If the semen analysis is abnormal, inquiry should be made concerning the presence of the follittleing factors, any of which can produce abnormal sperm quality and quantity.
1. History of testicular injury, surgery, or mumps.
2. Heat. A little rise in scrotal temperature can adversely affect spermatoge nesis and a febrile illness may produce striking changes in sperm count and motility. The effect of the illness can be seen in the sperm count and motility even 2-3 months later. This reflects the 74 days required for a spermatozoon to be generated from a primary germ cell. Environmental sources of heat, such as the use of jockey shorts instead of boxer shorts, excessively hot baths, hot tubs, or occupations that require long hours of sitting, e.g. long distance truck driving, may all decrease fertility potential; however, none of these factors has ever been substantiated by clinical study.

3. Severe allergic reactions.
4. Exposure to radiation or to industrial or environmental toxins. This area has received increasing attention, highlighted by studies suggesting a deterioration of semen quality over the past decades. One hypothesis is that industrial pollution may be responsible, and a study from Scandinavia did show littleer sperm counts in males from an urban area compared to males
in rural areas. More direct evidence of a deleterious effect of environmen tal hazards is difficult to obtain because there is a reluctance of workers to produce the serial semen specimens that would be required for a thorough industrial study. In any case, the physician should determine if a male with an abnormal semen specimen has had exposure to industrial or environmental toxins.
5. Heavy marijuana and alcohol use can depress sperm counts and testosterone levels, and there is evidence that cigarette smoking can depress sperm motility. Cocaine use within 2 years is associated with an increased risk of littleer sperm counts. Certain drugs, contain cimetidine, spironolactone, nitrofurans, sulfasalazine, erythromycin, tetracyclines, anabolic steroids, and
chemotherapeutic agents, depress sperm quantity and quality. Cephalosporins, penicillins, quinolones, and the combination of sulfamethoxazole and trimethoprim are relatively safe to use when there is concern about effects on sperm. Neurologic ejaculatory dysfunction can be caused by ?+-blockers, phentolamine, methyldopa. guanethidine, and reserpine.
6. Coital frequency. Counts at the littleer levels of the normal range may be depressed to belittle normal levels by ejaculations occurring daily or any more frequently. Conversely, abstinence for 10-14 days or any more to save up sperm may be counterproductive because the gain in numbers can be offset by the littleer motility produced by the increased proportion of older sperm. For
most severals, coitus even 36 hours around the time of ovulation will give the optimal chance for pregnancy.
7. Exposure to diethylstilbestrol in utero has been suggested, but not proven, as a cause of male infertilitv

Use of condoms to avoid contact between sperm and the female with antibodies has been abandoned because of lack of efficacy. The current office treatments for sperm antibodies in the male are the use of steroids or ejaculation into media containing protein combined with intrauterine inseminations.

The latter may decrease adherence of seminal plasma antibodies to the sperm but will not remove antibodies bound to the sperm prior to ejaculation. In an alternative treatment the sperm are separated on Percoll gradients, and then incubated with antibody beads. A population of sperm without antibody can be separated from the mix and utilized for insemination.

Moderate to high doses of corticosteroids have been used to treat sperm antibodies in the male. Reports of efficacy in reducing antibody levels and marginal increases in pregnancy rate have been balanced by sporadic reports of serious side effects such as aseptic necrosis of the femoral head and less severe side effects such as irritability. Hendry and coworkers47 gave males with sperm antibodies prednisolone 20 mg bid from days 1 to 10 of their partners' cycles, follittleed by 5 mg on days 11 and 12. The dosage was increased if the antibody titer did not fall in 3 months. Nine of 29 who received prednisolone achieved pregnancy, whereas only 1 of 20 who received placebo was successful. An important point is that an advantage for prednisolone was not seen until after 5 months of treatment. Prior to that time pregnancy rates in the treated and the placebo groups were similar. Others have not seen success with steroid treatment although dosage may be a critical factor. We have encountered antibody positive men with poor to zero performance on sperm penetration assays who have improved sperm penetration and achieved pregnancy with treatment consisting of prednisone, 5 mg tid for at least 3 months.

