Abstract
At least 50% of couples referred for infertility investigation and treatment are found to have a contributing male factor. Male factor infertility can represent a variety of defects, which result in abnormal sperm number, morphology or function. Detailed analysis of sperm assessment and function are important for accurate diagnosis, and are described in detail in numerous textbooks of practical andrology and semen analysis. A comprehensive review of semen analysis is beyond the scope of this book, and only details relevant to assisted conception treatment will be described here.
Introduction
At least 50% of couples referred for infertility investigation and treatment are found to have a contributing male factor. Male factor infertility can represent a variety of defects, which result in abnormal sperm number, morphology or function. Detailed analysis of sperm assessment and function are important for accurate diagnosis, and are described in detail in numerous textbooks of practical andrology and semen analysis. A comprehensive review of semen analysis is beyond the scope of this book, and only details relevant to assisted conception treatment will be described here.
The World Health Organization (WHO) Laboratory Manual (5th edition, 2010) describes standard conditions for the collection of semen samples, their delivery and the standardization of laboratory assessment procedures. This manual represents a major revision over the previous (WHO, 1999) edition and includes new ‘Reference Values’ to allow decisions to be made about patient management; the latest edition proposes that ‘normal’ should potentially be defined as the 5th centile of a population of men whose partners conceived within 12 months of stopping contraceptive use. Therefore, the lower reference limits (5th centile and 95% confidence intervals) are 15 × 106/mL (12–16) for concentration, 32% (31–34) for good forward progressive movement within 60 minutes of ejaculation and 4% (3–4) for normal morphology (see WHO Manual, 2010: appendix A1.1). The introduction of external quality control and quality assurance schemes in semen assessment has highlighted the fact that accurate analysis of seminal fluid is notoriously difficult to standardize, with many technical variables, and the quality of semen analysis in different laboratories can be highly variable (Matson, 1995; Pacey 2006). This implies that diagnosis and treatment modality chosen for a patient could differ according to the laboratory carrying out the assessment. Without accurate semen analysis data, patients may be offered inappropriate treatments or no treatment at all; it is essential that an assisted reproductive service should ensure that laboratory personnel are adequately and correctly trained in basic semen assessment techniques according to WHO guidelines. Even the most confident of laboratories should have a discipline of monitored standards. The routine application of intracytoplasmic sperm injection (ICSI) provides effective treatment for even the most severe cases of male infertility which were previously felt to be beyond hope, and the fact that fertilization can be achieved from semen with ‘hopeless’ sperm parameters has forced a review of standard semen analysis and sperm function testing. This chapter will address only the basic principles required in the practical features of sperm preparation procedures for assisted conception techniques.
Semen Assessment
Sample Collection and Handling
(a) Record information: Before sample production the patients should be asked to confirm their personal details and, if necessary, provide suitable identification. They should be asked when they last ejaculated, and for a history of recent illness, medication taken, smoking and alcohol consumption; this information should be noted on the final report form. Once the sample has been produced, the patient should sign a consent form agreeing to the use of that sample for analysis or treatment.
(b) Provide adequate instructions: patients should be given written instructions about the process involved, including precise details about the location and time that their sample will be required. They should be informed of the need to abstain from sexual intercourse or masturbation for between 3 and 5 days before their sample is to be produced. Care should be taken over language difficulties and for patients with special needs.
(c) Methods of sample production: The sample should ideally be produced by masturbation after the required period of abstinence. However, it is acknowledged that this is not possible for all patients. A small number of men can only produce a sample at intercourse and in these cases they should be provided with a silastic (nonspermicidal) condom. No other condoms are permissible. Samples produced by coitus interruptus without a condom should not be accepted for analysis.
(d) Location of production: Whenever possible, all samples for analysis or treatment should be produced on site. It is unacceptable to ask a patient to produce a sample in a lavatory, and there should be a special room set aside for this purpose. Where it is not possible to produce a sample on site, a sample produced at home should be brought to the laboratory within 1 hour of production.
(e) Specimen container: Samples should be collected into a preweighed wide-necked plastic or glass container. All specimen containers should be cytotoxically tested as some plastics are detrimental to sperm motility (see WHO, 2010, box 2.1, for details). Samples previously assessed as having high viscosity benefit from collection into pots containing 1 mL of medium. Prior to production, the patient should be asked to pass urine and then rinse his hands and penis.
(f) Treatment of samples post-production: Once the sperm sample has been produced, the patient and a member of staff should check that the sample container is identified with the patient’s name or identification number, and the time of sample production. The samples should be placed in an incubator at 37°C for up to 1 hour to allow liquefaction.
