Ejaculated semen is processed to separate viable from non-viable sperm for artificial insemination (AI), intrauterine insemination (IUI), or for assisted reproductive technology (ART). Sperm concentration is one of the major semen parameters to be evaluated before semen processing. The clinical diagnosis of male partner fertility relies primarily on the outcome of standard semen analysis. It is therefore imperative that the concentration of sperm be determined accurately and reliably. Although the initial sperm concentration of a semen sample has been shown to have a relatively low value postpreparation, concentrations are routinely used for the accurate insemination of oocytes in vitro. The parameter can be measured manually or by a computer-assisted semen analyzer system (CASA). Several reusable and disposable chambers are available for both manual and computerized evaluation. Before a chamber can be used for the routine evaluation of sperm concentration, its accuracy and precision has to be evaluated by comparison to a hemocytometer (Neubauer, Germany), Makler chamber (Sefi Medical Instruments, Israel) or disposal Leja chamber (Leja, Nieuw-Vennep, The Netherlands) (Figures 15.1-15.4).
SPERM SEPARATION METHODS
(TABLES 15.1 AND 15.2)
Separation of viable from non-viable sperm
The semen of both human and animal ejaculates contains a mixture of viable and non-viable spermatozoa; the non-viable cells can exceed 80% of the total, consisting of dead, immotile or abnormal cells. In cases where the proportion of viable cells is low, problems regarding the fertility of the subject arise, using both natural and AI. Therefore, it is necessary to develop methods of enchancing the proportion of viable cells present when AI is to be attempted using a donor semen.
Figure 15.1 Microscopic examination using hemocytometer to count sperm in the ejaculate (a) and (b). Note an automatic counter at the bottom of (b) to count simultaneously viable and non-viable sperm, leukocytes, debris and other cell contaminants. (c) Micropipetting for evaluation, counting and storage in cryopreservation straws for sperm and eggs
Figure 15.2 Model tray for the collection of a split ejaculate. The tray consists of three cups (C) which are attached to each other, a handle (H) and a tray cover. Each cup is oval and rounded with a cross-sectional diameter of 6 cm to allow entry of the glans penis. Approximately 1 cm from the bottom of the tray the cup comes to a point. The depth at the point of each cup is 6 cm. Volume markings are present at the bottom of each cup for easy volume measurement. Each cup is numbered to indicate the ejaculate fraction (I,II or III).The tray is made from clear plastic with no spermicidal properties.The handle is 12cm long and rounded,with a diameter of 3 cm, so that it fits easily into the hand.The cups are covered with coverslips after ejaculation so that spillage is avoided (designed by Hafez, Zaneveld and Polakoski)
Separation of human sperm
Sbracia et al. (1996) applied the following technique to separate human sperm:
(1) Nycodenz isotonic solution (27.6% w/v Nycodenz) is supplemented with 1 mg/ml human serum albumin.
(2) Ham’s F-10 medium containing 1 mmol/l calcium lactate, 20 mmol/l NaHCO3, 5 mmol/l KHCO3, 0.5 mmol/l MgSO4, 50 mg penicillin G, 50 mg/ml streptomycin and 1 mg/ml human serum albumin is prepared.
(3) Four aliquots of Nycodenz solution are prepared by dilution of the stock solution with the F-10 medium.
(4) The aliquots are carefully layered into 12-ml centrifuge tubes (under-layering will preserve the interface between the layers more efficiently).
(5) Liquified semen is diluted with 1-2 volumes of medium, loaded on top of the gradient and centrifuged at 350 g for 10 min.
(6) After centrifugation the seminal fluid is removed together with the 9.7% and 13.8% layers and any cells that had settled on the 13.8/17.9% interface; these aliquots are discarded.
(7) The 17.5% layer is collected, diluted with medium and mixed gently by inversion.
(8) After centrifugation at 350 g for 10 min, the supernatants are discarded and the pellets containing the motile cell fraction are gently resuspended in fresh medium.
Calculation of sperm cell concentration
Establish average sperm count from two chambers.
Dimensions of the large central area of the Neubauer counting chamber are 1 mm (width) x 1 mm (height) x0.1 mm (depth) for a volume of 0.1 mm3. Since sperm cell concentration is normally reported in sperm number per milliliter (ml), the sperm count must be multiplied by a factor of 10 000. Since the semen is diluted at a 1 : 100 ratio prior to the sperm count, the final sperm count must be multiplied by an additional factor of 100.
