection of motile normal spermatozoa showed – 40% PureCeption= 4.0 ml isotonic PureCeption to be an effective substitute for PureCeption + 6.0 ml sperm wash medium Percoll.5-7 In the following section we present the • Take a conical centrifuge tube (Falcon 352095) procedures for separation of spermatozoa using and transfer 1.0 ml of 80% PureCeption solution PureCeption density gradient. in the bottom. Very gently overlay 1.0 ml of 40% PureCeption on the top of 80% solution. Take ONE STEP DENSITY GRADIENT care not to disturb/mix the two layers. (Recommended for normozoospermic semen • Gently overlay 1.0 ml of liquefied semen on the top of the 40% PureCeption. samples; Recommended method of Note: if the semen is viscous, treat with insemination-IVF or ICSI) chymotrypsin as described earlier. • Take the reagents out from the refrigerator and • Centrifuge at 400 × g for 30 minutes. warm to 37oC. • Gently aspirate the upper column of seminal • Take 9.0 ml of isotonic (100%) PureCeption plasma and PureCeption without disturbing the solution and add 1.0 ml sperm wash medium. sperm pellet. This is 90% PureCeption solution. • Add 0.1 ml fresh culture medium to the pellet, • Transfer 1.0 ml of 90% PureCeption solution to mix gently and transfer the sperm suspension to a conical centrifuge tube (Falcon, 352095). a fresh conical tube. Add 2.0 to 3.0 ml sperm • Gently layer 1.0 to 1.5 ml of liquefied semen on wash medium. the top of the 90% PureCeption using a sterile • Centrifuge at 400 × g for 10 minutes. 156 A Practical Guide to Setting Up an IVF Lab, Embryo Culture Systems and Running the Unit

• Discard the supernatant and re-suspend the • Gently overlay the suspended pellet on the top pellet in 0.1 to 0.5 ml of culture medium. of the 40% PureCeption layer. • Perform postwash sperm count, motility and • Centrifuge at 400 × g for 45 minutes. morphology as per laboratory’s standard • Using a sterile Pasteur pipette, gently aspirate protocol. the upper layers of the gradient while leaving the sperm pellet in the bottom of the tube THREE LAYER DISCONTINUOUS DENSITY undisturbed. GRADIENT OR MINI-DENSITY GRADIENT • Transfer the sperm pellet along with small PROCEDURE volume of residual medium to a new conical centrifuge tube. (Recommended for oligozoospermic and/or • Add 2.0 ml sperm wash medium and mix the asthenozoospermic semen samples) suspension gently. This procedure is based on the method described • Centrifuge at 400 × g. by Ord and colleagues8 in 1990 for isolating sperm • Discard the supernatant and resuspend the for IVF from poor semen samples. It was used (with sperm pellet in 0.1 ml of sperm wash medium. limited success) in pre-ICSI era to obtain enough • Perform postwash analysis. motile sperm for IVF from oligo and asthenozoospermic samples. With the advent of ICSI, sperm n Retrograde Ejaculation preparation by three-step gradient method seems extensive, laborious and superfluous. This method Normally following orgasm, during the process of may however be used in certain situations (e.g. ejaculation, spermatozoa stored in the distal part of patients with oligospermia who may prefer IVF to the epididymis travel through the vas deferens and ICSI for insemination) and is therefore presented. en route are mixed with the secretions of prostate gland and seminal vesicles. The mixture of sperm, Method prostatic fluid, seminal vesicular fluid and secretions • Warm the reagents to 37oC. of bulbourethral glands pass through the urethra • Perform a simple sperm wash on liquefied and the semen is released from the urethral opening. semen as described earlier as resuspend the As a normal physiologic process, during ejaculation pellet in 0.3 ml sperm wash medium. the sphincter in the neck of the urinary bladder • Prepare three different PureCeption density constricts to prevent urine leakage into the urethra. solutions (40%, 60%, 80%) as follows: However, in some men, instead of passing through – 40% PureCeption= 4.0 ml isotonic the urethra the semen reflexes back (retrograde ejaculation) into the urinary bladder. This condition PureCeption + 6.0 ml sperm wash medium is also sometimes referred as “dry ejaculation” since – 60% PureCeption=6.0 ml isotonic the male experiences the orgasm but apparently no PureCeption + 4.0 ml sperm wash medium