Macroscopic Evaluation
Once the process of liquefaction has occurred (usually within 30–60 minutes of ejaculation), the sample should be examined macroscopically, with evaluation of:
Appearance and consistency: Semen should be a grayish opalescent liquid with a neutral odor. Any unpleasant smell or discoloration (e.g., contains blood), or the presence of mucus or jelly should be reported.
Liquefaction and viscosity: Although semen is ejaculated as a coagulum, it should liquefy within 30 minutes. If a sample fails to liquefy or is highly viscous after liquefaction, this should be noted.
Volume: Volume is measured by weighing the container in which the specimen was ejaculated and subtracting the full weight from the empty weight. The volume can be inferred from the weight assuming the density of semen to be 1 g/mL. The use of volumetric methods to measure semen volume is no longer recommended.
pH: The most convenient way to measure the pH of a sample is to use pH paper.
Macroscopic anomalies can provide important information about the patient and should not be ignored. For example, a low pH can indicate infection of the genital tract, and a low volume could suggest a retrograde ejaculation, a leakage from the sample container or that the patient failed to collect the entire sample he produced.
Sperm Motility
Since sperm motility decreases with increasing exposure to seminal plasma, this should be the first assessment carried out. There are three important aspects to correctly estimating sperm motility:
Observation chamber: A variety of types are available, but this should have a minimum depth of 20 µm to allow the sperm to move freely. A number of companies produce disposable chambers designed for motility observation (e.g., Microcell), but an alternative strategy is to place a 10-µL drop of semen on a glass microscope slide and cover it with a 22-mm diameter coverslip. NB: the depth of a Makler chamber (10 µm) makes it unsuitable for accurate motility measurements. However, when the purpose of assessment is the selection of an appropriate method of preparing the sample for assisted conception procedures, the Makler chamber does allow simultaneous judgment of approximate motile and immotile concentrations, and a quick assessment of type of sperm motility.
Temperature: The microscope slide should be maintained at 37°C on a heated stage during motility assessment for correct identification of motility grade.
Microscope: Observe the sample at ×200 or ×400 magnification using a phase-contrast objective.
Grading system: Approximately 200 sperm should be examined and each sperm classified as belonging to one of four motility grades. Figure 10.1 outlines the difference between the motility grades, with a flow chart explaining how to classify them. A percentage for the number of sperm belonging to each category should be calculated.
Aggregated or Agglutinated Spermatozoa
A high number of aggregated or agglutinated spermatozoa can make accurate motility assessment impossible. A motility count should then be performed only on the free-swimming portion, with this noted in the report.
Sperm Concentration
Methods used to determine sperm concentration have long been a subject of debate. Andrologists tend to agree that using a hemocytometer is the most appropriate, as it provides the most reproducible result with the lowest coefficient of variation when used properly. Since it normally relies upon the use of fixatives to kill spermatozoa before they are placed on the counting chamber, its use is often thought to be in conflict with the principles of trying to reduce chemical contaminants in the IVF laboratory. Many embryologists, however, use water instead of fixative in which to dilute the spermatozoa, and the osmotic shock is sufficient to immobilize sperm sufficiently for a count to be undertaken. Others prefer to use a Makler chamber or disposable chambers such as the Microcell. If an alternative chamber is chosen, then it is important that its accuracy is regularly checked using a thorough internal quality control system. This should preferably be checked against a hemocytometer as the gold standard. Whatever chamber is chosen, it is important to pay special consideration to samples in which no spermatozoa are observed. These samples should be centrifuged at >3000 g for 15 minutes; a sample can be classified as truly azoospermic only if no sperm are observed in the pellet after centrifugation.
Before performing the count, note the presence of agglutination and type if present (H-H, T-T, H-T), and any debris and cells other than spermatozoa, such as red or white blood cells.
Examine the counting grid and count the number of motile sperm in 20 squares. If the count appears on initial observation to be less than 10 million/mL, all 100 squares should be counted. Count the total number of sperm in the same group of squares and calculate motility:
Sperm density in millions/mL = the number of sperm in 10 squares of the grid
The sperm concentration should be reported in millions per milliliter.
