Monoclonal antibodies to porcine zona pellucida that block the initial stages of fertilization.

Zona pellucida (ZP) is thought to be of utmost biological importance in the early stages of fertilization and implantation. Current hybridoma technology was used to produce monoclonal antibodies (MAbs) against specific antigens to porcine ZP. Two monoclonal antibodies (4F2 and 2D9) were raised that reacted against ZP antigens shared by human and porcine ZP. These antibodies were shown to block fertilization of human oocytes in in vitro fertilization (IVF). It is likely that MAb 4F2 recognized a protein epitope localized on the outer surface of ZP. These antibodies may be quite useful immunologic probes for studying the precise mechanisms of the early events of fertilization in mammals.

Common egg envelope antigens are limited to the animal class.

The antigens of the egg envelope (zona pellucida) in mammals are of special interest because of their possible involvement in immunoinfertility and as candidate targets for immunocontraception. Conserved zona epitopes from divergent species may present a suitable source and an animal model for investigation of the above factors. We compared egg envelope antigens from 6 species of vertebrates belonging to 3 different classes in order to demonstrate the existence of shared antigens. Egg envelopes from the trout, carp, turtle, hen, duck and quail were isolated and heat-solubilized. They were tested with rabbit polyclonal antisera against carp, trout and duck egg envelopes by the enzyme-linked immunosorbent assay. The results showed significant cross-reactions among egg envelopes of fish and birds. The examined solubilized preparations did not show cross-reactivity with egg envelopes from any other class, suggesting that divergent species did not share common egg envelope antigens, and that their use may not be appropriate in the investigation of immunoinfertility and immunocontraception in humans.

A novel approach to the generation of antibodies against phylogenetically preserved sperm antigens.

Conventional methods for immunization of laboratory animals against human spermatozoa proved not to be efficient enough to identify phylogenetically conserved sperm-specific antigens. A combination of vasectomy and subcutaneous administration of autologous testis homogenates was tested in 5 New-Zealand rabbits, and in 7 Long-Evans and 8 Spraque-Dawley rats in an attempt to induce an autoimmune response against such antigens. This experimental procedure resulted in a generation of sperm autoantibodies cross-reactive with human, rabbit and rat spermatozoa, as demonstrated by sperm-agglutination, ELISA and flow cytometry (FCM). No specific binding to human seminal plasma was detected by ELISA, indicating that intrinsic sperm membrane antigens rather than sperm-coating antigens were involved in establishing cross-reactivity with human spermatozoa. This suggestion was confirmed by the finding that rabbit autoantisera reacted more strongly against epididymal than against ejaculated human spermatozoa as shown by FCM. Humoral antispermatozoal response correlated well with impaired spermatogenesis in rabbits. The autoimmunized rats revealed severe alterations in reproductive tissues, including testicular and epididymal sperm granulomas; however, they showed a lower incidence of circulating antibodies. The results indicate that the established experimental model in rabbits can be further used to identify and characterize evolutionary preserved intrinsic sperm membrane autoantigens, which are desirable candidates for contraceptive vaccine development.

[Therapeutic approaches to the treatment of infertility associated with antisperm antibodies production]. / Terapevtichni podkhodi pri bezplodieto, cvurzano s produktsiia na spermoantitela.

A lot of extensive investigations have been conducted to evaluate the potential role of antisperm antibodies (ASA) in infertile couples. But many questions still remain on the role of ASA in reproduction, testing methods, significant levels of ASA and treatment of immunologically-mediated human infertility, due to ASA. The aim of the present critical review was to the analyze the literature data, published in the last 20 years, concerning the most frequently used in the international centers methods for treatment of human immunological infertility associated with ASA production.

[The use of SIT and ELISA to detect serum sperm antibodies in infertile patients]. / Prilozhenie na SIT i ELISA za otkrivane na serumni spermoantitela u bezplodni patsienti.

The existence of natural sperm antibodies in fertile subjects requires the application of additional methods in certain cases of significant, but near to the borderline agglutinating titers, in order to distinguish between pathological and natural sperm antibodies. We applied the sperm immobilization test (SIT) of Isojima and an enzyme-linked immunosorbent assay (ELISA) with sera from infertile patients (n = 66) that have been positive in at least one of the agglutination methods of Kibrick and Friberg. The data were compared with the results for a control group of blood donors (n = 50). It was demonstrated that the positive, but low-titer sera in the test of Friberg were often highly positive in ELISA, while the high-titer sera displayed lower OD values. No such correlation was established by SIT. Probably, it would be practical to use ELISA in cases, where no convincing laboratory data exists for immunity against spermatozoa--relevant, but low agglutinating titers, which can be found in fertile subjects. Besides, SIT and ELISA can recognize antigen-antibody systems different from those identified by the agglutinating tests.

[Methods for the detection of antisperm antibodies associated with immunologically-mediated human infertility]. / Metodi za dokazvane na spermoantitelata, svurzani s imunologichno obuslovenoto bezplodie u choveka.

