Over 200 laboratory directors and technical supervisors attended the Embryology and Andrology Review Course sponsored by the American Association of Bioanalysts at the Rush Medical Center in Chicago, Illinois on August 27-28, 1994. With sessions in Laboratory Management, Male and Female Reproductive Physiology, Andrology, and Embryology, the course was designed to prepare these individuals for the newly required certifying examination to be given by the American Board of Bioanalysis. The andrology scientific program featured four lectures, providing a comprehensive review of andrology laboratory techniques.
The andrology session began with a discussion by Dr. Gail Prins of the components and techniques for a complete semen analysis. She encouraged the participants to refer to The World Health Organization Laboratory Manual for the Examination of Human Semen for procedural details, universal standards, and normal ranges. Initial semen analysis begins with an assessment of semen quality. Factors such as sexual abstinence, technique of sample production, container for sample collection, temperature, and time to analysis can greatly affect the accuracy of a semen analysis. The time to liquefaction, viscosity, volume, color and pH must be noted. It is important to distinguish between incomplete liquefaction and hyperviscosity but, as Dr. Prins noted, either may be treated with a 0.2% alpha-amylase solution. Abnormalities in any of these semen parameters will help in the diagnostic process of male fertility.
Next, Dr. Prins discussed the importance of sperm morphology in the assessment of male fertility. A minimum of 100 stained sperm, preferably 200, should be classified into 5 categories: normal, head abnormalities, neck/midpiece abnormalities, tail abnormalities and immature. She recommends the Papanicolaou modified-staining procedure because it provides excellent nuclear and cytologic detail. The WHO considers 0-70% abnormal forms the normal range for human sperm. Strict criteria for the analysis of sperm morphology were also reviewed. Recent claims indicate that morphology determined by the strict measurement of head size and shape correlates more closely with IVF outcome. Dr. Prins concluded her lecture with a review of methods for sperm quantitation. She noted that manual counts using a hemacytometer require a dilution in saline or culture medium, which not only can increase the risk of error but also affects motion characteristics. Therefore, a motion analysis must be made on a separate, undiluted aliquot of semen. The Makler counting chamber eliminates the problems associated with dilution but tends to overestimate the count when compared to the hemacytometer. Computer Assisted Semen Analysis (CASA) is an additional option for sperm quantitation and motion analysis, which provides an objective, consistent analysis and expands the description of sperm motion characteristics. Parameters such as linearity, curvilinear velocity, and lateral head displacement can be measured. However, the lack of normal ranges and universal CASA standards limits the value of such information.
The second lecture, given by Dr. William Baird, reviewed the methods of sperm preparation and cryopreservation. The purpose of any sperm processing method should be to recover a high percentage of motile sperm while maintaining the sperm's normal function. Dr. Baird reviewed four categories of sperm preparation, all of which were designed to remove not only seminal fluid but also round cells, cellular debris, and immotile sperm. These procedures included dilution and washing, sperm migration (swim up), selective washing (Percoll and Nycodenz) and adherence techniques (glass beads and glass wool). Semen parameters such as initial concentration, motility, and viscosity will dictate which method should be used, thus requiring andrology laboratories to be proficient in several protocols.
One point of concern raised by Dr. Baird was sperm exposure to oxygen free-radicals encountered in the process of centrifuging motile sperm, immotile sperm, and debris together into a single pellet. The result is an increase in oxygen radicals and a decrease in sperm functionality. Percoll separation avoids this problem because motile sperm are centrifuged away from immotile sperm and cellular debris. Although the use of Percoll may have certain benefits, several members of the audience expressed concern that Percoll may introduce endotoxins since it has neither been tested nor approved for human use. Cryopreservation was the final topic of Dr. Baird's lecture. He stated that the stepwise addition of TEST citrate egg-yolk buffer or glycerol for cryoprotection and slow cooling and slow thawing provide the best results for the recovery of a motile population of sperm.
Although the semen analysis provides valuable information, it cannot assess the ability of sperm to penetrate cervical mucus and function within the female reproductive tract. The topics of sperm-cervical mucus interaction were next reviewed by Dr. Grace Centola. Several tests can be employed to evaluate the sperm-mucus interaction including the post-coital test, the slide or Kurzrock-Miller test, the spermcervical mucus contact test, the capillary penetration or Kremer test, or the crossed hostility test. Dr. Centola reviewed each of these tests, but questioned the usefulness of all but the post-coital test. She noted that physicians will perform the post-coital test, but with a poor outcome proceed to testing for antisperm antibodies or intrauterine insemination.
Impaired sperm movement such as immobilization or agglutination in the cervical mucus or abnormal semen analysis may indicate the presence of antisperm antibodies. Dr. Centola described two specific diagnostic tests for antisperm antibodies: the Mixed Antiglobulin Reaction Test (MAR Test) and the Immunobead Binding Test (IBT Test). The MAR test can detect IgG antibodies on the sperm surface through the use of sensitized red blood cells but because sperm antibodies can be found not only on the sperm surface but also in cervical mucus, serum and follicular fluids, the IBT test is preferred. The andrology session concluded with a lecture by Dr. Christopher DeJonge on sperm function testing. Detailed protocols were given for four sperm function assays: the Hamster Zona-Free Penetration Test, the Hemi-Zona assay, the Hypoosmotic Swelling Test and Acrosome Status testing. Each of these tests has been shown to provide definitive diagnostic information about fertilization potential.
Dr. DeJonge discussed each test in terms of what information each can and cannot provide. For example, the Hamster Zona-Free Penetration test evaluates the ability of sperm to undergo capacitation, acrosome reaction, fusion with the oolemma and nuclear condensation but does not give information about the interaction between the sperm and the zona pellucida, which the Hemi-Zona Assay does. The Hypoosmotic Swelling test determines the functional and physical integrity of the sperm membrane. And the Acrosome Status testing can identify an inability to initiate a normal acrosome reaction. He noted in conclusion that no single sperm function test can predict fertilization potential given the multifactorial nature of the process.
The syllabus for the Embryology and Andrology Review Course can be obtained from: American Association of Bioanalysts, 818 Olive Street - Suite 918, St. Louis, MO 63101-1598, Telephone: (314) 241-1445, Fax: (314) 241-1449.
Jonna Frasor, Sue Tarchala and Richard G. Rawlins, USA