Example
Sperm count of diluted semen in chamber 1 =240
Sperm count of diluted semen in chamber 2 = 250
Average sperm count=245
Sperm cell concentration = 245 x 104 x 102/ml or 245 x 106/ml
Figure 15.3 Measurement of sperm concentration using a hemocytometer (a), and (b), the semen suspension is placed under the coverslip of the hemocytometer, (c) the sperm count is conducted under approximately 10x magnification, (d) and (e) sperm are counted in squares. CELL-VU® (not shown) is a disposable hemocytometer that can be used for all manual cell counting needs, from blood (full blood count) and sperm to body fluids, such as cerebral spinal fluid
Figure 15.4 (a) Makler® counting chamber used for rapid semen analysis from undiluted specimens. For rapid results the number of spermatozoa counted in ten squares of the grid indicates their concentration in million/ml.The grid is built into the cover glass. There is no need to insert a grid into the microscope eyepiece. The optimal depth of 10 gm eliminates blurring and enables free sperm movement for easy motility evaluation. Sperm count, motility evaluation and morphology determination can all be undertaken in one chamber, which is economical, reusable and easily cleaned with disinfectant solution. Calibration is unnecessary and repeated use does not alter the accuracy. (b) Cross-section of the Makler chamber with a drop of semen placed on the center of the lower part; (c) when covered with the coverslip which is placed upon the four slightly elevated pins the drop becomes 10-gm thick. Sperm cells are spread in one focal plane where they can move. (d) One of the 0.2 x 0.2 mm squares, bounded by double lines and subdivided into 16 small squares in which sperm are counted, the sperm with white heads are ignored. (e) Makler insemination device designed to seal the cervix preventing the leakage of injecting semen (courtesy Sefi Medical Instruments Ltd., Haifa, Israel)
Sperm filtration
Human periovulatory mucus is an effective barrier against seminal fluid containing decapacitating factors, non-motile or dead sperm, leukocytes, prostaglandin and various infective agents. Cervical mucus is particularly selective in allowing only progressively motile sperm of normal shape and size to penetrate and migrate through the cervix.
Sperm processing methods that mimic this periovulatory-mucus selectivity may therefore favorably influence the outcome of oocyte fertilization. The simplest sperm washing techniques are those which merely concentrate the most favorable sperm and remove seminal plasma; however, these may not be adequate owing to their imprecise nature. Other sperm preparations have been developed that more closely mimic cervical mucus selectivity. Such procedures specifically recover a select population of normal motile sperm from the raw ejaculate.
Psychosocial parameters of infertile couples
Infertile couples are characterized by various psychosocial parameters, including anger, grief, guilt, frustration, depression, feelings of isolation and powerlessness, depression, marital problems, separation and divorce. However, sporadic conception is noted in infertile couples after or during romantic holidays, after adoption, or after abandoning infertility-clinic treatment. Spontaneous pregnancy occurs in 10% of untreated infertile couples with oligozoospermic men, in patients who have been on a waiting list for in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) for 2 years. ‘Psychogenic infertility’ is associated with chronic and acute stress with transitory azoospermia or oligospermia.
SPERM PREPARATION FOR ASSISTED REPRODUCTIVE TECHNOLOGY
Various procedures have been applied to maximize the use of naturally ejaculated fresh semen and stored, cryopreserved semen (Tables 15.3-15.5):
(1) In vitro fertilization (IVF);
(2) Gamete intrafallopian transfer (GIFT);
(3) Intracytoplasmic sperm injection (ICSI);
(4) Intrauterine insemination (IUI);
(5) Epididymal sperm aspiration/testicular biopsy sperm extraction;
(6) Testicular/epididymal sperm cryopreservation;
(7) Cryopreservation of specimens with extremely low sperm count or motility.
The female reproductive tract naturally induces sperm capacitation. Thus IVF and GIFT procedures, although requiring fewer viable sperm than IUI, nonetheless require in vitro means of capacitation.
