Are antisperm antibodies indeed sperm-specific?

Antisperm antibodies (ASA) are present in a high percentage of infertile patients. The development of ASA in the male depends on the sequestration of antigens on germ cells, the testis being an immune privileged region. In this study, we investigated the specificity of ASA binding to sperm antigens by exposing a number of organ preparations to ASA. In none of the organ homogenates was a significant difference between the results of the Western blot with ASA-containing fluids, neither serum nor seminal plasma, and those without ASA observed. We conclude from our results that the ASA tested in our study obviously are sperm-specific. ASA as an autoimmune are thus restricted to spermatozoa. The antigens are suitable for trials in immune therapy, they may also serve for the development of antigen-specific diagnosis and treatment in infertility. They also indicate cognate antigens of possible immune contraceptive agents.

Localization of binding sites of naturally occurring antisperm antibodies on human spermatozoa by immunofluorescence.

Antisperm antibodies (ASA) may affect sperm motility, acrosome reaction, sperm penetration of cervical mucus, binding to the zona pellucida, and sperm-egg fusion. We investigated the localization of ASA of infertile men or men after vasectomy bound on the sperm surface using an immunofluorescence method. Binding occurred in the acrosomal region, midpiece, and tail. Most of the ASA in both groups of patients bound to the midpiece alone or in combination with other regions of spermatozoa. Only few ASA samples showed binding to all the three sperm regions. A combination of binding to the acrosomal region and to the midpiece was never observed. In infertile patients with ASA, the binding site was compared with sperm parameters. ASA binding to the sperm head influenced the acrosome reaction. Binding of ASA on tail and/or midpiece was not associated with a significant alteration of viability and motility. Immunofluorescence appears to be a valuable tool in the diagnosis of immune infertility, in particular when impairment of the acrosome activity is suggested.

Cellular and biochemical markers in semen indicating male accessory gland inflammation.

Leucocytospermia is considered to be a sign of male accessory gland inflammation. The leucocytes in semen are mainly polymorphonuclear neutrophilic granulocytes. Leucocytospermia is not associated with the presence of bacteria and antibiotic treatment does not significantly lower the extent of leucocytospermia. A higher frequency of elevated herpes simplex antibodies titres were found in men with leucocytospermia. The concentration of inflammatory cytokines, interleukin-6 and -8, is closely correlated with the number of leucocytes. Their determination does not provide additional information. Reactive oxygen species (ROS) are generated at least in part by seminal leucocytes in response to stimulating factors. Purified leucocytes produce high levels of ROS. The determination of ROS appears to represent a parameter of functional activity of leucocytes. The role of chlamydiae in male accessory gland infection is unclear. Their determination in semen by DNA amplification and by immunological tests does not provide reliable results.

Male infertility and genital chlamydial infection: victim or perpetrator?

The role of Chlamydia trachomatis in the cause of male infertility is under discussion. This paper attempts to summarize data from the literature, which support the role of C. trachomatis in male infertility or oppose this suggestion. The following observations are based on a survey of the literature: 1) Chlamydia trachomatis is a frequent pathogen in male genital inflammation, the micro-organisms are rarely present in healthy men. 2) Without doubt, C. trachomatis causes inflammations of the male urethra and the epididymis. Prostatitis and glandulitis vesicalis are discussed controversially. 3) Chlamydia trachomatis antigen or DNA is not demonstrable in secretions of the male accessory glands including the semen with sufficient reproducibility. However, it is easily demonstrable in urethral swabs and the urine. 4) Determination of chlamydial antibodies in serum or semen does not conclusively indicate a current infection with C. trachomatis. 5) There are no conclusive studies showing that men infected with C. trachomatis are less fertile than uninfected men. 6) The male genital chlamydial infection is a threat to the female genital organs, because C. trachomatis infection of the female genital organs may be deleterious to female fertility.

Serum levels of inhibin B in men of different age groups.

The decline of testosterone levels in aging men is well documented. However, it is unclear to what extent the Sertoli cell marker inhibin B changes during aging. Herein we report on the determination of serum levels of inhibin B, follicle stimulating hormone (FSH), luteinizing hormone (LH) and testosterone in 906 patients from 16 to 89 years of age, presenting to our department for different complaints. There was a weak but significant correlation of the levels of inhibin B with age (r = 0.064, p < 0.05). A significant negative correlation of FSH and inhibin B was documented in all age groups (r = -0.423, p < 0.01). Also, the LH levels increased significantly with low inhibin B levels (r = -0.289, p < 0.01). Testosterone levels showed no significant correlation with inhibin B. We conclude from our study that Sertoli cell function as documented by serum levels of inhibin B is stable throughout life. In addition, the ongoing correlation of FSH and inhibin levels also indicates no significant decline of Sertoli cell function in the aging male.

