[Experience in the treatment of autoimmune male infertility in patients with category 4 chronic prostatitis].

Administration of longidaza at a dose of 3000 IU intramuscularly twice a week after 1 month of treatment leads to the statistically significant reduction of antisperm antibodies (ASAB) at the surface of sperm cells to 23% (7-48%) for MARIgG and to 14.5% (3-34%) for MARIgA. Maximum reduction of ASAB, however, was observed after third month of treatment, mean MARIgG was 2% (1-26,5%) and MARIgA - 1% (0-11.5%). ASAB level has not reduced less than 50% only in one patient (1.67%). At follow-up three months after the cancellation of Longidaza, only 17 (28.33%) men showed an increase of ASAB IgG and (or) IgA more than 50%. Inthe study group, during the observation, spontaneous pregnancy occurred in 6 (10%) pairs, and IVF was successfully performed in 3 (5%) pairs. Thus, we consider it necessary to appoint Longidaza in patients with category 4 chronic prostatitis and elevated levels of antisperm antibodies on sperm cells, who preparing for assisted reproductive technologies, or preparing for natural pregnancy, as a high-effective pathogenetical agent for the treatment of autoimmune infertility.

Co-expression of two distinct polysialic acids, α2,8- and α2,9-linked polymers of N-acetylneuraminic acid, in distinct glycoproteins and glycolipids in sea urchin sperm.

Naturally occurring polysialic acid (polySia) structures have a large diversity, primarily arising from the diversity in the sialic acid components as well as in the intersialyl linkages. In 2004, we demonstrated the presence of a new type of polySia, 8-O-sulfated N-acetylneuraminic acid (Neu5Ac) capped α2,9-linked polyNeu5Ac, on the O-glycans of a major 40-80 kDa sialoglycoprotein, flagellasialin, in sea urchin sperm. In this study, we demonstrated that another type of polySia, the α2,8-linked polyNeu5Ac, exclusively occurs on O-glycans of a 190 kDa glycoprotein (190 kDa-gp), whereas the α2,9-linked polyNeu5Ac is exclusively present on flagellasialin. The 190 kDa-gp is localized in both flagellum and head of sperm. We also demonstrated that polysialogangliosides containing the α2,8-linked polyNeu5Ac are present in sperm head. Thus, this study shows two novel features of the occurrence of polySia in nature, the co-localization of polySia with different intersialyl linkages, the α2,8- and α2,9-linkages, in a single cell and the occurrence of α2,8-linked polyNeu5Ac in glycolipids. Anti-α2,8-linked polyNeu5Ac antibody had no effect on fertilization, which contrasted with the previous results that anti-α2,9-linked polyNeu5Ac antibody inhibited sperm motility and fertilization. Based on these properties, distinct functions of α2,8- and α2,9-polySia structures are implicated in fertilization.

[The diagnosis of an ABO(H) ABSe group in the traces of saliva, sperm and vaginal secretion using mixed agglutination reaction].

Experimental spots of the saliva, sperm, vaginal secretion from persons with groups Ase, Bse, ABSe and Abse were studied with mixed agglutination reaction (MAR) using hemagglutinating sera anti-A and anti-B (heteroimmune, isoimmune), monoclonal antibodies. MAR with monoclonal antibodies was able to diagnose ABSe group not only in the spots of saliva, but also in the spots of sperm and vaginal secretion where heteroimmune sera failed.

Immunological evidence for the localization of sialoglycosphingolipids at the cell surface of sea urchin spermatozoa.

The localization of sialoglycosphingolipids in the plasma membrane of sea urchin spermatozoa was studied by employing immunological methods including immunolysis of liposomal model membranes. The antibodies produced against these complex lipids were found to agglutinate various sea urchin spermatozoa differently. Both species differences and species similarities in the agglutination were found in spermatozoa of the echinoderm, the sea urchin and the starfish. The agglutination of the sea urchin spermatozoa was inhibited specifically by ceratain carbohydrates. Only a limited number of molecular species of sialoglycosphingolipid were localized at the surface of the plasma membrane of sea urchin spermatozoa cells. Moreover, topographical differences were found in the localization of the sialoglycosphingolipids at the cell surface of spermatozoa.

Sensitization to inorganic mercury could be a risk factor for infertility.