The various techniques of sperm preparation for assisted reproduction are directed to the separation of sperm from seminal fluid and from other cellular components of the semen as well as to the concentration of the motile sperm population. The most frequently used techniques are: sperm ‘washing’ in various media; ‘swim-up’ or ‘swim-down’ preparations, in which the motile sperm population migrates away from the non-motile elements of semen; and centrifugation of sperm through various density gradients exploiting the higher density of mature sperm that completed cytoplasm extrusion during spermatogenesis (Figures 15.5 and 15.6).
Concerns about the adverse ‘iatrogenic’ effects of sperm preparation techniques have focused on three issues regarding the choice of sperm preparation media, the mechanical damage owing to centrifugation/resuspension, and the propagation of lipid peroxidation during sperm pelleting owing to the close vicinity of already-damaged leukocytes and spermatozoa that may generate reactive oxygen species.
Table 15.5 Physiological action and clinical applications of products (courtesy Nidacon International AB, Goteborg, Sweden)
|
Product |
Physiological action |
Clinical application |
|
Sperm |
Cryopreserving |
Freezing solutions containing egg yolk, high concentrations of glycerol can be toxic to sperm; |
|
CryoProtectTM |
human sperm |
thus, it is free from egg yolk and contains a reduced concentration of glycerol. It is an optimized solution for protecting human sperm from damaging freezing/thawing effects. The solution contains osmotically active ingredients to reduce intracellular water, and cryoprotectants to reduce injury caused by ice crystal formation. Human serum albumin is added to serve as a cryoprotectant, counteract the detrimental effects of reactive oxygen species (ROS), and prevent sperm aggregation/adherence to laboratory equipment It can be also used as a cryoprotectant for untreated ejaculates. However, when untreated ejaculates are used, the number of motile sperm post-thawing will be reduced Seminal plasma, a source of prostaglandins, ROS and pathogens, should be removed from the sperm suspension before intrauterine insemination (IUI) |
|
PureSperm®100 |
Improved stock suspension |
Improved formulation so the gradient works even more efficiently and cleanly than before PureSperm® stock suspension should be diluted with PureSperm® Buffer to provide layers of different densities required for the gradient Sperm pellets are washed in PureSperm® Wash. These three products, PureSperm 100 diluted with PureSperm Buffer, and PureSperm Wash, form an optimized system to prepare sperm for assisted reproductive technologies (ART) |
|
Ready SwimTM |
Solution for optimized swim-up |
Balanced salt solution that maximizes sperm survival during ‘swim-up’ In vivo, motile sperm quickly migrate from liquefying semen into the cervix, thereby removing themselves from the presence of both seminal plasma and ROS arising from cell debris. In vitro, this process is imitated by allowing sperm to swim into a layer of medium, thereby separating them from remainder of ejaculate |
Table 15.5 (Continued)
|
Product |
Physiological action |
Clinical application Ionically balanced solution to avoid premature hyperactivation. Sudden pH and osmolatity changes during the sperm movement from semen sample to the fertilization medium are minimized. Avoiding premature hyperactivation conserves energy resources and improves fertilization potential During swim-up, motile sperm move away from extraneous cells and damaging factors in the ejaculate: ROS/seminal plasma. Damage to sperm DNA and structural molecules caused by ROS is minimized by removal of immature sperm/lymphocytes, key sources of ROS Motile sperm separated from immotile sperm, epithelial cells/cell debris.When unopened can be stored for 1 year at room temperature.The use of borosilicate Type I glass bottles ensures that Na+ ions do not leach into the preparation.This prevents any changes in Na+ levels and osmolality which can impair sperm survival and function, and cause premature hyperactivation |
|
PureSperm® Wash |
Washing sperm pellet |
A balanced salt solution containing EDTA/human serum albumin. Designed specifically for washing sperm pellet obtained from a PureSperm gradient |
|
Rapid method to prepare human sperm |