Sperm Antibody Detection
Antibodies directed against sperm can be detected by two methods, the mixed antiglobulin reaction (MAR) test and the immunobead test. They differ slightly in their approach and methodology, but their interpretation is similar in that they rely upon the identification of motile spermatozoa with adherent latex spheres or beads. Kits are available for antisperm antibody screening in semen samples; the MAR test will nonspecifically detect IgG, IgA or IgM antibodies, and specific latex particle immunobeads can be used to distinguish between the different categories of antibody. Although the results of the two tests do not always agree, it is generally considered that a test is clinically significant only if >50% of sperm have antibodies directed against them. In cases where the test cannot be performed due to an insufficient number of motile sperm, the sample can be tested indirectly by using the sperm of a donor (known to have no sperm antibodies in his semen) as part of the test: the donor sperm acts as a reagent in the assay. The percentage of spermatozoa with adherent particles should be recorded on the report form after evaluating 200 sperm.
Sperm Morphology Estimation
Sperm morphology assessment is one of the most controversial measures in semen analysis. This is due partly to several changes in reporting the dimensions of normal spermatozoa in successive editions of the WHO manual, but is also due to the technical difficulty of making accurate morphology measurements without the aid of a computerized system. All morphology measurements should be made using fixed smears stained by the Papanicolaou method or the Diff-Quick or Shorr stains (prestained slides are also available). Stained slides should be examined by bright-field optics using an oil-immersion objective at ×1000 magnification. At least 200 spermatozoa should be examined; an eyepiece graticule can be used to measure individual spermatozoa if necessary. The normal head has an oval shape with a length:width ratio of 1.50:1.75. A well-defined acrosomal region should cover 40–70% of the head area. No neck, midpiece or tail defects should be evident, and cytoplasmic droplets should constitute no more than one-third the size of a normal sperm head. Figure 10.2 illustrates some examples of typical sperm abnormalities. Count the number of sperm that display:
1. Abnormal heads
2. Tail abnormality
3. Midpiece abnormalities
4. Immature forms.
All borderline forms are classified as abnormal. Calculate the percentage of each abnormal form, and add together the percentages to yield the total percentage of abnormal forms in the sample.
Figure 10.2 Common abnormalities found in human sperm morphology.
A normal, fertile semen sample contains a very high proportion of morphologically abnormal forms, and the significance of abnormal sperm morphology is not entirely understood. Although sperm of abnormal morphology evidently have reduced fertilizing potential, the true anomalies present in abnormal sperm cells have been only partially characterized; a correlation has been found with specific deficiencies such as poor zona pellucida binding and penetration, poor response to agonists that modulate intracellular calcium concentrations, and with biochemical markers such as reactive oxygen species production and enhanced creatine phosphokinase activity. The significance of sperm morphology is discussed further in Chapter 13.
Other Cells in Semen
Other (non-sperm) cells can sometimes be observed during the semen analysis, either in the wet (motility) preparation or in the stained morphology slide. These include epithelial cells from the urethra, erythrocytes, germ cells and leukocytes. Whilst epithelial cells and erythrocytes are easily identifiable from their morphology, germ cells and leukocytes can easily be confused. Therefore, specific stains are needed to discriminate between the two cell types and to correctly enumerate their concentration on the semen analysis report. Leukocytes can be identified using a peroxidase-based stain, or with the use of specific monoclonal antibodies. The concentration of any non-sperm cell can be calculated relative to the sperm concentration using the equation c = n × s/100, where n is the number of a given cell type in the same field as 100 sperm, and s is the sperm concentration in millions per milliliter.
Internal and External Quality Control Procedures
A final but important part of the semen analysis is the application of internal and external quality control procedures to the semenology laboratory. Several studies have shown that samples analyzed in different laboratories can give rise to radically different results, in some cases leading to an inappropriate diagnosis for the patient. Many techniques have been outlined that can be used to monitor the performance of a laboratory, and these are described in more detail in the 5th edition of the WHO manual. Any laboratory involved in making diagnoses should have such protocols in place and should be members of an external quality assessment scheme for andrology.
Sperm Kinematics
In the early 1980s several studies used time-lapse photography to analyze detailed movement characteristics of sperm in time and space (sperm kinematics). The motion of spermatozoa can be described in a number of different ways (see Mortimer, 1994):
VSL = straight line velocity
VCL = curvilinear velocity
VAP = average path velocity
ALH = amplitude of lateral head displacement.
These early data led to the suggestion that specific patterns of sperm motility behavior were advantageous. For example, only spermatozoa with a high degree of lateral head displacement are able to penetrate cervical mucus. The development of computerized systems allowed such measurements to be made more rapidly as well as allowing the analysis of more sophisticated behavior patterns, such as sperm hyperactivation. Although the measurement of sperm hyperactivation has been controversial, it has been linked with IVF success. However, this technology is not used routinely as the prognostic value is poor, and the high cost of the machines precludes their use in all but the most specialized laboratories. (See Tomlinson et al., 2010 for review.)