semen is noticeable (aspermia). Retrograde – 80% PureCeption=8.0 ml isotonic ejaculation may be caused by surgical interventions PureCeption + 2.0 ml sperm wash medium related to the prostate or urethra or by certain medical These solutions can be prepared in advance, conditions such as diabetes. Certain medications labeled, stored in the refrigerator (4oC) and used used to treat hypertension or benign prostate up to one week. enlargement, as well as some psycho-altering drugs • Take a conical centrifuge tube (Falcon 352095) have also been reported to be a causative factor in and pipette 0.3 ml of 80% PureCeption solution retrograde ejaculation. in the bottom. On the top of this gently layer 0.3 The exposure to urine is nonphysiologic and ml of 60% PureCeption solution. Take care not detrimental for spermatozoa. Usually the low to mix the two gradients. Now taking utmost (acidic) pH and high osmolality of urine adversely care, layer 0.3 ml of 40% PureCeption solution affects the viability of ejaculated sperm. In order to on top of 60% column. isolate spermatozoa from urine for insemination Sperm Processing and Selection Techniques in an IVF/ICSI 157

procedure, it is important to minimize the pH and touch the inside of the containers. Some patients osmotic stress as well as the duration of exposure of experience partial retrograde ejaculation. This is spermatozoa to urine. This is done by calibrating the marked by the appearance of a few drops of semen urinary pH to slightly alkaline range (7.5–8.0) and at ejaculation (antegrade) while most of the osmolality between 260 and 350 mOsm at the time ejaculate reflexes into the bladder. If any antegrade of ejaculation. ejaculate is expelled, it should be collected in one of the two containers and not mixed with the urine. n Procedure for Isolation of Method Spermatozoa from the Urine of Patients with Retrograde • Measure the volume of the urine using a sterile pipette. Ejaculation • Divide the urine aseptically into several conical centrifuge tubes (Falcon 352095). PATIENT PREPARATION • Centrifuge all the tubes together at 400 × g for 15 minutes. The patient is instructed to abstain from ejaculation • Discard the supernatant from all the centrifuge for at least 3 days. On the evening of the day before tubes. providing the sample, the patient is asked to drink a • Collect the pellets from all the tubes and glass of water (250 ml) containing 5 g sodium resuspend in 5.0 ml culture medium. bicarbonate (NaHCO3). In the morning, after waking • Centrifuge the sperm suspension for 10 minutes up the patient may empty his bladder to void at 400 × g. concentrated urine and drink another glass of water • Discard the supernatant. (250 ml) with sodium bicarbonate (5 g). In about an • Resuspend the washed pellet in 0.5 to 1.0 ml of hour or so when the patient starts to feel the urge to sperm wash medium. empty the bladder, he should provide a sample of • Perform postwash analysis. urine (without emptying the whole bladder) in a Note: Several variations of the sperm isolation sterile specimen container. The pH and osmolality technique from retrograde urine exist. In one of the urine sample is determined and if these are such method the patient is provided with sterile in the acceptable range (pH between 7.2 and 8.2 and containers that are prefilled with 30 to 40 ml of concentration between 260 and 350 mOsm/L), prewarmed protein supplemented sperm wash patient is ready to provide the sample as described medium or TEST-YOLK buffer. The patient then below. In case, the pH is lower than acceptable (in directly voids into the container with the culture the acidic range), the dosage of sodium bicarbonate medium thus reducing the deleterious effects of should be increased. Patient may drink another exposure of sperm to urea, salts and other glass of water with sodium bicarbonate and wait for excreted metabolites. an hour to provide another sample. Alternatively, if The spermatozoa isolated from retrograde the sperm are to be cryopreserved for later use, it ejaculate often exhibit suboptimum vitality and may be more feasible to increase the sodium motility presumably due to a combination of pH, bicarbonate dosage to 10 g each in the evening and osmotic and solute (especially urea) shock. Even the morning