‘Swim-up’ does not remove sperm with abnormal morphology or damaged DNA, and does not separate out bacteria and viruses from the sperm preparation.There is commercially available ‘Speedkit’, a kit containing sufficient sperm preparation material for five patients, together with glass centrifuge tubes, five semen collection tubes, one for each patient, and empty tubes to balance the centrifuge rotor PureSperm is a single layer of colloid, ready-to-use in the centrifuge tube: medium added to 1.5 ml of patient semen and centrifuged.The sperm pellet is then transferred to the centrifuge tube containing SpermAssistTM which is a washing solution and maintenance medium.After centrifugation, most of the supernatant is removed. The sperm pellet is then resuspended in the last 0.5 ml of the washing solution, creating a sperm preparation ready to transfer directly to the patient. Instructions included in pack insert |
|
|
Sperm CatchTM |
Used for ICSI |
A natural alternative to polyvinyl plastic for slowing sperm ICSI The only products available for slowing sperm motility contained polyvinylpyrrolidone; injecting this artificial product into the oocyte along with the sperm may cause damage to acrosomal, mitochondria plasma membranes and chromosomal DNA It contains only substances normally found in mammalian reproductive tract, it slows sperm, and mimics the naturally occurring sequence of events during IVF |
|
NidOilTM |
Selected for its purity and suitability for use with embryos |
Some procedures for IVF and ICSI use an oil overlay to protect small volumes of culture medium from evaporation in the incubator and to help dampen the effects of sudden changes in temperature, gas saturation and pH. Likewise,gamete manipulation under the microscope requires the gametes to be held in small drops of medium under oil, to maintain a constant environment within the culture medium NidOil, a paraffin oil product designed for gametes/embryos is neither sticky nor too viscous, to facilitate pipetting, but is sufficiently viscous to prevent movement of drops of media around culture dish |
|
Sperm Assist™ |
For IUI |
HEPES-buffered salt solution formulated specifically to resuspend sperm for IUI;with ionic balance to meet sperm requirements avoiding premature hyperactivation Sperm survival/fertilizing ability and pregnancy rates improved, avoiding media which cause premature hyperactivation Does not contain any coloring or preservatives, which can be toxic to sperm Used to suspend sperm prepared on a PureSperm® gradient, (made from either PureSperm® Buffer or ready-to-use PureSperm® 40 + PureSperm® 80) and washed with PureSperm® Wash gives optimal results. Yield of motile sperm is maximized, sperm survival is increased |
Figure 15.5 PureSperm® product range that uses integrated and optimized sperm preparation for assisted reproductive technology (Nidacon International AB, Goteborg, Sweden). IVF, in vitro fertilization; ICSI, intracytoplasmic sperm injection; CRYO, cryoprotectant; IUI, intrauterine insemination
Sperm processing for intrauterine insemination
Several methods have been applied to separate and eliminate decapacitation factor(s) and contaminants from the seminal plasma. These may be in the form of cellular debris (gelatinous pieces, epithelial cells), bacteria, mycoplasmas, chlamydia, trichomonads and various blood components (leukocytes/erythrocytes). This is accomplished through filtering the motile sperm, and recovery of the most viable sperm. The three methods that are reliable, repeatable and simple to perform are sperm migration, column adherence and density gradient centrifugation (swim- up and swim-down).
Gamete interaction and intracytoplasmic sperm injection
Extensive investigations have been conducted on the micromanipulation techniques used to enhance fertilization and successful pregnancy in sheep and goats with specific emphasis on direct sperm injection into oocytes. Exogenous oocyte activation was not mandatory for fertilization, and subsequent normal fetal development. Several techniques have been applied for domestic and exotic mammalian species.
Gamete interaction involves recognition between a sperm receptor located in the plasma membrane and an oocyte receptor located in the zona pellucida (ZP3). ZP3 induces the acrosome reaction. Closer interaction occurs between ZP2 and an inner acrosomal membrane receptor. Certain acrosomal proteins are necessary for this process. The acrosome is necessary for the sorting and correct organization of plasma membrane proteins. Immotile sperm used for ICSI give results comparable to those of motile sperm if the initial ejaculate contained > 20% viable sperm and specific criteria were used to select the immotile sperm.
Testicular sperm processing method
Little is known about processing testicular sperm in men. Figure 15.7 shows the cystoscopic view after transurethral resection of the ejaculatory ducts, the appearance of vasal anastomosis after inner-layer suturing, the epididymis after epididymal tunic aperture creation, and the longitudinally or transversely incised scrotum.