INTRODUCTION: Heavy metals can negatively influence the reproduction due to the fact that they are able to impair the immune reactions including autoantibody production in susceptible individuals. In such a way the infertility could be also caused by altered pathologic immune reaction. AIM OF THE STUDY: To investigate the in vitro lymphocyte reaction after stimulation with metals and production of gamma interferon and antisperm antibodies in supernatants after lymphocyte stimulation in patients with infertility and with proven antisperm antibodies in their serum. The cause of antisperm antibodies presence was not determined. METHODS: The diagnosis of metal allergy was performed by the lymphocyte proliferation method modified for metals (MELISA) in supernatants of tissue cultures of lymphocytes without the antigen stimulation and after stimulation with mercury chloride, the in vitro production of gamma interferon and antisperm antibodies was studied by ELISA. RESULTS: More than 50% of patients were reacting to mercury, iron, aluminium and silver as mean by lymphocyte reactivity. When compared the lymphocyte reaction in patients with and without mercury allergy we found that the lymphocytes of patients with mercury intolerance produced less gamma interferon and more antisperm antibodies in supernatants after mercury stimulation of their lymphocytes. CONCLUSION: In patients with metal intolerance diagnosed by the MELISA test the release of metal ions from dental materials can be one of the stimulating factors which may adversely affect fertility.

Autoantibodies against spermatozoal antigens detected by immunoblotting and agglutination. A longitudinal study of vasectomized males.

Sera taken pre- and post-operatively at regular intervals within a year from 16 men undergoing vasectomy were analysed for autoantibodies against spermatozoal proteins by immunoblotting. The reaction patterns were compared with the results of sperm agglutination tests. Immunoblotting revealed the presence of autoantibodies against various spermatozoal polypeptides in all sera taken pre-operatively and post-operatively. On average, seven polypeptides showed reaction. During the post-operative period two patients developed spermatozoal agglutinins in moderate titers (greater than 16) but in immunoblotting no change in band reactivity was observed for these two patients. However, scanning of the immunoblotting results revealed that one of the patients, although without sperm agglutinins, during the post-operative period showed an increasing band colouring of a polypeptide of Mr 31,500, reflecting an increased level of the corresponding antibodies.

PIP: Sera taken at the postoperative intervals of 10-21 days, 1-2 months, 3-5 months, 9-12 months from 16 men undergoing vasectomy were analyzed for autoantibodies against spermatozoal proteins by immunoblotting. After recording the natural pattern of antibody binding, qualitative and quantitative changes were identified and related to the sperm agglutinins. On average, an observation time of 6 months was obtained. All the men had been proven fertile prior to surgery and had azoospermia after surgery. Sera also were taken from 7 normal controls with at least 3 month intervals during 1 year to determine whether changes of the spectrum of the antibody specificities occurred. A figure shows an immunoblotting analysis of IgG autoantibodies to SDS-PAGE separate spermatozoal antigens with 5 serum samples from 1 person taken preoperatively and 1, 2.5, 3, and 8 months after vasectomy. Even prior to the operation at a time when the patient was fertile, an IgG binding was demonstrated. This observation held true for all 16 vasectomized males and the group of 7 controls. The staining pattern of the consecutive serum samples was constant. Only 2 patients reacted after vasectomy with a moderate increase in antibody titre (greater than 16), but neither of these patients, nor the rest of the group, developed new bands in immunoblotting for IgG. The immunoblotting analysis for IgM antibodies in sera from the 2 men with sperm agglutinins did not show variation with time. The 7 control men did not show any qualitative or quantitative changes in their IgG reaction pattern during the observation period nor did they reveal the presence of any autoantibodies in the agglutination tests. Scanning of the immunoblotting results revealed that 1 of the patients, although without sperm agglutinins, showed an increasing band coloring of a polypeptide of Mr, 31,500 during the postoperative period, reflecting an increased level of the corresponding antibodies.

Localization of human seminal plasma No. 7 antigen (Ferrisplan) in accessory glands of male genital tract and spermatozoa.

The establishment of a hybridoma (1C4) producing sperm immobilizing monoclonal antibody to human seminal plasma No. 7 antigen (HSP No. 7 Ag.) and the isolation of the pure antigen by immunoaffinity chromatography bound monoclonal antibodies have been reported previously. In the present investigation, HSP No. 7 Ag. has been termed 'Ferrisplan' and its distribution in male genital organs, spermatozoa and body fluids has been studied. The amount of Ferrisplan in the body fluids was determined by radioimmunoassay. Large amounts were detected in seminal plasma and milk, trace amounts in saliva, and none in the serum and urine. The concentration of Ferrisplan was highest in the seminal plasma of azoospermic patients and gradually decreased from oligospermia to normospermia. Using an immunofluorescent method with anti-Ferrisplan monoclonal antibody, strong staining was observed on the epithelial layers of human seminal vesicles, no staining on testes and bright staining on the post-nuclear cap and mid-piece segment of spermatozoa. These results indicate that Ferrisplan is excreted mainly from the seminal vesicle and adheres to the post-nuclear cap and mid-piece of the spermatozoa as a sperm-coating antigen.