and reschedule the appointment. If the though IVF using retrograde ejaculated sperma- urine is concentrated (>350 mOsm/L), the patient tozoa is feasible and has been employed success- should drink water (250 ml) and provide another fully, nowadays ICSI is the recommended method test sample in 30 to 60 minutes. Once the pH and for inseminating oocytes. osmolality are within acceptable range the patient is ready to provide the postejaculatory retrograde urine. n Testicular Sperm Patient is provided with two sterile specimen containers and instructed to collect urine soon after Soon after the introduction of ICSI it became evident ejaculation. Patient should follow caution not to that various sperm parameters in the ejaculated 158 A Practical Guide to Setting Up an IVF Lab, Embryo Culture Systems and Running the Unit

semen had little bearing on fertilization and testicular suspension to a hypotonic solution successful outcome. If viable sperm (as evidenced effectively removes erythrocytes with no harmful by motility, tail movement or hypo-osmotic swelling effect to spermatozoa. The RBC-lysis buffer is test) were available in the semen, fertilization and prepared as follows: pregnancies could be achieved. Report of successful NH4Cl (Sigma Cat # A 0171) 0.829 g 9 pregnancy by the use of testicular sperm in 1993 KHCO3 (Sigma Cat# P 9144) 0.100 g launched a new era for the treatment of infertility EDTA (Sigma Cat # ED2SS) 0.074 g in men with certain types of azoospermia. The Dissolve in 100 ml of tissue culture grade water incidence of azoospermia in general population has and adjust the pH to 7.2. Filter sterilize using 10 been reported to be between 1 and 2%. In men Nalgene syringe filters (0.22 µ) attached to 50 ml with nonobstructive azoospermia, the cause can syringe. The RBC-lysis buffer may be stored in often be attributed to microdeletions in the Y- refrigerator (4oC) for one month. chromosome. The spontaneous mutation induced loss of AZFa and AZFb regions on the long arm of n Procedure for Preparation of the Y-chromosome have been reported to result in absolute azoospermia. In comparison, the micro- Testicular Sperm deletions in the adjacent AZFc region on the same • Wash the testicular tissue with 1 to 2 ml of arm of the Y chromosome affect spermatogenesis culture medium to remove red blood cells. less dramatically resulting in severe oligospermia. • Transfer the tissue to a petridish and add a few Sometimes patients with AZFc microdeletion show drops of culture medium to keep the tissue complete absence of sperm in the ejaculate, how- moist. Using a pair of sterile scissor, mince the ever upon further investigation focal spermato- tissue. Add 1.0 ml of culture medium to the finely genesis in isolated seminiferous tubules may be minced/squeezed sample. Aspirate the 11 evident. suspension and gently pass it through a 21 gauze Men with obstructive azoospermia have normal needle attached to a 3.0 ml syringe. Repeat this spermatogenesis. Surgical correction may restore process 2 to 3 times. The passage through the fertility in some men however patients with certain needle breaks down the seminiferous tubules in conditions such as congenital bilateral absence of small pieces and also dislodges the sperm from vas deferens (CBAVD) are left with the only option the lumen. of testicular sperm extraction for the treatment of • Place 5 to 10 ul of suspension in petridish and male factor infertility. Spermatozoa retrieved from overlay with mineral oil. Observe and record the testis are mostly nonmotile but viable and may show number of spermatozoa per high power field weak tail movement. Epididymal sperm on the other (200 × g). hand when retrieved are mature with good motility. • Transfer the suspension to a conical tube and The detailed procedure for the extraction and let it stand at room temperature for 5 minutes. preparation of spermatozoa from testicular and This allows the pieces of seminiferous tubules epididymal source is presented in the following and large tissue clumps to settle down. section. • Transfer the supernatant to another conical tube and centrifuge (300 × g) for 10 minutes. n Procedure for Isolation of • Discard the supernatant. Testicular Sperm • If the pellet exhibits the presence of red blood cells (as it invariably does), add 2.0 ml of RBC- PREPARATION OF RBC-LYSIS BUFFER lysis buffer to the tube and resuspend the pellet. (155 mm Ammonium Chloride, 10 mm • Centrifuge at 300 × g for 5 minutes. Potassium Bicarbonate, 2 mM EDTA; pH 7.2) • Discard the supernatant and wash the pellet with culture medium (1.0 ml). Varying quantity of red blood cells is observed in the • Resuspend the pellet in 0.1 ml of culture testicular biopsy samples. A brief exposure of medium. Sperm Processing and Selection Techniques in an IVF/ICSI 159