DNA Fragmentation
As detailed in Chapter 3, spermatogenesis is a complex and dynamic process of proliferation and differentiation, involving mitosis, meiosis, changes in cytoplasmic architecture, replacement of histones with transition proteins and the final addition of protamines, leading to a highly packaged chromatin. It is not surprising therefore that ejaculated spermatozoa have a variety of abnormalities at the nuclear, cytoskeletal and organelle levels. There is now evidence to suggest that sperm DNA integrity may be useful in predicting male fertility potential. The first manuscript describing in-situ detection of sperm DNA fragmentation was published more than 30 years ago, and a surge in published reports about sperm DNA fragmentation then appeared between 2005 and 2010. These studies provide strong evidence that semen samples in which more than a third of the DNA is fragmented have a reduced chance of resulting in clinical pregnancy (Sakkas and Alvarez, 2010). Sperm DNA fragmentation may result from aberrant chromatin packaging during spermatogenesis, apoptosis before ejaculation, excessive production of reactive oxygen species in the ejaculate, exposure to environmental or industrial toxins, genetics, oxidative stress, smoking, etc. Current standard sperm preparation techniques depend on a sedimentation or migration approach to separate spermatozoa based on their motility or density with molecular events being overlooked. Thus, the use of sperm with DNA damage during IVF may be one of the reasons for suboptimal pregnancy and low live birth rates.
Sperm Chromatin Assays
Sperm condensation quality and sperm morphology studies suggest that the quality of chromatin packaging in human sperm, as assessed by its binding capacity for specific dyes and fluorochromes, can be used as an adjunct to the assessment of morphology. Sperm of poor morphology may possess loosely packaged chromatin, and this may contribute to a failure in sperm decondensation during fertilization.
Damaged chromatin will take up the following dyes:
1. Chromomycin (CMA3) staining
Fix prepared semen samples or semen smears in 3:1 v/v of methanol/glacial acetic acid, at 4°C for 5 minutes.
Treat each slide for 20 minutes with 100 mL CMA3 solution: 0.25 mg/mL in McIlvane’s buffer, pH 7.0, containing 10 mM MgCl2.
Evaluate the slides using fluorescent microscopy.
2. Aniline blue staining (AAB)
Fix the samples in 3% buffered glutaraldehyde for 30 minutes.
Stain the slides with 5% aqueous aniline blue and mix with 4% acetic acid (pH 3.5) for 7 minutes.
Three classes of head staining can be noted: unstained (gray/white), partially stained, entire sperm head dark blue intensity.
Detection of DNA Fragmentation by the TUNEL Assay
Kits for DNA fragmentation analyses are commercially available from a number of different companies, each with its own protocol to be followed.
Wash a semen aliquot containing 1–2 × 106 spermatozoa with phosphate-buffered saline (PBS) by centrifugation at 500 g at room temperature for 5 minutes.
Remove the seminal plasma and wash the pellet twice in PBS with 1% bovine serum albumin (BSA).
Suspend the pellet in 100 μL of PBS/BSA 1%, and fix it in 100 μL of 4% paraformaldehyde in PBS (pH 7.4) for 1 hour at room temperature, with agitation.
Wash the cells again in PBS/1% BSA, spot a 10-μL aliquot onto a demarcated area on a clean microscope slide and allow this to air dry.
Rinse the slides twice in PBS, and then permeabilize using 0.1% Triton X100 in 0.1% sodium citrate for 2 minutes on ice.
Wash again twice with PBS, add terminal deoxyribonucleotidyl transferase (TdT)-mediated dUTP nick-end label in order to allow DNA elongation, and incubate the slides in a humidified chamber at 37°C for 60 minutes.
Rinse slides twice in PBS and counterstain with 1 mg/mL 4,6 diamidino-2-phenylindole (DAPI) to visualize the nucleus.
Include negative (omitting TdT from the reaction mixture) and positive (using only DNAse I, 1 mg/mL for 30 minutes at room temperature) controls in each sample tested.
Evaluate a total of 500 sperm per sample using fluorescence microscopy for fluorescein isothiocyanate (FITC). Count the number of sperm per field stained with DAPI (blue); the number of cells with green FITC fluorescence (TUNEL positive) is expressed as a percentage of the total sample.