Levels of interleukin-6 and interleukin-8 in seminal fluid of men attending an andrological clinic.

The presence of interleukins (IL) and other cytokines in seminal plasma was demonstrated in the literature. In particular, the levels of IL-6 were found to be related to male accessory gland inflammation. The close correlation to leucocyte count indicates a production of interleukins from the leucocytes and by the prostate gland. No relation of IL-6 levels to spermatogenic activity was quoted in the literature. We measured IL-6 and IL-8 in 454 men and compared the values with seminal parameters. The mean values of IL-6 30.7 +/- 101.2 pg ml-1 and IL-8 2023 +/- 1721 pg ml-1. The correlation analysis revealed a significant correlation of IL-6 and/or IL-8 to age, total fructose, immunoglobulin G (IgG) concentration and leucocyte count. The significant correlation of IL-6 and fructose levels indicates that also the seminal vesicles take part in the production of seminal IL-6. No correlation of the two interleukins measured to sperm parameters occurred. The calculation of a single harmonic trend revealed a significant trend over the year of the levels of IL-6 with a maximum in December and a peak-to-trough variation of 33% of the mean. It may be the consequence of a higher frequency of seminal tract inflammations in autumn and winter.

Computer-assisted analysis of sperm morphology with the aid of lectin staining.

Lectins are useful for staining the acrosome, which is a pre-requisite for the assessment of acrosome reaction in vitro. We tested wheat germ agglutinin, peanut agglutinin and pisum sativum agglutinin. The determination of the categories of normal and abnormal spermatozoa as defined by the World Health Organization, including an additional category 'acrosome-less cell', was performed with the aid of a system of computer-assisted semen analysis (CASA) in comparison with visual estimation. The acrosome reaction in vitro was induced by calcium ionophore A23187. Incubation with A23187 decreased the percentage of normal sperm heads and increased the number of acrosome-less sperm heads in comparison with the control samples. This shift was demonstrable with all three lectin staining procedures. No significant differences were observed in the comparison of sperm classes obtained by visual assessment or determination by CASA with two of the lectin staining procedures. After staining with pisum sativum agglutinin the classes of normal and of acrosome-less spermatozoa were significantly different between visual and CASA estimation. Our results indicate that estimation of the acrosomal status in vitro is possible by CASA when lectin staining is used.

[Inhibin B as a marker of spermatogenesis. A new dimension in andrology]. / Inhibin B als Marker der Spermatogenese. Eine neue Dimension in der Andrologie.

In 1932, McCullagh postulated the existence of a specific hormone of the testis, regulating FSH secretion. He named this hormone inhibin. As we know today, several proteins of the inhibin family are produced in the Sertoli cells. The major product is inhibin B, consisting of a alpha- and a beta-subunit. It is also present in spermatogonia, spermatocytes and early spermatids. The serum levels of inhibin B increase in puberty. In the adult man they are correlated positively to sperm count and testis volume, but negatively to FSH levels. These correlations exist also in OAT syndrome independent of the cause of spermatogenic failure. In azoospermia high serum levels of inhibin B predict the existence of spermatozoa in the testis. This improves the prediction of the success of a TESE. It may be that the determination of inhibin B is superior to that of FSH.

Differences in the antigen pattern recognized by antisperm antibodies in patients with infertility and vasectomy.

PURPOSE: Antisperm antibodies may impair sperm fertilizing capacity. They are found in infertile patients and in men after vasectomy. Little is known to date of the biochemical nature of the antigens that induce the production of antisperm antibodies. MATERIALS AND METHODS: Sperm membrane proteins were prepared from donor spermatozoa, separated by 1-dimensional polyacrylamide gel electrophoresis and exposed to seminal plasma samples of 36 infertile men and 34 after vasectomy containing antisperm antibodies. RESULTS: Ten antigenic protein bands with different molecular weight were recognized by antisperm antibodies. Antisperm antibodies binding to the antigen band at 55 kDa. were significantly more common in infertile men, while those binding to the 72 kDa. band were more common after vasectomy. Significant differences also occurred in the incidence of detecting the 55 kDa. antigen band by the antisperm antibodies of patients with and without varicocele. Comparing antisperm antibodies from patients with or without a history of genital diseases or trauma did not reveal significant differences in the antigens detected. CONCLUSIONS: It seems likely that the development of antisperm antibody binding to different antigens is related to the mode of antibody induction. Since the antigenic properties of spermatozoa change during passage through the epididymis, the antigens detected by antisperm antibodies from men with vasectomy are mostly related to epididymal passage. The identification of human sperm antigens is essential for understanding the mechanism by which antisperm antibodies influence the fertilization capacity of spermatozoa. It is also necessary for the potential development of reliable diagnostic methods for antisperm antibodies that are relevant to infertility.