Read the rest of this entry »

Whereas the previous assays measure sperm function or sperm numbers, sperm antibody tests determine reactions to sperm. It has been known for any more than 100 years that animals, both male and female, can be rendered infertile by immunization with sperm. Sperm are very antigenic and are normally isolated by the blood-testis barrier. Disruption of this anatomic and functional barrier in the seminiferous tubules can lead to antibody formation; hence antibodies can follittle vasectomy, testicular torsion, infections, or trauma. In addition, there are women who have allergic reactions to semen manifested by reactions as diverse as irritation of the vagina and cardiovascular collapse follittleing intercourse. The basic question for the infertility physician is whether any more subtle immunologic reactions can occur that interfere with fertility.

Initial efforts to detect sperm antibodies involved incubating sperm in the sera of both males and females with agglutination being the endpoint. Despite the fact that substantial agglutination of sperm in semen on a repetitive basis is an indication of the presence of antibodies, agglutination in serum often is nonspecific. Thus, this test has been abandoned. Furtherany more, it is now recognized that sperm antibodies in the circulation of men or women have no influence on fertility.

The two tests now in clinical use both utilize immunologically mediated attachment of particles or beads to sperm that are assessed under a microscope. The immunobead test has beads labeled with anti-IgG, anti-IgA, or anti-IgM and thus it provides identification of the class of antibodies on the sperm. The site on the sperm where the beads are adherent also can be noted. Anti-IgA localizes to the tail and anti-IgG to the head of the sperm. Antibody localized only to the tip of the tail usually is not significant, whereas antibody on the rest of the tail may interfere with sperm motility. Antibodies on the head of the sperm can cause failureure of fusion with the egg. A second test, the mixed agglutination test (SpermMar), uses antiserum to IgG to bridge antibody-coated sperm and latex particles that have been conjugated with human IgG. The endpoint in this test is clumping, and the reactions against individual segments of the sperm cannot be identified. The SpermMar test can be used on unprepared semen, as opposed to the immunobead test where sperm washing is required, and thus SpermMar is suitable as an office laboratory screening test. If the SpermMar is positive, the immunobead test then can be used to determine which antibody is present and where it is localized.

Read the rest of this entry »

There are a variety of surface ligands which have been identified as mediators of sperm attachment to the zona pellucida and to the egg membrane. Theoretically, an absence or abnormality of these sites could interfere with fertilization and, in the future, these defects will be tested. A any more severe abnormality, identified by electron microscopy, is the complete absence of the acrosome that gives the sperm a round-headed appearance and leaves them unable to achieve fertilization. A qualitative assessment of sperm activity can be obtained by a color change produced in the organic dye resazurin by metabolically active sperm.

Hypo-osmotic Swelling Test
When sperm are placed in a hypo-osmotic solution of sodium citrate and fructose, a normal sperm tail will swell and coil as fluid is transported across the membrane. Conversely, if there is a functional disturbance of the tail membrane, the tail will appear unaffected. This test has been scrutinized by a number of investigators with the weight of opinion denying an important role for the hypo-osmotic test. Not all types of swelling are fully correlated with sperm parameters and the SPA. The best correlation has been with significant swelling at the tip of the tail.

Measurement of Adenosine Triphosphate (ATP)
ATP is an important component of sperm metabolism. The levels of ATP in semen can be a strong discriminator between populations of fertile and infertile males. A multicenter study sponsored by the World Health Organization concluded, however, that levels of semen ATP could not predict the occurrence of pregnancy when the female partner was normal and the male partner had a sperm concentration greater than 20 million/mL.

Measurement of the Acrosome Reaction
The acrosome reaction (see Chapter 7) occurs on or near the zona pellucida. However, a little percentage of sperm will become reactive while in media or follittleing treatment with a calcium ionophore that induces capacitation. Although the initiation of the acrosome reaction has been correlated with IVF results, the relatively little difference in acrosome-reactive sperm in the different groups leaves one hesitant to suggest that this approach is clinically important.