SEMEN PROCESSING PRODUCTS (TABLE 15.5)
Several commercially available products are optimized for purification of human sperm for assisted reproductive technology. All batches are strictly tested to assure high levels of sterility, low endotoxin levels and optimal performance. Ready-to-use products, such as 40% and 80% dilutions (PureSperm® 40 and PureSperm® 80, respectively), save preparation time in the laboratory. PureSperm 40 and PureSperm 80 are formulated to minimize sudden pH and osmolality changes during the transition of sperm from the semen sample to the fertilization medium. This helps to avoid premature hyperactivation and improves the fertilization potential. They maintain a pH between 7.4 and 7.8 at room temperature and at 37°C, thus providing suitable conditions for good sperm motility, survival time and fertilization potential. The preparation contains glucose as a usable energy substrate for normal cell metabolism and sperm function.
Figure 15.6 Preparation of PureSperm® density gradients for human sperm. It is recommended to prepare two gradients for each ejaculate in steps A-F (Nidacon International AB, Goteborg, Sweden)
Figure 15.7 Testicular sperm processing methods. (a) Cystoscopic view after transurethral resection of the ejaculatory ducts, showing obstructing ductal calculi within lumens, bilaterally. (b) Appearance of the same vasal anastomosis after inner-layer 10-0 nylon sutures have been tied and cut. The inner layer has been completed, and the anastomosis is watertight. (c) View of the epididymis after epididymal tunic aperture creation. Note pearly white and dilated tubule appearance. (d) Scrotum longitudinally or transversely incised.Tunica vaginalis longitudinally deepened. Tunica albuginea is exposed surface. From Brannigan et al., 2003, with permission
Nicotine (systematic name, 5-(N-2,3-dihydrox- ypropylacetamido)-2,4,6-tri-iodo-N, N' bis (2,3- dihydroxypropyl)isophthalamide) is a non-ionic iodinated gradient medium which readily dissolves in water to produce non-toxic autoclavable solutions. To separate viable cells in a morphologically intact state by isopycnic centrifugation, it is necessary to use a gradient medium that provides gradients of an appropriate density/tonicity. It is important to maintain isotonic conditions throughout the gradient; otherwise, the cells will change in volume and, therefore, density, and large changes in tonicity across the gradient can damage the cells irreversibly. The properties of iodinated gradient media make them suitable for separating cells and sperm (Ford and Rockwood, 1982).
‘Accudenz’ facilitates a higher rate of recovery of sperm and motile sperm. Sperm motility is lower in Accudenz compared to Percoll pellets; however, the long-term retention of sperm motility is substantially improved in Accudenz at 24 h. Sperm activation status monitored by chlortetracycline fluorescence indicated that after 4 h of incubation the incidence of fully acrosome-reacted spermatozoa in the Accu- denz versus Percoll pellets was 6.2 ± 0.3% versus 13.1 ± 1.0%, a 100% increase in Percoll.
Accudenz yields a higher concentration of motile sperm with improved retention of motility, velocity and acrosomal integrity and without an increase of sperm with diminished cellular maturity.
TEST yolk treatment
TEST (TES and Tris buffer) yolk treatment enhances sperm quality by increasing the ability of sperm to penetrate zona-free hamster oocytes. Such treatments also enhance the ability of sperm to bind to the human zona pellucida, further improving IVF outcome. These sperm enhancement functions have been substantiated (and with no apparent deleterious effects on sperm). To enhance fertilization potential further, the treated sperm sample can be additionally processed using column adherence methods.
SPERM PREPARATION EQUIPMENT
Table 15.6 shows the equipment, separation media and techniques for sperm preparation and IVF.
Real-time sperm separation
Wang tubes (Figures 15.8 and 15.9)
This system encompasses the application of newly designed separation tubes and innovative methods. Several biological and biophysical phenomena, and basic and theoretical principles, have been applied in the real-time sperm separation system:
(1) Non-pathological spermatozoa do not transfer microorganisms.
(2) The motility pattern and swim-up capacity of infected sperm or pathological sperm are limited or disturbed.
(3) The faster the sperm migrate and the greater distance they cover, the more normal are the sperm.
(4) Spermatozoa that are different from what is expected for their stage of development and sperm with morphological or physiological disorders can be identified by characteristics such as different migration velocities and distances.