Blot-immunobinding test for the detection of anti-sperm antibodies.

A blot-immunobinding test was used to detect anti-sperm antibodies in human sera and to identify the corresponding auto- or iso-antigens on human sperm. A high proportion of sera at a 1:100 dilution from fertile persons, as well as infertile patients, contains antibodies reactive with sperm. This phenomenon might be physiological. At 1:2,000 dilution, a higher binding capacity was detected in the sera from infertile groups, but a few fertile persons were also positive. Antibodies to a single antigenic determinant with Mr of approximately 14,000 were found in a significantly higher proportion among males with unexplained infertility.

SpermCheck: a simplified screening assay for immunological infertility.

SpermCheck (Bio-Rad Laboratories, Hercules, CA), a new screening test for regional surface antibodies on motile sperm, uses monodispersed latex microspheres of uniform size as a vehicle to link rabbit antihuman immunoglobulins (IgA, IgG, IgM) and provides both negative and positive control sera, as well as sufficient buffer for sperm preparation in ambient CO2 atmosphere. When compared with reference data available for the immunobead test (IBT), the direct protocol (semen) for SpermCheck yielded 94.4% sensitivity with 100% specificity; the indirect protocol (serum) provided a sensitivity of 100% with 94.7% specificity. The microspheres of SpermCheck maintain a nearly uniform concentration per volume, with none to negligible clumping. The greater difference between the optical densities of latex and cytoplasm allows use of a light microscope for the rapid assessment of the percent of regional binding rather than the phase-contrast microscope required for the IBT. SpermCheck eliminates many difficulties encountered with the IBT, making SpermCheck a convenient screening assay for use in the physician's office.

Evaluation of polyacrylamide gel as substitute for human cervical mucus in the sperm penetration test.

OBJECTIVE: To compare polyacrylamide gel as synthetic medium with human cervical mucus (CM) for the in vitro sperm-penetration test during infertility investigation. PATIENTS: One hundred sixty-nine randomly chosen couples with a median duration of infertility of 4 (range, 1 to 16) years presenting at the infertility unit of the Women's University Hospital of Heidelberg, Germany. MAIN OUTCOME MEASURES: Evaluation of sperm migration in polyacrylamide gel used in four different concentrations (1.5%, 1.6%, 1.7%, 1.8%) in the capillary tube test in parallel with CM of patients' female partners and CM of fertile donors, obtained under standardized conditions. Correlation of migration test results with outcome of semen analysis including microbial cultures and testing for local antisperm antibodies by means of the mixed antiglobulin reaction, postcoital testing, and the subsequent pregnancy rate after control for female infertility factors in a prospective study. RESULTS: Sperm ability to penetrate the synthetic medium (concerning all concentrations) correlated significantly with the penetration of human CM, although polyacrylamide proved to be a stronger barrier. Sperm velocity and duration of progressive motility were markedly reduced in polyacrylamide. Polyacrylamide results correlated with the outcome of standard sperm analyses but not with sperm antibody testing. No clear differentiation was obtained with regard to subsequent fertility (19% after 6 months), although adequate sperm migration in polyacrylamide 1.8% was significantly more frequent in the fertile group. CONCLUSIONS: In analyzing the intrinsic motility, penetration testing with polyacrylamide gel provides important information not obtained by routine sperm analysis. However, particularly with regard to immunological factors and fertility prognosis, human CM should be preferred whenever possible.

The effect of fixatives and/or air-drying on the plasma and acrosomal membranes of human sperm.