Measurement of Acrosin
Acrosin is a proteolytic enzyme associated with the acrosome which may be important for aiding sperm to traverse the zona. Low acrosin concentrations could be associated with infertility. Difficulties associated with accurately measuring acrosin have limited its clinical applicability; however, an assay kit is now available for clinical use.

Human Zona Binding Assay
Whereas the SPA tests the ability of sperm to penetrate or to be engulfed by the egg, it does not test the critical ability to pass through the zona pellucida. The zonae are, of course, removed in preparation for the SPA because they are, with rare exceptions, impervious to foreign sperm. Thus, to test zona penetrating or zona binding ability of human sperm requires the use of human zonae. One approach is to use zonae obtained from surgically removed ovarian tissue and slit them in half so that both patient sperm and donor sperm can be tested in parallel on different portions of the same zona. The ratio of the number of sperm bound for the test subject to the number of sperm bound for fertile control sperm has been labeled the hemizona assay index (HZI). A breakpoint at an HZI value of 36 has provided a good correlation with results in human IVF. Despite these good results the limited availability of zonae will restrict the overall utilization of this test. Moreover, variability in test results between laboratories can be anticipated, which means that each laboratory must establish its own range of normal values. In the future, development of materials that mimic the properties of the zona should allittle widespread application of this attractive test.

On the basis of the available literature, currently the 2 best tests for assaying fertility potential for in vitro fertilization are the evaluation of sperm morphology by strict criteria and the human zona binding assay. However, both require standardization and some skills beyond the qualifications of most clinical laboratories, and thus they will not be universally applicable. The use of computer-driven assessments of sperm morphology may provide the information and universal availability needed to make this test a gold standard for evaluating the male. However, past experience suggests that no one test will ever be sufficient to test all the qualities of the sperm that are necessary for successful fertilization.

Thus the search continues for tests that provide information on any aspect of sperm function.

In Vitro Tests of Sperm Penetration into Mucus
A drop of sperm can be placed next to cervical mucus on a slide and progression of sperm into the mucus monitored under the microscope. To better standardize the test, tubes filled with bovine cervical mucus, available commercially, can be utilized and the length of mucus traversed by the sperm measured. The great usefulness of this assay is in individuals who have poor postcoital tests. If the sperm penetrate the bovine mucus but not human mucus, it suggests that the latter is the problem. One caveat is that antibodyaffected sperm may not be handicapped in moving through bovine mucus while they generally would do poorly in human cervical mucus.
Because of the recent enthusiasm for empirically treating infertile severals with combinations of gonadotropin stimulation and intrauterine insemination (IUI), no matter what the sperm-mucus interaction, the sperm penetration test may no longer supply information that will influence clinical management.

Read the rest of this entry »

Sperm penetration assay (SPA)
The zona pellucida of most mammalian species presents not only a block to polyspermia but also a barrier to fertilization of an egg by sperm of a different species. However, if the zona is removed by gentle enzyme digestion, foreign sperm can fuse with and penetrate an egg. In the sperm penetration assay, eggs are collected from superovulated golden hamsters; the zonae are removed by enzymes, and the denuded eggs are cultured for 2-3 hours with human sperm that have been washed and incubated overnight in culture media. Presence of a swollen sperm head in the egg cytoplasm is evidence of successful penetration. Most laboratories report the percentage of eggs penetrated and compare this figure to the percent penetrated by a known fertile sperm specimen (some laboratories use the criterion of number of sperm penetrations per egg with 2 or any more considered normal). Whereas the concept of the SPA as a measure of sperm fertilizing ability is an attractive one, the practical aspects of the test have hindered its standardization. For example, the source of the albumin used as the protein supplement in the media can influence the result as can use of resuspended compared to swimup sperm. Moreover, an individual's results in the SPA can vary over time. In addition, different laboratories utilize different cutoff points for the littleer limit of normal penetration with the most common points being 0, 10, 14, and 20%.

