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. A review of semen analysis is beyond the scope of this blog post, and only details relevant to ART will be described here.
Without accurate semen analysis data, patients may be offered inappropriate treatments or no treatment at all. It is essential that an ART should ensure that an IVF laboratory personnel are adequately and correctly trained in basic semen assessment techniques. Even the most confident of IVF 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.
Sample Collection and Handling
Record information: Before sample production the patients should be asked to confirm their personal details and, once 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.
Provide adequate instructions: Patients should be given precise instructions about the process involved, including 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 three and five days before their sample is to be produced.
Methods of sample production: The sample should ideally be produced by masturbation after the required period of abstinence.
Location of production: Whenever possible, all samples for analysis or treatment should be produced on site. There should be a special room set aside for this purpose. Where it is not possible to produce a sample on the site, a sample produced at home should be brought to the IVF laboratory within one hour of production.
Specimen container: Samples should be collected into a pre-weighed wide-necked container. All specimen containers should be cytotoxically tested as some plastics are detrimental to sperm motility. 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.
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 ID 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.
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 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.
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.
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 x200 or x400 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. 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.
Less than 50% of the Spermatozoa Are Motile
If less than 50% of the spermatozoa are motile, a vitality test, such as a hypo-osmotic swelling (HOS) test, is recommended in order to determine whether the nonmotile sperm is dead or alive.
Methods used to determine sperm concentration have long been a subject of debate. Andrologists tend to agree that using a hemocytometer be 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 be 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 azoospermia only if no sperm is observed in the pellet after centrifugation.
Sperm Antibody Detection
Antibodies directed against sperm can be detected by two methods, a) the mixed antiglobulin reaction (MAR) test and b) the immuno-bead 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.
Although the results of the two methods 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 donor sperm 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 Assessment
Sperm morphology assessment is one of the most controversial measures in semen analysis. All morphology measurements should be made using fixed smears. 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.
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 has 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.
Other Cells in Semen
Other (non-sperm) cells in semen 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.
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 IVF laboratory. Many techniques have been outlined that can be used to monitor the performance of the IVF laboratory. Any IVF laboratory involved in making diagnoses should have such protocols in place and should be members of an external quality assessment scheme for andrology.
The motion of spermatozoa can be described in a number of different ways:
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 hyper-activation.
Although the measurement of sperm hyper-activation 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 IVF laboratories.
Sperm DNA Fragmentation
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 that ejaculated spermatozoa have a variety of abnormalities at the nuclear, cyto-skeletal and organelle levels. There is no evidence to suggest that sperm DNA integrity may be useful in predicting male fertility potential. Sperm DNA fragmentation may result from improper chromatin packaging during spermatogenesis, apoptosis before ejaculation, excessive production of reactive oxygen species in the ejaculate, exposure to environmental toxins or industrial substances, 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 de-condensation during fertilization.