Andrology

Laboratory scientists working in the andrology field perform tests that help evaluate the potentially infertile male patient. Among these tests are the semen analysis and sperm antibody assay.

Semen Analysis (SA)

A significant amount of clinically useful information can be obtained if all the reported parameters are carefully considered. Because potentially significant fluctuations can occur, a single SA may not be representative of the total clinical status of the male patient. Hence, at least two SA are recommended during the course of a work-up, especially if the initial SA shows abnormalities.

A comprehensive SA should include an assessment of many semen parameters regarding seminal quality, cytology and sperm quantitation.

I. Seminal Quality

A. Sample production - sample should be collected 2-7 days after last ejaculation. Shorter periods of abstinence may result in lower than average sperm concentration and samples with immature spermatozoa. Longer periods of abstinence may result in samples with reduced motility or viability. Recommended technique for sample production is masturbation without lubricants (most are spermicidal).

B. Liquefaction - following ejaculation, seminal plasma immediately forms a coagulum due to the presence of coagulating factors produced by the seminal vesicles. This coagulum is liquefied by enzymes within the ejaculate which originate from the prostate gland. The normal time frame for this process is 10-30 minutes. Absent, incomplete or prolonged time of liquefaction must be noted since it may be clinically relevant. Entrapped sperm may lead to subfertility.

C. Viscosity - notation of the specimen's viscosity is important since hyperviscosity may impair sperm motility.

D. Volume - normal volume ranges between 1 and 5 ml and is a function of the accessory gland activity.

E. Color - normally, semen which contains spermatozoa is a grey to white opalescent fluid. Different colors may indicate a problem.

F. pH - semen is slightly basic with a pH range of 7.2-8.0. Seminal fluid has a significant buffering capacity and it buffers the acidic vaginal environment which is detrimental to spermatozoa.

II. Cytology

A. Sperm morphology - morphologic characterization of sperm has significant correlations to male fertility. Many laboratories follow the World Health Organization (W.H.O.) guidelines to classify spermatozoa. The spermatozoa can be classified into one of five categories: normal, head abnormality, neck/mid-piece abnormality, tail abnormality or immature. The W.H.O. now considers the normal range for human sperm to be > 30% normal forms. Their previously published normal range was > 50% normal forms.

Another method of assessing sperm morphology, STRICT CRITERIA, has recently been introduced by Dr. Thinus Kruger and colleagues from Tygerberg Hospital in South Africa. This classification system correlates more closely with invitro fertilization outcome than the W.H.O. classification. The defects considered are the same as those with the W.H.O. classification. The difference is that very strict criteria must be met for a sperm to be classified as normal. According to these criteria, > 14% normal forms is considered a normal, fertile specimen. The rest are broken into two groups:

5-14% Normal Forms = "G" Pattern or Good Fertilization Prognosis

0-4% Normal Forms = "P" Pattern or Poor Fertilization Prognosis

We rely more on the results from Dr. Kruger's classification system for decision making on an appropriate assisted reproductive technology course of action. Our andrology personnel have been individually trained to perform strict criteria sperm morphology assessments by Dr. Thinus Kruger personally.

B. Pyospermia - the normal amount of white blood cells in human semen is < 1 x 106/ml. Since pyospermia, white blood cells in semen, is potentially indicative of a genital tract or urethral infection, samples should be cultured when the semen white blood cell concentration is elevated.

C. Microbiology - semen samples are not sterile. Many microbiologic organisms may be present, some of which are associated with infertility and others which have little significance. Chlamydia Trachomatis is a cause of epididymitis and may result in duct obstruction, maturation impairment and increased antisperm antibodies.

Genital tract infections and pyospermia may alter fertility in many ways and require treatment, therefore the presence of increased bacteria should be noted on the SA report. Infections may be passed to the female partner causing female tract infections and subsequent infertility. Again, semen samples should be cultured whenever indicated.