Influence of antisperm antibodies on the acrosome reaction as determined by flow cytometry.

OBJECTIVE: To evaluate the influence of antisperm antibodies on the acrosome reaction (AR). DESIGN: Clinical study. SETTING: University of Marburg, Department of Andrology, Clinical Training Center of the European Academy of Andrology. PATIENT(S): Spermatozoa from a pool of healthy donors were incubated with 30 seminal plasma samples from infertile men containing antisperm antibodies; they were compared to a control group of 10 samples without antisperm antibodies and five samples with buffer only. INTERVENTION(S): The spontaneous acrosome reaction (SAR) and the induced acrosome reaction (IAR) by calcium ionophore A23187 were observed and determined by means of a flow cytometer. Flow cytometric double-staining estimates of acrosomal integrity were determined by using a monoclonal antibody (TUS 19), marked with a secondary fluorescein isothiocyanate-labeled antibody. Cell viability was determined by counterstaining with propidium iodide (PI). MAIN OUTCOME MEASURE(S): Number of acrosome reacted spermatozoa. RESULT(S): The spermatozoa treated with antisperm antibodies showed significantly higher SAR and IAR responses than the control group. CONCLUSION(S): Some of the antisperm antibodies from individual patients are able to enhance the acrosome reaction in donor sperm, but none of them appeared to inhibit acrosome reaction.

Isolation and identification of sperm membrane antigens recognized by antisperm antibodies, and their possible role in immunological infertility disease.

Antisperm antibodies (ASA) are the main cause of immunological infertility, as they impair sperm function by binding to the sperm membrane. In this study, we isolated highly enriched sperm membrane proteins by two-dimensional (2D) gel electrophoresis. Isoelectric focusing, as a first dimension, was performed on precast DryStrip IPG 4-7. The second dimension was carried out on 12% sodium dodecyl sulphate-polyacrylamide gels. A total of 18 antigens were identified by the subsequent 2D Western blotting using ASA from seminal plasma samples of infertile patients. Six of the recognized proteins were isolated and analysed by means of mass spectrometry and peptide matching. They were identified as heat shock proteins HSP70 and HSP70-2, the disulphide isomerase ER60, the inactive form of caspase-3 and two subunits of the proteasome (component 2 and zeta chain). The biochemical identification of these proteins will be helpful in understanding the mechanisms by which ASA impair both sperm function and fertilization. Thus, these proteins may also be useful in the development of reliable methods for ASA detection.

Why do we determine alpha-glucosidase activity in human semen during infertility work-up?

alpha-Glucosidase is a normal constituent of human semen, produced mainly in the epididymis. It is significantly correlated to sperm count. Its activity is low in cases of epididymal obstruction. We evaluated alpha-glucosidase activity in 653 semen samples of patients, who attended our department for marital infertility, with respect to associations to clinical and other seminal parameters. The normal range (mean +/- 2 SD) in samples with normal parameter values was 7.2-46.4 mU ml-1. The determination in patients with azoospermia revealed mean values of 7.7 +/- 9.5 mU ml-1 in obstructive azoospermia, and 15.8 +/- 11.5 mU ml-1 in nonobstructive azoospermia. The difference was not statistically significant in that the sensitivity of determination with respect to the presence of obstruction was only 0.66, and the specificity 0.83. A significant correlation (r = 0.34) of alpha-glucosidase activity with log sperm count was observed. The mean alpha-glucosidase activity was not significantly different in groups formed according to sperm motility, according to leucocyte count or according to semen volume. A difference between smokers and nonsmokers with comparable sperm count, as reported in the literature, did not occur. We conclude from our results that the determination of alpha-glucosidase activity does not give additional information of the fertility status exceeding that of other clinical investigations or parameters of semen analysis.

The intra- and inter-assay variation of the indirect mixed antiglobulin reaction test: is a quality control suitable?

