Update On Clinical Andrology
Volume 1 Issue 2 Winter 1994

A Newsletter addressing contemporary issues in male fertility and male sexual function


Editor:
BRUCE R. GILBERT,M.D.,PH.D.
Associate Professor of Urology - Cornell University Medical College
Associate Professor of Urology - SUNY at Stony Brook
Diplomate American Board of Urology
Diplomate American Board of Bioanalysis
Fellow American College of Surgeons
email: bruce.gilbert@verizon.net

INSIDE THIS ISSUE

New Research Suggests How varicoceles make men infertile

Vasectomy : Is there a causal relationship between vasectomy and prostate cancer?

Treatment for Ejaculatory Disorders

Sperm function: High levels of antisperm antibodies alter sperm function.

New Research Suggests How Varicoceles make men Infertile

Varicocele is well known to be the most common associated cause of male factor subfertility. It is generally accepted that the presence of a varicocele is associated with an impaired semen quality and decreased fertility. However, how varicoceles impair sperm function is not known. Recent data has now suggested that sperm binding to the zona pellucida (measured indirectly by the sperm penetration assay) and pregnancy is increased after varicocele ligation. This data supports an effect of varicocele ligation on subsequent sperm capacitation, the post ejaculatory maturation process which normally occurs in the female tract. Upon capacitation sperm acquire fertilizing potential as defined by the appearance of the capacity for a species specific, receptor-dependent adhesion of the sperm head to the zona pellucida (zp) of the egg with subsequent ZP-induced acrosome loss. Data from our laboratory now demonstrates that the major human sperm zona recognition molecule is a mannose-specific lectin whose expression on the sperm surface from sub-plasma membrane stores is dependent upon capacitation-associated efflux of cholesterol from the cell membrane. Therefore, for the first time, it is possible to directly investigate the effects a varicocele may have on specific molecules critically involved ln human fertilization, The knowledge to be gained from such studies would be useful for infertility management and in the development of rational steps towards effective therapies.

In a prospective blinded, non-randomized (controlled) study supported by the American Foundation for Urologic Research (the research arm of the American Urologic Association) and Searle, we are evaluating two specific sperm membrane parameters (surface mannose lectin expression and cholesterol concentration) in men with documented varicoceles (by color flow scrotal ultrasound), a prolonged period of subfertility and multiple failed impregnation attempts by intrauterine insemination. To study specific changes in membrane parameters which may be associated with varicocele ligation, we are characterizing their expression in subfertile males both before and at intervals after varicocele ligation. Sperm from known fertile males and men not undergoing varicocele ligation will serve as controls. All specimens are examined immediately after ejaculation and after 18 and 72 hours of incubation in an albumin supplemented medium to mimic in vitro the effects of in vivo incubation in the female tract.

Known fertile sperm populations exhibit marked increases in the percent of spermatozoa showing mannose lectin expression and in spontaneous acrosome loss which are at plateau values by 18 hours and do not increase on extended incubation. In specimens from men with documented varicoceles, surface mannose-specific lectin expression did not significantly increase even after prolonged incubation, nor was there an increase in spontaneous acrosome loss. Sperm from a varicocele patient was de-membranated by vortexing, before and after capacitating incubations. His sperm were found to contain sub-plasma membrane stores of mannose lectins at all times. These stores are comparable to those seen only in fresh specimens from the normal control, thus indicating that there was a failure of trans-plasma membrane translocation on incubation. A similar analysis of sperm from one patient after varicocele ligation revealed that his sperm were essentially normal and the carbohydrate binding sites of these sub-plasma membrane mannose lectins seen in fresh sperm externalized transmembrane to the sperm surface after capacitating incubations.

As surface mannose lectin expression and spontaneous acrosome loss by sperm from fertile males are dependent on increased membrane fluidity during capacitation, our preliminary data suggests that the presence of a varicocele alters the character of the sperm membrane. This may be the result of the increased intrascrotal temperature found in men with varicoceles which may alter the sterol and lipid composition of the sperm membrane during spermatogenesis.
Vasectomy and prostate cancer is there a causal relationship?

Two recent articles in JAMA (Giovannucci et al,JAMA, 269:872&878,1993) have again stirred debate as to the long term effects of vasectomy. The last major debate was in the 70's and 80's when a study in monkeys found an increased risk of cardiovascular disease after vasectomy. It took ten years, 9 large scale epidemiologic studies finding no increased risk of cardiovascular disease after vasectomy and the inability to reproduce the results (even by the group that made the original claim) to provide compelling evidence that vasectomy did not increase the risk of cardiovascular disease. However, with the large number of vasectomies being performed in this country yearly, and the increasing incidence of prostate cancer, any possible association needs to be fully studied.