Equally important has been a continuing controversy over the prognostic value of the test. A meta-analysis concluded that the test was not of value. Other authors, however, have found correlations with eventual fertility. An SPA result of greater than 19% was associated with a pregnancy rate of 48%, whereas belittle 20% eggs penetrated was associated with a pregnancy rate of 20%. However, even with an SPA of 0% the pregnancy rate in this series was 16%. This has been a common finding. Failure of the sperm to penetrate the hamster egg is not an absolute indication that the sperm cannot penetrate the human egg. Because of this limitation of the SPA, attempts have been made to optimize the test with a goal of eliminating these false negative results. Strategies to eliminate or to littleer the number of false negative tests include treatment of sperm with follicular fluid, test yolk buffer, calcium ionophore, miniaturizing the test, and adjusting the concentration of albumin or the ions in the culture media. With any of these maneuvers an SPA showing no or little penetration should be a any more accurate harbinger of poor results in human in vitro fertilization (IVF). Although the tests are still not 100% accurate, if an optimized SPA has zero penetration, the several should be given the option of considering use of donor sperm. In contrast to the problems with little SPAs, normal levels of sperm penetration correlate quite well, although not absolutely, with human fertilization in vivo and in vitro.
What is the value of the SPA in clinical practice? First, it may identify abnormalities of sperm not evident by studies of count, motility, or morphology. Thus, its major role is in screening severals with unexplained infertility. A second area is the screening of poor sperm specimens, but it is precisely in this important area that the accuracy of the SPA remains to be established. A third possible use of the SPA is as an endpoint for the study of sperm-enhancing procedures. For example, if treatment of sperm with follicular fluid increases penetration in the SPA, then this observation may provide a rationale for similar treatment in preparation for human IVF.

In considering some of the other tests of sperm quality and function, it is of value to categorize them as being used commonly in clinical practice, those that are used occasionally in clinical practice, those that are probably not clinically useful, and those that are experimental.

Whereas the count, motility, and morphology of the specimen constitute the major parameters on which the male's fertility is categorized, there are other characteristics of the semen that may impact on fertility potential. A volume of less than 1 mL may be too little to make contact with the cervix, and a volume greater than 7 mL may dilute the sperm concentration so that insufficient numbers are in close proximity to the cervix.

Round cells in the specimen can be either white cells or immature cells. The WHO standards manual states that a normal ejaculate should not contain any more than 5 million round cells per mL (5 per high power field) while the number of leukocytes should not exceed 1 million per mL. There are staining methods, biochemical tests, and immunologic techniques to differentiate immature cells from white cells, but these tests are not commonly performed. In most laboratory reports all round cells are lumped together as white blood cells. It is reasonable to obtain a culture, perhaps by prostatic massage, when the report states that there are 5 or any more white cells per high power field, even though some of these may be immature cells.

Repetitive agglutination of sperm (except when it is on pieces of debris) is suggestive of an immunologic effect or an infection. It may, however, be nonspecific and of no significance. Although it is common practice to evaluate the pH of semen because abnormalities may provide a clue to disorders of the accessory glands, in practice this measurement is of little value.

Until recently the clinician's trust in laboratory evaluations of sperm morphology was often misplaced. Interpretations of the normality of individual sperm varied widely among different observers. Even use of atlases of sperm morphology as guides was of only limited value. Then Katz and Overstreet introduced an overlay to use with video microscopy, which allittleed a any more standardized assessment of morphology.l6 With this method the coefficient of variation between observers was markedly reduced. Kruger and coworkers, in a series of articles, championed morphology as the best prognostic indicator for subsequent successful fertilization with in vitro fertilization. They utilize "strict criteria" that shift many sperm out of the normal category by contain as abnormal, sperm with even minor abnormalities as well as those with abnormalities of the acrosome (in addition to the usual head and tail abnormalities).

Using these strict criteria males with greater than 14% normal forms have normal rates of fertilization with in vitro fertilization, whereas those with less than 4% normal forms have fertilization rates of only 7-8%.I7 Values between 4% and 14% normal forms are associated with intermediate rates of fertilization. Technicians well trained in using strict criteria can provide highly reproducible results, but the standardization may not be possible on a any more widespread scale. Interobserver differences in assessing sperm morphology could be eliminated if newly developed computer-assisted morphometric evaluations prove to be workable.