Preparation of Sperm for In-Vitro Fertilization
At the time of oocyte retrieval or intrauterine insemination (IUI), the laboratory should already be familiar with the male partner’s semen profile and can refer to features that might influence the choice of sperm preparation method used. Semen is a nonsterile body fluid that can transmit infection, and viral screening tests should be confirmed as negative before the sample is handled for preparation in the laboratory. Aseptic technique should be maintained throughout.
The choice of sperm preparation method or combination of methods depends upon the assessment of:
motile count
ratio between motile:immotile counts
volume
presence of antibodies, agglutination, pus cells or debris.
Ejaculated semen is a viscous liquid composed of a mixture of testicular and epididymal secretions containing spermatozoa, mixed with prostatic secretions produced at the time of ejaculation. This seminal plasma contains substances that inhibit capacitation and prevent fertilization. The purpose of sperm preparation is to concentrate the motile spermatozoa in a fraction that is free of seminal plasma and debris. Early IVF practice involved preparing sperm by simple washing and centrifugation, but this method also concentrates cells, debris and immotile sperm, which can jeopardize fertilization. Aitken and Clarkson (1987) demonstrated that leukocytes and dead sperm in semen can generate reactive oxygen species (ROS), and these can initiate lipid peroxidation in human sperm membranes. Peroxidation of sperm membrane unsaturated fatty acids leads to a loss of membrane fluidity, which inhibits sperm fusion events during the process of fertilization. When preparing sperm for assisted conception, it is advantageous to separate motile sperm from leukocytes and dead sperm as effectively and efficiently as possible. However, if ICSI is the treatment of choice, sperm fusion events are of course bypassed, and direct high-speed centrifugation of these suboptimal sperm samples does not appear to jeopardize fertilization by ICSI.
Sperm samples that show moderate to high counts (>35 × 106 motile sperm/mL) with good forward progression and motility can be prepared using a basic overlay and swim-up technique. Discontinuous buoyant density gradient centrifugation is the method of choice for samples that show:
1. Low motility
2. Poor forward progression
3. Large amounts of debris and/or high numbers of cells
4. Antisperm antibodies.
At the end of each preparation procedure, adjust the pH of the resulting samples by gassing gently with 5% CO2, and store the samples at room temperature until final preparation for insemination.
Standard Swim-Up or Layering
1. Pipette 2 mL of HEPES-buffered culture medium into a round-bottomed disposable test-tube.
2. Gently pipette approximately 1.5 mL of neat semen underneath the medium (being very careful not to disturb the interface formed between the semen and the medium).
3. Tightly cap the tube and allow it to stand at room temperature for up to 2 hours. (The ejaculate can also be divided into several tubes for layering if necessary.)
4. Harvest the resulting top and middle clouded layers into a conical test-tube and spin at 200 g for 5 minutes.
5. Remove the supernatant and resuspend the pellet in 2 mL of medium. Centrifuge again, discard the supernatant and resuspend the pellet in 1 mL of medium.
6. Assess this sample for count and motility, gas the surface gently with 5% CO2 in air, and store at room temperature prior to dilution for the insemination procedure.
Alternatively, 2 mL of medium can be gently layered over the semen sample in its pot, which provides a larger interface surface area. After 10–45 minutes, suspend an aliquot of this layer in 1 mL of medium and process as above. The time allowed for swim-up should be adjusted according to the quality of the initial sample: the percentage of abnormal sperm that will appear in the medium increases with time and continues to do so after normal motile density has reached its optimum level.
Pellet and Swim-Up
This method is used when the semen has been collected into medium or medium + albumin. It is also useful for viscous samples (once the viscosity has been decreased, by vigorous pipetting or syringing) and when the total volume of semen is very low. This method is not recommended when motility is very poor or when there is a large degree of cellular contamination and debris (the sperm will be concentrated with this prior to the swim-up).
1. Mix semen and medium and centrifuge once. Note: In some cases (i.e. oligo/asthenospermia) much more semen will need to be prepared, and the volume of medium used should be increased accordingly. As a general rule, be careful not to take far more of the semen than is required.
2. Carefully remove all the supernatant and then very gently pipette about 0.75 mL of medium over the pellet, taking care not to disturb it.
3. Allow the sperm to swim up into the medium. If the sample has poor motility, it sometimes helps to lay the centrifuge tube on its side.
10 minutes is sufficient for very motile sperm (activity 3–4).
1 hour plus may be required for poorly motile sperm.