It has been hypothesized that some fixatives and conditions of slide preparation expose internal sperm antigens and thus are not suitable for demonstrating surface-specific antigens by immunocytochemical assays. This study examined the ability of five fixatives (glutaraldehyde, acetone, methanol, paraformaldehyde, and periodate-lysine-paraformaldehyde [PLP]) and two conditions of slide preparation (air-drying or maintaining sperm in a liquid phase) to maintain the integrity of human spermatozoal membranes at the ultrastructural level as monitored by transmission electron microscopy. Regardless of the fixative employed, air-drying was detrimental to plasma and acrosomal membrane integrity. Acetone and methanol completely disrupted the plasma and acrosomal membranes over the entire sperm surface whether air-drying or liquid phase conditions were employed. Fixation with glutaraldehyde and paraformaldehyde (to a lesser degree) maintained the morphologic integrity of acrosomal and plasma membranes provided the sperm were maintained in a liquid phase. However, glutaraldehyde and paraformaldehyde fixation increased the postfixation binding of immunoglobulins to the sperm surface. We concluded that immunocytochemistry at the light microscopy level may imply erroneous interpretations regarding antigen/antibody interactions on the sperm plasma membrane surface unless care is taken to verify that the antibodies of interest are binding to the antigenically intact plasma or acrosomal membrane.

Clinical experience with an artificial spermatocele.

Artificial spermatoceles were implanted into three patients with congenital absence of the vas. In each case, the testicular biopsy demonstrated normal spermatogenesis, and the dilated epididymal tubule was packed with spermatozoa. The ciliated epididymal mucosa appeared normal despite the tubular dilatation. The spermatoceles were constructed of expanded polytetrafluoroethylene, and they were microsurgically implanted over the cut end of the epididymis. The grafts were aspirated monthly for up to six months, and the aspirates containing spermatozoa were used for artificial insemination. Spermatozoa were consistently retrieved from each patient, but no pregnancies have resulted. The most obvious finding was that the spermatozoa lacked motility. In the discussion, other problems related to artificial spermatoceles are reviewed, including epididymal development and sperm maturation, aspiration techniques, and sperm storage.

The immunoglobulin class of antispermatozoal antibodies in serum.

The aim of this study was to determine the immunoglobulin class of circulating antisperm antibody using a technique called the indirect immunobead test (IBT). In the IBT sperm bound antibodies are detected using polyacrylamide beads coated with rabbit antihuman immunoglobulin classes IgG, IgA, and IgM. Of the 20 infertile men with serum immobilizins, 100% were found to be positive for sperm-bound IgG, 50% positive for IgA, and 0% positive for IgM, using the IBT. Similarly, 20 infertile females with serum immobilizins showed 95% positivity for IgG, 60% for IgA, and 15% for IgM. Thus there was a good correspondence between the presence of serum immobilizins as determined by the sperm immobilization test (SIT) and the IBT. This study provides data that indicates that IgG and IgA are the two major immunoglobulin classes of sperm antibody in male and female immune sera as detected by a simple, sensitive immunological technique, the serum IBT.

Reversible immunosuppression of fertility in the rat following immunization by a liposome incorporated spermatozoal polypeptide fraction.

A rat spermatozoal polypeptide with 40 amino acid residues and an N-terminal amino acid residue of Lys was purified to homogeneity by recycling high pressure liquid chromatography. Following incorporation of the peptide into pure phosphatidylcholine liposomes and immunization of female Wistar rats, a significant reduction in fertility rate occurred (chi 2 = 6.4, p less than 0.01). Following a 4-month recovery period after immunization, fertility rates returned to normal. No adverse effects of the immunization on vital organs could be detected.

[Sperm antibodies and immunologic intolerance to metals in infertile couples]. / Protilátky proti spermiím a imunologická intolerance nekterých kovu u neplodných páru.

OBJECTIVE: Verification of the hypothesis of a relationship between the presence of antibodies against sperm cells and immunological reactivity to some metals in infertile couples by the MELISA test. SETTING: Department of Obstetrics and Gynecology, Medical Faculty, Charles University and Faculty Hospital, Plzen. METHOD: From 23 female patients and 21 men (a total of 44 subjects treated for infertility) with confirmed serum antibodies against sperm cells the authors isolated lymphocytyes from the peripheral blood stream, divided them into individual cultures and investigated them by the MELISA test using different metal compounds. RESULTS: The outcome of the MELISA test are values of the stimulation index (SI) by means of which the authors investigated the reactivity of the organism to the given metal. Special attention was devoted to compounds of organic and inorganic mercury. The SI values were subsequently compared with different data obtained from a detailed anamnestic questionnaire which was focused specially on contact with metals and on allergic reactions. In the investigated group of patients the authors detected a positive immune reactivity to inorganic mercury, Ag, Al, Fe. In some subjects they found a very high positive immune reactivity to inorganic mercury, Ni, Al, Cd and Ti. The control groups were formed by healthy fertile subjects without antibodies against sperm cells and with physiological SI values. CONCLUSION: The authors did not prove a direct relationship between the intensity of the laboratory reactivity to metals and the presence of antibodies against sperm cells which cause deterioration of fertility. An exogenous load of metals could in case of genetic predisposition be only one of the factors which participate in the formation of antibodies against sperm cells. The investigation proved that its is not essential, contrary to the view of many stomatologists, to eliminate metal compounds completely from dental practice.