Among others, immunological factors can be the reason for human infertility. The aim of the present critical review was to the analyze the literature data, published in the last 20 years, as well as our experimental and clinical results concerning the most frequently used in the international centers methods for the detection of antisperm antibodies associated with immunologically--mediated human infertility.

[Frequency of anti-sperm antibodies demonstrated by classical tests for sperm antibodies in patients with unexplained infertility]. / Chestota spermoantitelata dokazvani chrez klasicheskite testove za spermoantitela u patsienti s neiziasnena etiologiia na bezplodieto.

The aim of the present investigation was to establish the frequency of sperm antibodies in patients with etiologically unexplained infertility, and to compare the demonstrated frequencies with the results from y control group of y healthy blood donors, as well as with the results of other investigators. The gelatin agglutination test of Kibrick and the tray agglutination test of Friberg were applied to test 244 sera from infertile patients and 50 sera from healthy blood donors at the Laboratory of Immunology of Reproduction, Department of Biology, Medical University of Sofia. For the infertile patients, relevant sperm antibody titers were demonstrated in 2.5% (titer > or = 16) for the Kibrick method, and in 7% (titer > or = 32) for the Fiberg method. The test of Kibrick did not reveal significant antibody titers in the healthy controls, while the test of Friberg showed sperm antibodies in 2% of the blood donors. Fisher's exact test demonstrated extremely significant correlation (p < 0.0001) between the presence of sperm antibodies in sers of patients with unexplained infertility as revealed by the tests of Kibrick and Friberg. Most often mixed agglutinates were demonstrated in the Friberg test. In contrast with the results of other investigators head-to-head agglutinins were observed more often in male sera, while tail-to-tail agglutinins--in female sera. Finally, the results from the present investigation, as well as the analyzed literature data showed a low frequency of anti-sperm immunity in the Bulgarian population. The established high degree of correlation between the tests of Kibrick and Friberg, the good reproducibility of the results and the low cost of these methods confirm their appropriate use for the diagnosis of sperm antibodies in patients with unexplained infertility.

[The role of antisperm antibodies in the induction of immunologically-mediated human infertility]. / Roliata na spermoantitelata v induktsiraneto na imunologichno obusloveno bezplodie pri choveka.

Among others, immonulogical factors can be the reason for human infertility. Our critical review of English-Language literature published between 1979 and May 2001, as well as the analyzed literature data for the Bulgarian population, showed sufficient evidence that antisperm antibodies impair fertility in couple with unexplained infertility by different pathogenetic mechanisms.

Serum sperm antibodies after diarrhoeal diseases.

The origin of 'natural' anti-sperm antibodies found in fertile humans, virgin girls, and boys before puberty, is quite obscure. One hypothetical mechanism relates their existence to inflammatory gastrointestinal entities: as a result of the disease, cross-reactive antibodies produced against gastrointestinal flora bind spermatozoa. To test this assumption, we evaluated the level of serum sperm antibodies after diarrhoeal infections. Serum samples from 17 patients with shigellosis and 12 patients with salmonellosis were screened for anti-sperm antibodies directed against sperm surface antigens (gelatin agglutination test - GAT, tray agglutination test - TAT, sperm immobilization test - SIT), profound sperm antigens [enzyme-linked immunosorbent assay (ELISA)], and anti-bacterial antibodies (slide agglutination test - SAT) upon diagnosis (group A) and 4-35 days later (group B). The patients from group B demonstrated an increased sperm antibody incidence by GAT (20.7%), TAT (13.8%) and ELISA (31%) when compared to group A and to healthy controls, although statistically significant data were acquired only for the latter group. The absorption of positive sera with bacteria and/or spermatozoa revealed significant reactivity changes in the antibody values by GAT and TAT for shigellosis, and by TAT and ELISA for salmonellosis patients. These data demonstrate increased serum sperm antibody levels in salmonellosis and shigellosis patients.

[Not Available]. / [The teaching of biology at the Medical Faculty in Sofia]

The teaching of Biology at the Medical Faculty in Sofia started in 1918 with Botany and Zoology. Already in the next year, it was radically changed by Metodii Popov: 1. Instead of Botany he introduced General Biology, and instead of Zoology - Parasitology (including a general review of the evolution of non-vertebrate animals) and Comparative Anatomy of the Vertebrates; 2. He adapted the teaching of Biology to the needs of the medical education. Those changes were possible thanks to the considerable medical background of M. Popov - it started in 1911 with suitable specialization and research activities, and continued with the establishment of Department of Biology and the Medical Faculty, and the involvement of medics, besides biologists, in its academic staff. During the past years there have been a lot of changes in the curriculum both in its schedule and contents. Some of them were as a result of the development of the biological science and the integration with the other disciples, but some were forced by the administration. Today the students have 90 hours of lectures and a practical course of 90 hours as well. They have at their disposal textbooks on "Biology", "Parasitology" and "Comparative Anatomy of the Vertebrates", as well as, a "Textbook for the practical Course on Biology". Their knowledge is evaluated during the practical course, at two colloquia, and at a practical and theoretical (oral) exam at the end of the first year of education.