In general, do not leave for too long, as some cells and debris will become resuspended.
4. Carefully remove the supernatant from the pellet and place in a clean centrifuge tube.
5. Centrifuge again, resuspend in medium, assess count and motility, and gas with CO2 before storing at room temperature.
This method has the disadvantage of exposing motile sperm to peroxidative damage during centrifugation with defective sperm and white cells. Aitken (1990) has shown that unselected sperm exhibit higher levels of ROS production in response to centrifugation than the functionally competent sperm selected prior to centrifugation by the layering method. Sperm that are selected prior to centrifugation produce much lower levels of ROS, and their functional capacity is not impaired.
Discontinuous Buoyant Density Gradient Centrifugation
Buoyant density gradient ‘kits’ for sperm preparation are commercially available. These are based upon either coated silica particles, a mixture of Ficoll and iodixanol, or highly purified arabinogalactan. Individual experiences comparing the use of these products have reported no significant differences between them. Buoyant density gradients apparently protect the sperm from the trauma of centrifugation, and a high proportion of functional sperm can be recovered from the gradients. Discontinuous two- or three-step gradients are simple to prepare and highly effective in preparing motile sperm fractions from suboptimal semen samples. A single layer of 90–100% density can also be used for simple filtration by layering the sample on top of the column and allowing the sperm to migrate through the density medium, where they can be harvested from the bottom of the test-tube.
Methods
Manufacturers’ instructions should be followed for the different commercial preparations, but ‘recipes’ can be adapted according to each individual semen sample, in particular with respect to volumes, speed of centrifugation and length of centrifugation. In general, a longer centrifugation time increases the recovery of both motile and immotile sperm; normal immotile sperm are only decelerated by the particles, and after long spinning they will reach the bottom of the gradient. Higher centrifugation speeds increase the recovery of motile sperm, and also of lower density particles; therefore, if the gradients are spun at a higher speed, a shorter time should be used. Debris, round cells, and abnormal forms with amorphous heads and cytoplasmic droplets never reach the bottom of the tube because of their low density. Gradients with larger volumes result in improved filtration, but decreased yield. The three layers of a mini-gradient improve filtration, and the smaller volumes improve recovery of sperm from severely oligospermic samples. Large amounts of debris can disrupt gradients and prevent adequate filtration. There is a limit to the number of cells that can be loaded onto any gradient, and samples with high density or a large amount of debris should be distributed in smaller volumes over several gradients. Severely asthenozoospermic samples with a normal sperm density but poor motility can also be distributed over a series of mini-gradients.
The temperature of the prepared gradients also affects the ‘merging of gradients,’ which is improved at 37°C compared to room temperature.
Isotonic Gradient Solution
Mix, and filter this solution through a 0.22-mm Millipore filter.
Add 90 mL of density gradient preparation media.
Store at +4°C for up to 1 week.
Two-Step Gradient, 80/40
Can be used for all samples which contain> 4 × 106 motile sperm/mL.
Should be used for all specimens with known or suspected antisperm antibodies:
80%: 8 mL isotonic + 2 mL culture medium
40%: 4 mL isotonic + 6 mL culture medium.
1. Gradients: pipette 2.0–2.5 mL of 80% into the bottom of a conical centrifuge tube, and gently overlay with an equal volume of 40%.
2. Layer up to 2 mL of sample on top of the 40% layer.
3. Centrifuge at 600 g for 20 minutes.
Cells, debris and immotile/abnormal sperm accumulate at the interfaces, and the pellet should contain functionally normal sperm. Recovery of a good pellet is influenced by the amount of debris and immotile sperm, which impede the travel of the normal motile sperm.
4. Carefully recover the pellet at the bottom of the 80% layer, resuspend in 1 mL of medium and assess (even if there is no visible pellet, a sufficient number of sperm can usually be recovered by aspirating the bottom portion of the 80% layer).
5. If the sample looks sufficiently clean, centrifuge for 5 minutes at 200 g, resuspend the pellet in fresh medium and assess the final preparation.
6. If there is a high percentage of immotile sperm, centrifuge at 200 g for 5 minutes, remove the supernatant, carefully layer 1 mL fresh medium over the pellet and allow the motile sperm to swim up for 15–30 minutes. Collect the supernatant and assess the final preparation.
If at least 106 motile sperm/mL have been recovered, spin at 200 g for 5 minutes and resuspend in fresh medium. This will be the final preparation to be diluted before insemination, therefore the volume of medium added will depend upon the calculated assessment.
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