[Effect of heavy metals on immune reactions in patients with infertility]. / Vliv tezkých kovu na imunitní reakci u pacientu s prokázanou neplodností.

BACKGROUND: Heavy metals can negatively influence reproduction because in sensitive persons they are able to alter the immune reactions including autoantibodies production. The altered immune reaction can then cause infertility. METHODS AND RESULTS: The in vitro lymphocyte reaction after stimulation with metals, the production of interferon (IFN-gamma) and antisperm antibodies in supernatants after lymphocyte stimulation in patients with infertility and with the antisperm antibodies present in their serum were investigated. The cause of antisperm antibodies presence was not determined. The diagnosis of metal intolerance was performed by the proliferation method modified for metals (Melisa). In supernatants of tissue cultures of lymphocytes without the antigen stimulation and after stimulation with mercury chloride, the in vitro production of gamma interferon and antisperm antibodies was studied by Elisa. More than 50% of patients did not tolerate mercury, iron, aluminium and silver. When the lymphocyte reaction was compared in patients with and without mercury intolerance we found that lymphocytes of patients with mercury intolerance produced less gamma interferon and more antisperm antibodies in supernatants after mercury stimulation of lymphocytes. CONCLUSIONS: In patients with metal intolerance diagnosed by the Melisa test, metal ions released from the dental materials can represent a factor, that does not cause infertility but is able to influence it negatively.

[The possibility of detecting soluble HLA antigens in traces of blood and excretions]. / Vozmozhnost' vyiavleniia rastvorimykh antigenov sistemy HLA v sledakh krovi i vydelenii.

A modified lymphocytolysis inhibition test revealed that specific results may be attained in 50-60% of examinations of blood, spermatic, and salivary spots if highly specific HLA sera are used. HLA antigens were found to be present in spermatic and salivary spots for a year (follow-up period). Recommendations were developed for practical use of HLA antigens in examinations of blood, spermatic, and salivary spots by lymphocytolysis inhibition test in forensic medical expert evaluation.

[The possibilities for using anti-A, anti-B and anti-H immunoreagents obtained by new technology in direct and indirect immunofluorescence reactions]. / Vozmozhnosti ispol'zovaniia immunoreagentov anti-A, anti-B i anti-H, poluchennykh po novoi tekhnologii, v reaktsiiakh priamoi i nepriamoi immunofliuorestsentsii.

The authors propose to prepare group-specific immunoreagents anti-A, anti-B, and anti-H for direct and indirect immunofluorescence from non-precipitated antisera of the appropriate specificities by a new technology making use of routine bioadsorbents and synthetic oligosaccharides. The method has been developed at Research Center of Forensic Medical Expert Evaluations of the Russian Ministry of Health. The immunoreagents were tried on a vast scope of experimental and expert specimens at several institutions.

[The investigation of blood and secretion stains by the absorption-elution reaction using anti-H monoclonal antibodies]. / Issledovanie piaten krovi i vydelenii reaktsiei absorbtsii--éliutsii s pomoshch'iu monoklonal'nykh antitel anti-H.

Possible use of monoclonal antibodies anti-H in absorption-elution reaction was studied. Blood and secretion stains on gauze were analysed. Practical usefulness of monoclonal antibodies anti-H for investigation of human blood and secretions was stated. Differences in interaction of monoclonal antibodies with traces of different origin were found.

[The use of immunoenzyme analysis for determining ABH antigens in traces of saliva and sperm]. / Primenenie immunofermentnogo analiza dlia opredeleniia ABH-antigenov v sledakh sliuny i spermy.

The authors suggest a simple sensitive technique for enzyme immunoassay (EIA) of ABH antigens in saliva and semen. A two-staged dot blot solid-phase EIA on nitrocellulose membranes was employed with anti-ABH monoclonal antibodies obtained in immunization of mice with human red cells. 4-chloro-1-naphthol substrate solution was used to visualize the peroxidase label. The results of analysis of salivary and spermatic samples obtained from donors of various groups evidence that this EIA variant may be useful in forensic medicine.