III. Sperm Quantitation

A. Sperm count - a good quality counting chamber developed specifically for a SA should be utilized. Our center utilizes a Makler counting chamber. A 5 ul drop of well mixed semen is placed on the chamber well and the cover-piece with grid is positioned on top providing a 10 ul depth sample. The cover-piece contains a central grid with 100 squares. Using a 20x objective, the number of motile cells in 10 squares is counted followed by the number of immotile cells in the same area. These numbers are totaled and provide the concentration of sperm/ml as well as % motility.

Motile sperm and total sperm concentrations/ml of semen are reported as well as motile sperm counts/ejaculate.

B. Sperm motion analysis - subjective determination of sperm motion or sperm kinetics is made by grading the average quality of motion on the following scale:

0 - immotile

1 - weak movement with no forward progression

2 - weak to moderate forward progression

3 - good forward progression; active tail movement

4 - rapid forward progression; vigorous tail movement

Viability testing is indicated when the percent of motile sperm is abnormally low. A supravital stain such as eosin can be used to differentiate immotile from dead cells. Eosin will penetrate all dead cells and leave the live cells clear. A minimum of 100 cells are counted to estimate the live:dead ratio.

Comment

It is apparent from the above that a significant amount of useful information can be obtained from a SA if it is performed correctly. Likewise, misleading information may be given if the SA is performed without rigid attention to detail, quality control, technical training and reporting of results.

Specialty centers that have laboratory scientists working in andrology are more likely to provide the patient with a comprehensive semen analysis than laboratories that do not have specially trained personnel.

Antisperm Antibodies

A significant number of couples presenting with idiopathic infertility are found to be positive for sperm antibodies. Antisperm antibodies can be immobilizing, that is, they cause immotility or low motility, or can be of the agglutinating type, where clumping or agglutination of the spermatozoa is induced. Cytotoxic antibodies cause a loss of sperm liability or cell death. Male antisperm antibodies can be present in the serum, seminal fluid and directly on the sperm surface. In the female, antisperm antibodies can be found in the cervical mucous, genital tract secretions, utero-tubal fluids and follicular fluid.

In the male, isolation of the testicular seminiferous tubules from the circulation and surrounding tissue by the blood-testis barrier prevents the development of autoimmunity to sperm. Breakdown of this barrier as a result of trauma or infection, as well as vasectomy, may result in spermatozoa being exposed to the circulating components of the immune system (plasma cells), resulting in production of antibodies directed at the spermatozoa. Alteration in the balance between the immune system and sperm antigens and natural immunosuppressive factors may, in theory lead to development of antisperm antibodies.

Antisperm antibodies impair a man's fertility potential primarily by impairing sperm transport through the female reproductive tract. Immobilizing sperm antibodies can reduce sperm motility or cause complete immobilization of spermatozoa, whereas agglutinating antibodies cause clumping of groups of spermatozoa, thereby reducing the numbers of sperm that are available to penetrate the cervical mucous, and migrate into the fallopian tubes. Thus, transport through the female reproductive tract is impaired by virtue of the low motility or clumping, as well as the reduction in freely motile spermatozoa capable of transport through the tract.

Various sensitive and specific tests for detection or sperm antibodies are available. In our center, we use the immunobead test. The principle behind the assay is the use of commercially available microscopic polyacrylamide spheres, ranging in size from 2 to 10 um, coated with anti-human immunoglobulins against human IgG, IgA or IgM. The advantage of this test allows the determination of regional distribution of the antibodies over the surface of the sperm: head-directed, mid-piece directed, tail-directed, or tail-tip directed. Immunoglobulin binding to the tail-tip only may have little or no significance.

Intrauterine insemination (IUI) is a logical approach to treat not only possible cervical mucous-sperm hostility, but also cervical mucous antibodies. Intrauterine insemination bypasses the hostile cervical mucous, thus inhibiting or preventing antibody binding to the sperm. Also, by placing the sperm in the uterine cavity, giving the sperm a head start may help to locate the sperm closer to the sight of fertilization, and thus reduce the effect of sperm immobilizing or agglutinating antibodies.

If IUI fails, other assisted techniques, such as IVF or GIFT might be considered. These procedures eliminate the inhibition of sperm transport through the female tract to reach the site of fertilization. By placing the sperm and oocyte in close vicinity, there may be a greater chance of sperm binding to the oocyte and subsequent penetration.