The editorial that followed the two articles in JAMA (summarized below) by Dr. Stuart Howards and Dr. Herbert Peterson (JAMA,269:91,1993) highlights several of the concerns in labeling this a causal relationship.

Neither of these studies were initiated with the primary purpose of evaluating vasectomies risk factor for prostate cancer. The adjusted relative risk of 1.56 for the Nurse's Health Study had 1.66 for the Health Professionals follow up study, (which were the two studies they presented), are statistically considered to be weak associations and may be due to chance, bias or a causal relationship. Dr. Howards and Peterson agree that although chance is an unlikely explanation it should not be dismissed as a possible explanation. However,, in another analysis of the Nurse's health study, the risk of dying from prostate cancer was not statistically significantly increased, but the risk of dying from lung cancer was. An association between vasectomy and lung cancer, not previously been reported, is also implausible.

We also must consider, that in the United States, most vasectomies are performed by urologists and most prostate cancers diagnosed by urologists. Urologic examinations enables the Urologist to evaluate various genitourinary tract symptoms. Therefore, men who have had vasectomies, and are followed by urologists, might have a higher detection rate of prostate cancer then those not having vasectomies and who are not usually followed by urologists. Thus, selection bias might exist.

The most likely non-causal explanation for the findings as pointed out by Dr. Howards and Peterson is unmeasured, confounding. Approximately one in every eleven men in the United States will develop prostate cancer, the majority of these men have not undergone vasectomy. Since the causes of the prostate cancer remain unknown, it is impossible to know whether or not the true risk factors for prostate cancer are equally distributed between men who have undergone vasectomy and men who have not. In fact, in the Nurse's Health Study, men who have underwent vasectomy had a lower total mortality than those men who did not. This would imply that men who underwent vasectomy in that study must have different characteristics than men who did not. These characteristics in themselves might increase the risk of prostate cancer.

As was pointed out by Howards and Peterson the studies by Giovannucci et al are well designed and analyzed. However, these results also have to be compared with other epidemiologic studies, in fact, three other studies published to examine the relationship between prostate cancer and vasectomy, have not found an association between vasectomy and prostate cancer. In addition, is a relationship between vasectomy and prostate cancer plausible? To date none of the changes known to occur with vasectomy have been implicated in the biologic mechanisms that can be used to explain prostate cancer.

In 1991 the World Health Organization convened a conference to discuss relationship between vasectomy and cancer of the prostate and testes, the group concluded, "That on the basis of existing biological and epidemiological evidence, any causal relationship between vasectomy and the risk of prostate or testicular cancer was unlikely. And no changes in family planning policies concerning vasectomy are justified".

The high caliber of the work by Giovannucci et al however, suggests the need for further research. The recommendations suggested by Dr. Howards and Peterson, are:
1. Pre-sterilization counseling should include information regarding vasectomy and health, particularly the issues raised on vasectomy and prostate cancer.
2. Alternate methods of contraception should always be discussed, and the fact that all methods of contraception carry some risk.
3. A documentation of this discussion should be included as part of the consent for the procedure.

The standard recommendation for digital examination and determination of serum prostate antigen level between the ages of 50 and 70 years remains unchanged. Certainly much more research is needed prior to demonstrating any causal relationship between vasectomy and prostate cancer.

Studies are needed to study the safety of vasectomy and should be encouraged. As stated by Drs. Howards and Peterson, "..The issues at stake are clear. If vasectomy is a risk factor for prostate cancer, people need to know and informed choices need to be made......if associations are spurious but believed to be real, the popularity of a highly effective contraceptive method will be reduced, opportunities to reduce unintended pregnancies will be lost, and maternal and infant mortality and morbidity will increase".

after Howards and Peterson (JAMA,269:91,1993)

Treatment for Ejaculatory Disorders

Although originally used exclusively in spinal cord injured males (SCI), electroejaculation is now utilized in those individuals unable to ejaculate because of testicular cancer surgery, diabetes, multiple sclerosis or other neurologic impairments (NSCI). Clinical trails involving artificial insemination of partners of these men desiring pregnancy are currently underway (BRG).

For SCI patients no anesthesia is required. For NSCI patients a general anesthesia is used. The procedure consists of insertion of a probe into the rectum of the patient. A current is then applied and an erection, potentially followed by ejaculation, is produced. A general anesthetic which lasts approximately 15-20 minutes is administered in order to avoid discomfort during the brief procedure.