The test most commonly used to detect sperm antibodies is the mixed antiglobulin reaction (MAR), standardized by the World Health Organization. The indirect MAR test detects soluble sperm antibodies in seminal plasma by using healthy donor spermatozoa as antigen. In this study we systematically investigated the influence of donor spermatozoa and the source of sperm antibodies upon the results of the indirect MAR test, and calculated the intra- and inter-assay variations. Using one individual seminal plasma and the same donor semen, results of the indirect MAR test are highly reproducible (low intra-assay variation). Two dimensions of inter-assay variation must be considered: (i) serial ejaculates of an individual donor may be used at different times; (ii) different donors may be applied to identical antibody sources. Donor spermatozoa strongly influenced the results of the indirect MAR test. Using multivariate statistical tests, highly significant main effects between the different donors (P < 0.001) and specific reciprocal effects between donor spermatozoa and seminal plasma samples (P < 0.001) were observed. The high inter-assay variation of the indirect MAR test will lead to incorrect results. There is urgent need of a reliable and reproducible test for sperm antibody detection to improve quality control of the methods.

Serum levels of inhibin B in men with different causes of spermatogenic failure.

Inhibin B appears to be the physiological feedback signal for FSH. Herein the determination of serum levels of inhibin B, FSH, LH and testosterone in 148 infertile patients and their association with clinical findings and seminal parameters are reported. A significant negative correlation of FSH and inhibin B (r = -0.60) was found. LH levels showed a significant inverse correlation (r = 0.37), but a weak regression (c0 = 0.01). No correlation with testosterone levels occurred. A significantly positive correlation was observed between testis volume and inhibin levels (r = 0.39) as well as between sperm count and inhibin levels (r = 0.39). To evaluate whether the secretion of inhibin B depends on the nature of damage to the Sertoli cells, inhibin levels in 23 patients with varicocele; eight after cryptorchidism, and 16 after hemiorchiectomy were compared to those of other patients without these diseases, but identical sperm count. No significant differences were found. In 20 men undergoing testicular biopsy, inhibin levels were compared to histology. Although the men with Sertoli-cell-only syndrome had significantly lower levels ((15.83 +/- 12.2) pg ml-1) than those with normal spermatogenesis ((183.8 +/- 112.3) pg ml-1), a distinction between patients with hypospermatogenesis from those with normal spermatogenesis was not possible on the basis of inhibin levels. Between these groups, the distinction was better achieved by the FSH levels (sensitivity of 85%). We conclude that inhibin B levels are a serum marker of Sertoli cell function, but the prediction of the quality of spermatogenesis is not superior to that of FSH levels.

The characterization of human spermatozoa membrane proteins--surface antigens and immunological infertility.

The aim of this study was to isolate and characterize highly enriched membrane proteins by two-dimensional (2-D) electrophoresis and to identify surface antigens binding sperm autoantibodies (SpAb). The presence of SpAb may reduce fertility by affecting sperm motility and acrosome reaction. The presence of the SpAb was shown to prevent sperm penetration of cervical mucus, to inhibit sperm-zona pellucida interaction, and to interfere with the sperm-egg fusion. The swim-up method was used to separate mature and motile sperm. Sperm membranes were obtained by hypoosmotic swelling, homogenization and sonication. Membranes were further isolated by differential centrifugation steps. The highly purified human sperm membrane proteins were separated by two-dimensional gel electrophoresis and electrotransferred to polyvinylidene difluoride (PVDF) membrane. The antigens were identified by bound SpAb, the sources of which were seminal plasma samples of infertile patients or of patients following vasectomy. Fourteen surface antigens were detected. Their identification may be (i) important for understanding the mechanism by which SpAb impair sperm fertilization capacity, (ii) suitable as a basis of new methods of fertility regulation, and (iii) helpful in developing reproducible and reliable methods for determinations of SpAb.

DAZ (Deleted in AZoospermia) genes encode proteins located in human late spermatids and in sperm tails.

We analysed the location of proteins encoded by the DAZ (Deleted in AZoospermia) genes in human testis tissue and in mature spermatozoa. The DAZ genes are known to be expressed exclusively in the human male germ line, and are candidate genes for the expression of the azoospermia factor AZFc mapped recently to distal Yq11. They encode testis-specific RNA binding proteins, the function of which is not yet known. Immunostaining experiments with antibodies prepared for the specific peptide domain encoded by the DAZ2 transcript (formerly SPGY1) revealed the presence of DAZ proteins in the innermost layer of the male germ cell epithelium and in the tails of spermatozoa. This suggests a function for DAZ proteins in the RNA metabolism of late spermatids, presumably in the storage or transport of testis-specific mRNA, the translation of which is repressed until the formation of mature spermatozoa. Deletion of DAZ genes is supposed not to interfere with human sperm maturation but to result in a gradual reduction of mature spermatozoa.