The use of ELISA to evaluate human antibody binding to epididymal sperm from different species.

PROBLEM: The impact of antibodies to epididymal sperm antigens in human infertility has been poorly understood. Cross-reactivity of human antibodies with animal epididymal sperm has been previously observed, however, only by means of qualitative methods. Moreover, it has been always compared to reactivity against human ejaculated rather than human epididymal sperm. METHOD: Following a screening study of 940 infertility patients, sperm agglutinating and immobilizing sera as well as sperm antibody negative controls were used to standardize an ELISA employing human ejaculated sperm. Nine sera positive in ELISA were further tested against epididymal human, guinea pig, rat, and hamster sperm. Differences among groups were evaluated by factorial analysis of variance (ANOVA). RESULTS: The specificity and sensitivity of ELISA were shown to be 85.1% and 61.18%, respectively. Eight out of nine antisperm antibody-positive sera from infertile subjects reacted relatively stronger with epididymal than with ejaculated human sperm. All tested infertility sera showed strong although variable cross-reactivity with sperm from guinea pig, hamster, and rat. CONCLUSION: ELISA has definite potential in sperm antibody research, allowing quantitative assessment of the results and immotile sperm employment. The suggested predominant role of epididymal sperm antigens in immune responses related to fertility needs further investigation. Some of these antigens are obviously phylogenetically preserved, and possibly in a quantitative aspect present differently on epididymal spermatozoa from various mammalian species.

Structure of the plasmalemma of human spermatozoa after treatment with antispermatozoa serum and concanavalin A.

Human ejaculated spermatozoa were treated with antispermatozoa serum as well as with the same serum and concanavalin. A. At both experimental conditions clusters of intramembranous particles both on P-face and on E-face in the acrosomal and postacrosomal regions were seen. The clusters were with different form and size and were built up from different number of intramembranous particles. Large areas with single particles or in some cases devoid of these particles were found out.

Soybean agglutinin-binding sites on the human sperm surface.

Some differences in the distribution of lectin-binding sites on the surface of the plasma membrane of acrosomal and postacrosomal regions and tail of ejaculated human spermatozoa were demonstrated by means of horseradish peroxidase labeled soybean agglutinin. The acrosomal region was more densely coated with labeled lectin in comparison with the postacrosomal region. Hapten inhibition with N-acetyl-D-galactosamine was used as control.

Detection of antibodies toward epididymal sperm antigens--an obligatory step in evaluation of human immunologic infertility?

PROBLEM: To test the relative impact of epididymal versus ejaculated sperm in immunologic infertility. METHOD: Human antibody binding to epididymal and ejaculated spermatozoa was compared by flow cytometry (FCM) since it allows quantitative analysis of viable sperm while ignoring nonsperm cells. To select sera for FCM, GAT, TAT, and ELISA were applied on 145 sera from fertile men, idiopathically infertile and varicocele patients. RESULTS: All GAT/TAT-positive infertile patients, a representative group of varicocele patients and the fertile control, were assessed by FCM. Higher reactivity toward epididymal sperm revealed 18/22 sera while only four out of them bound to ejaculated sperm stronger than the control. All varicocele sera were positive against epididymal while negative against ejaculated spermatozoa. CONCLUSIONS: Epididymal sperm antigens may play a predominant role in some cases of immunologic infertility. Such patients might not be adequately diagnosed and respectively treated due to the limitations of diagnostic procedures applying only ejaculated spermatozoa.

Sperm, nuclear, phospholipid, and red blood cell antibodies and isotype RF in infertile couples and patients with autoimmune rheumatic diseases.

PROBLEM: To determine if measuring of nonorgan-specific autoantibodies is useful for better understanding and management of unexplained infertility. METHODS: Sera were obtained from 70 infertile couples, 57 rheumatic patients, and 76 fertile donors. Sperm antibodies (SA) were detected by the tests of Kibrick and Friberg, anti-histones, anti-cardiolipin antibodies, and RF isotypes by ELISA, antinuclear antibodies by indirect immunofluorescence, and anti-red blood cell antibodies by Capture-R. RESULTS: Multiple autoimmune reactivity (both partners positive and/or more than one type of autoantibody involved), higher than naturally occurring in fertile individuals, was found in 55% of the idiopathically infertile couples. IgA-RF was the dominant autoimmune marker. SA revealed similar rates in patients with rheumatic diseases and in infertiles with or without other autoantibodies. CONCLUSION: Although no single autoimmunity marker could predict occurrence of SA, the coincidence of enhanced polyclonal autoimmunity in both partners of infertile couples might potentiate their negative effect on reproduction.