The preoperative evaluation consists of a history and physical edam, blood tests, and signing of an informed consent.

The following are some important facts about the Electroejaculation Program:

1. A commitment of at least 4 trials (one day a month for 4 months) is necessary.

2. The spouse or significant other should begin daily temperature charts on entry into the Program.

3. The male patient must be checked for retrograde ejaculation (sperm that goes back into the bladder at the time of orgasm) and have been given a one-month trial of Ephedrine, Sudafed, and/or Imipramine, if not hypertensive or allergic, prior to treatment with electroejaculation. If either of these manipulations results in an ejaculate, electroejaculation may not be necessary.

4. Fertility history of patients, including previous pregnancies, ejaculations, ability of achieve erections, is necessary. We also require copies of records detailing previous operations and chemotherapy when applicable.

5. Electroejaculation is a new procedure. Unfortunately, the desired results are not always obtained. Not all ejaculates are sufficient for insemination (sperm quality is impaired). In addition, not all patients produce an ejaculate.

6. A Reproductive Endocrinologist (female fertility specialist) will evaluate all female partners. They will also assess temperature charts, perform ultrasound exams and prescribe medications (as needed).

For more information please contact B.R. Gilbert

High Levels of Antisperm Antibodies Alters Sperm Function

In theory, antisperm antibodies (ASA) may act at three levels: (1) Pre-fertilization events at the level of sperm transport to the site of fertilization; (2) Peri-fertilization events involving the binding of spermatozoa to the zona pellucida, the acrosome reaction, sperm oolemma adherence and fusion; (3) Post-fertilization events such as pre-implantation embryo growth and nidation. Although there is a body of experimental evidence that antibodies raised against sperm can react with eggs and embryos and be damaging, whether such effects occur clinically remains controversial. This review will focus on the loci of action by which antisperm antibodies alter the behavior of spermatozoa and will develop a clinical strategy as regards the treatment of immunologically mediated infertility.

Effects of antisperm antibodies on cervical mucus penetration
The ability of spermatozoa coated with antibody to penetrate and survive within human cervical mucus is often impaired, as can be observed clinically on post-coital testing . The extent of impairment is dependent upon: (1) The proportion of sperm coated with immunoglobulin; (2) The amount of antibody coating the sperm surface; and (3) The immunoglobulin class. The greater the proportion of sperm within the ejaculate coated with antibodies, the fewer that enter cervical mucus. When all sperm are coated over the majority of their surface, it is rare to see any motile sperm within the cervical mucus, despite well timed postcoital testing and normal semen parameters. Circumstantial evidence suggests that this impairment is mediated through the Fc portion of the immunoglobulin molecule coating the sperm surface. Sperm exposed to Fab preparations of purified antisperm IgG, in which the Fc portion has first been removed enzymatically, are able to swim through cervical mucus. Conversely, those sperm labelled with intact antibodies do not. In addition, if the Fc portion of immunoglobulins bound to the sperm surface is liberated proteolytically, such protease treated sperm exhibit an improved ability to penetrate cervical mucus in vitro and to sustain motility therein. For example, when IgA labelled sperm were treated with a specific IgA protease, the percent of sperm penetrating a column of human cervical mucus in vitro increased from 8.9 + 10.6 (x + SD) before treatment to 23.3 + 13.9 after. In contrast, there was no improvement in the ability of antibody negative spermatozoa to penetrate cervical mucus following protease treatment (25.4 + 14% vs 21.7+6.3%).

Studies of the immunologic consequences of vasectomy indicate that antisperm antibodies of the IgA class appearing in the ejaculate following vasovasostomy are more likely to impair sperm mucus penetrating ability than IgG. Recently, Meinhertz et al (Fert.Steril:54:315,1991) studied the ejaculates of 26 men following sterilization reversal using a mixed agglutination reaction (MAR). The conception rate was 85% in a subgroup of men with a pure IgG response, while only 43% of men who also had IgA on their sperm fathered children. The conception rate was reduced even further when 100% of sperm were coated with IgA (21.7%). The combination of IgA on all sperm and a high serum titer (greater than 1:250) as detected by agglutination test, was associated with a zero conception rate.

These observations suggest than men who exhibit a high level autoimmunity to sperm, whether induced by vasectomy or idiopathic should be considered functionally oligospermic. Although these men may exhibit normal sperm concentrations and high total sperm outputs in their ejaculates, their spermatozoa have difficulty exiting semen, following its intravaginal deposition during coitus, and exhibit impaired entry of preovulatory cervical mucus.