Note: If the pieces of seminiferous tubules are ICSI, it is not an absolute requirement for achieving obtained by TESA , the sperm may be released livebirths. Several methods for isolating in the medium by gently squeezing the tubules spermatozoa from ejaculate and other sources are with a 27 gauze needle attached to a tuberculin available. The choice of sperm wash method should syringe or a flexible embryo handling pipette. depend on several factors including the quality of The squeezing of tubules should be done in the sample and the method of insemination. several flat droplets of culture medium preferably under oil. The culture medium drops n References containing testicular sperm suspension are aspirated using a 200 ml pipette and transferred 1. Mortimer D. Biochemistry of spermatozoa and to a conical bottom tube. The tube is centrifuged, seminal plasma. In: Practical Laboratory Andro- supernatant is discarded and the sperm pellet logy, Oxford University Press; 1994.pp.89-109. re-suspended in 50 to 100 ml culture medium. 2. Palermo G, Joris H, DeVroey MD, Van Steirteghem The sperm obtained from testis can only be used AC. Pregnancies after intracytoplasmic injection for insemination by ICSI. of single spermatozoon into an oocyte. Lancet. 1992;340:17-8. PREPARATION OF SPERM FROM 3. Chandra A, Surti N, Kesavan S, Agarwal A. EPIDIDYMAL ASPIRATES Significance of oxidative stress in human reproduction. Arch Med Sci. 2009;5:S28-S42. • Multiple aspirates from PESA (percutaneous 4. Mortimer D. Sperm recovery techniques to epididymal sperm aspiration) or MESA (Micro- maximize fertilizing capacity. Reprod Fert Dev. epididymal sperm aspiration) are provided by 1994;6:25-31. the urologist. 5. Classens OE, Menkveld R, Harrison KL. Evalua- • Scan a small fraction from each aspirate for the tion of three substitutes for Percoll in sperm presence of sperm. isolation by density gradient centrifugation. Hum • Those aspirates that contain spermatozoa are Reprod. 1998;13:3139-43. pooled together in a conical centrifuge tube. 6. Mousset-Simeon N, Rives N, Masse L, Chevallier • Add 2.0 to 3.0 ml sperm wash medium to the F, Mace B. Comparison of six density gradient tube containing pooled epididymal aspirates. media for selection of cryopreserved donor Mix gently. spermatozoa. Androl. 2004;25: 881-4. • Centrifuge at 300 × g for 10 minutes 7. Allamaneni SSR, Agarwal A, Rama S, • Discard the supernatant. Ranganathan P, Sharma RK. Comparative study • Resuspend the pellet in 0.5 ml sperm wash on density gradients and swim-up preparation medium. techniques utilizing neat and cryopreserved spermatozoa. Asian J Androl. 2005;7:86-92. n Conclusion 8. Ord T, Patrizio P, Marcello E, Balmaceda P, Asch RH. Mini-Percoll: A new method of semen The development of the technique of in vitro preparation for IVF. Hum Reprod. 1990;5:987-9. fertilization was a major breakthrough deemed 9. Schoysman R, Vanderzwalmen P, Nijs M, Segal worthy of the 2010 Noble Prize in Physiology and L, Segal-Bertin G, Geerts L, et al. Pregnancy after Medicine to Dr Robert G Edwards. Later fertilization with human testicular spermatozoa. developments such as ICSI paved the way for Lancet. 1993;342:1237. treatment of severe forms of male factor infertility. 10. Jarow P, Espeland MA, Lipschultz LI. Evaluation Theoretically for ICSI one viable spermatozoon per of the azoospermic patient. J Urol. 1989;142:62- oocyte is all that is needed for achieving fertilization 5. and pregnancy. Even though normal sperm 11. O’Flynn O’Brien K, Varghese AC, Agarwal A. The morphology may have some correlation with genetic cause of male infertility: A review. Fertil fertilization and successful pregnancies following Steril. 2010;93:1-12.

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