Figure 15.8 Wang tube system is the most effective method of obtaining a high-degree of sperm isolation and purification, which separates motile sperm from microorganisms, highly motile cells from less-motile or non-motile cells in a mixed sample, and double-stranded (normal) from single-stranded (abnormal) DNA sperm, while retaining an adequate volume of tissue culture medium, with an absence of cell debris, seminal plasma or other impurities in semen
(5) Highly motile sperm can be easily separated from other live cells such as leukocytes, red blood cells, lymphocytes and cellular debris.
(6) Tissue culture medium has a low viscosity, which does not favor the adhesion of the microorganisms to highly motile sperm.
(7) The specific gravity of a sperm pellet is greater than that of the tissue culture medium, so that the pellet will fall steadily to the bottom of a Wang tube, allowing the motile sperm to migrate.
(8) The velocity and direction of sperm migration are quite different from the random active or passive movement of microorganisms.
(9) The Wang tube system is a multidirectional chal- lenge/migration (MCM) technique. It is not just a simple swim-up technique. The spermatozoa are allowed to migrate along multiple directions and pass through carefully designed, computerized curvatures of critical angles. The challenge facilitates the selection of high-quality sperm.
(10) These formulae are based on the theories of Bernoulli’s equation and application of hydrodynamics.
GENETIC PARAMETERS
Gender selection
An Egyptian myth relates how the goddess Isis was once compelled by the fertility god Osiris to make all her progeny male by sorcery. This idea has not been entirely discarded and has recently re-emerged as advances have been made in separating X from Y spermatozoa. With increasing success, sex preselection has become a matter of general concern. Khatame et al. (1999) evaluated the success of gender selection using semen samples separated by a modified swim-up technique. They retrospectively compared the gender outcome of two treatments (A and B) for either male or female offspring with those who conceived spontaneously. The treatment groups consisted of 52 pregnancies for couples who conceived with the separation technique. Of these 52 participants, 15 desired female offspring and were placed into treatment group A and 37 desired male offspring and were placed into treatment group B. The control groups consisted of 162 women who presented with initial consultation for gender selection and conceived spontaneously. Control group A consisted of 80 women who initially chose female offspring, and control group B consisted of 82 participants who initially chose male offspring. In treatment group A, one timed IUI was performed using the bottom 0.5 ml of the separated semen on cycle days 12-14, when the follicle was 18-22 mm. Patients in this group were also administered clomiphene citrate and human chorionic gonadotropin. In treatment group B, one timed IUI was performed with the top 0.5 ml of the separated semen, when the follicle was 18-22 mm. The gender outcome of the pregnancies in the treatment and control groups was evaluated based on the known desired gender. The success rate for conceiving a female child after intervention (treatment group A) was 86.7% effective (p = 0.0002) as compared to control group A. Couples seeking a male child (treatment group B) were 89.2% effective (p = 0.0002) as compared to control group B. It would appear that the modified swim-up method with additional monitoring results in statistically significant gender preselection.
Chromosomal anomalies and genetic deletions
Genetic infertility includes chromosomal anomalies, genetic deletions such as Y deletion and cystic fibrosis.
Figure 15.9 Flow chart for selection of correct Wang tube. (1) More than six white blood cells in raw semen under high-power field; (2) selection of Wang tube for gamete intrafallopian transfer, zygote intrafallopian transfer or tubal embryo transfer is identical to that for in vitro fertilization (IVF); (3) more Wang tubes are suggested for more sperm preparations. IUI, intrauterine insemination
Oligozoospermic men have a high risk (5%) of sex chromosome and autosome anomalies. There are some 250 genes pertinent to spermatogenesis on the autosomal chromosomes. Most miscarriage/pregnancy failure in older women is a result of chromosome anomalies arising in the embryo. Most common genetic disorders associated with primary infertility in females are chromosome anomalies, e.g. Turner’s syndrome (monosomy X) and Stein-Leventhal syndrome (polycystic ovarian disease). Causes of genetic male infertility include Klinefelter’s syndrome, X-autosome reciprocal translocation, Y-chromosome microdeletion, congenital absence of the vas deferens, cystic fibrosis and obstructive azoospermia in Young’s syndrome. Successful pregnancy can be initiated in most of these syndromes by oocyte donation, ICSI or related assisted reproductive technologies. Disorders associated with specific potential dysgenic outcome of ICSI include sex chromosome numerical anomalies (45X, 47XXY), Y-chromosome microdeletion and cystic fibrosis.