Effects of antisperm antibodies on their fertilizing ability
Antisperm antibodies (ASA) could, in theory, impair sperm function through alterations in the timing of the acrosome reaction. ASA that cause loss of the plasma membrane and acrosomal contents leading to a premature acrosome reaction, could impair the ability of sperm to bind to and fertilize eggs. Several investigators have found evidence in support of this thesis.

We have observed that certain sperm head-directed antibodies, in the presence of complement, promote an acrosome reaction ultrastructurally similar to that seen following incubation of spermatozoa in medium containing human serum albumin at concentrations that support a spontaneous acrosome reaction. The mechanism of plasma membrane shedding and release of acrosomal contents in these experiments appeared to be different from that noted in the "classical" acrosome reaction of mammals, in which the fusion of the plasma membrane with the outer acrosomal membrane occurs. An increased proportion of acrosome reacted sperm has also been observed in ejaculates of some men with autoimmunity to sperm. Silverberg et al (AFS Meeting Nov 1992) have also recently documented that ASA promoted an acrosome reaction in vitro.

Sperm head directed ASA's may also promote or inhibit the penetration of zona-free eggs by mechanisms that do not involve an alteration in the proportion of acrosome reacted sperm. These experiments were performed with spermatozoa from the same fertile donor. Effects of each ASA-positive serum was judged against the ASA-negative control. ASA were transferred to donor sperm in vitro, and then antibody-labeled spermatozoa were washed free of serum before overnight incubation and insemination of zona-free hamster eggs.

These observations suggest that some antisperm antibodies promote the adherence of antibody-labeled sperm to the oolemma of zona-free hamster eggs independently of the acrosome reaction. Two different mechanisms could account for this phenomenon. A common epitope might be present on sperm and egg surfaces, which would allow antibody bridging between gametes. Alternatively, the oolemma might possess Fc receptors for the immunoglobulin coating the sperm surface. Evidence for both mechanisms exists. Immunization of laboratory animals with sperm can lead to the production of antibodies that react with unfertilized eggs, fertilized eggs, and pre-implantation embryos as well as sperm.

Zona-free eggs and spermatozoa were initially mixed and then 3-5 eggs were concurrently recovered at intervals from each treatment group, washed and stained with acridine orange, as whole mounts and examined under indirect immunofluorescent illumination for sperm enumeration. No difference in acrosomal status was noted between antibody labelled and unlabelled sperm, when scored with PSA staining.

We have shown that zona-free hamster and human eggs bind monomeric IgG Fc fragments as well as heat aggregated IgG. The presence of Fc gamma receptors on these eggs was further suggested by indirect immunofluorescence, using monoclonal antibody Fab preparations of anti-Fc gamma receptor antibodies. That oolemma Fc receptors play a role in the sperm to the egg is indicated by the abrogation of this effect when eggs were preincubated with IgG Fc fragments.

If antisperm antibodies enhance sperm adherence to the egg surface and promote penetration, they may increase the risk of polyspermy. Triploid human eggs have diminished developmental competence during their pre-implantation growth, as well as a higher likelihood of post-implantation loss, increasing the possibility of both occult as well as clinically manifest spontaneous abortion.

Antisperm antibodies also have effects at the level of the zona pellucida. We used bisected hemizonas from salt-stored human eggs to assess the effects of labelling sperm from fertile men with antisperm antibodies on their ability to attach to the zona pellucida. Immunobead binding was used to confirm that nearly all sperm were labelled with immunoglobulin over their heads. A wide range in effect was observed, several sera markedly lowered the number of tightly bound sperm observed, while others were completely without effect. In no case was sperm zona binding completely inhibited, suggesting that clinical in vitro fertilization as a treatment of immunologically mediated infertility was likely to succeed, even in the face of high levels of antibodies. These results further emphasize that the functional effects of antisperm antibodies in different individuals vary greatly, despite their common regional localization of binding to the spermatozoan surface.



Conclusions
The impact of autoimmunity to sperm on spermatozoal function is complex. The additive effects of four loci of action (sperm transport, the kinetics of the acrosome reaction, interaction with the zona pellucida and the oolemma) must all be placed within a complex equation. These varying and perhaps opposing effects should be more clinically predictable for each individual, once the specificities of the various antisperm antibodies for their antigenic determinants are known. Currently, however, given our inability to predict the effects of these antibodies on sperm function, the most practical clinical treatment approach is an empiric one, utilizing assisted reproductive techniques to enhance the likelihood of gamete contact and fertilization.


adapted from: Richard Bronson, M.D.The Andrology Report, April 1993