Production of Stem Cell Antigen 1 Sca-1/Ly-6A/E by Freshly Isolated NK Cells Cultured with Relevant Cytokines.

Activated NK cells in appropriate conditions are known to express stem cell antigen 1 (Sca-1/Ly-6A/E). To investigate its production, NK cells isolated from mouse spleens were incubated ex vivo in the presence of different combinations of cytokines (IL-12, IL-15, IL-18, and IFNγ). Expression of Sca-1 was found to be considerably higher in NK cells incubated in the presence of IL-18, IL-15, and IL-12 than in those treated with IL-15 and IL-18 only. To test the hypothesis that the effect of IL-12 was due to stimulation of IFNγ production, we replaced IL-12 with IFNγ in some samples and added specific anti-IFNγ antibody to some samples cultured with IL-15/IL-18+IL-12. In the subpopulations incubated in the presence of IL-15/IL-18 with added IFNγ instead of IL-12, the expression of Sca-1 was not increased. By contrast, in samples treated with IL-15/IL-18+IL-12 and anti-IFNγ antibody, the expression of Sca-1 was activated to a similar extent as in those stimulated by IL-15/IL-18+IL-12 combination without the antibody. The obtained data suggest that IL-12 activates the production of Sca-1 by NK cells through an IFNγ-independent mechanism.

[Antibodies Against Immunologically Sequestered Antigens in Patients with Polycystic Ovary Syndrome (PCOS).]

Polycystic ovary syndrome (PCOS) is a leading cause of anovulatory infertility. In recent times, autoimmune processes are supposed to play a role in a number of conditions with unexplained etiology. This includes some cases of human infertility. The attempts to detect antiovarian antibodies in patients with PCOS generally produced negative results. Presence of non-organ specific autoantibodies in patients with polycytosis has been reported, but the data are till inconsistent. Meanwhile, a number of recent sludies reveal a correlation between PCOS, hypothyroidism (manifested or subclinical) and autoimmunity. In this respect, many authors recommend thyroid function to be regularly checked in patients with PCOS. There are data that certain categories of women with polycystosis show an elevated concentration of antisperm antibodies. The problem is an interesting example how a pathological process can elucidate the complex interactions between the immune system and various compartments of the endocrine system that include immunologically isolated antigens.

[Genomic study in patients with idiopathic azoospermia and oligoasthenoteratozoospermia].

Genome disbalances are related to the different types of infertility and they play a role in the treatment of human infertility. Comparative genome hybridization (CGH) combined with microchips is a modern high resolution technique for all human chromosomes investigations. We analysed the genome disbalances in 16 blood samples of men with an idiopathic oligoastenozoospermia or azoospermia using CGH and microchips for the whole human genome. Our data showed a few affected loci, including: 3q26.32 deletion accompanied by 14q11.1 deletion, 9q12 deletion, 5q35.1 amplification, 7p22.3 amplification and 17q12-17q21.2 amplification. In this study we have match the deletions: in two patients in the same area in the 8 chromosome, as well as in 5 patients in 14 chromosome. The deleted region contains 25 genes. Two of them (SPAG11B and SPAG11A) are associated with stages of spermatogenesis and in particular formation and maturation of the spermatozoa. These genes play a role during spermatogenesis and fertilization. Loss. chr.14q11.2 (EDDM3A and EDDM3B) affected the proteins that are synthesized and secreted by epididymal epithelial cells that has been found up-regulated in epididimis of nonobstructive azoospermic men. Our results displayed the significance of CGH and microchip analysis as a promising area of research with serious clinical application for resolving the problems of the male infertility and still have an important annex for selecting the most appropriate methods for the treatment in these patients as a perspective scientific field of investigations with a clinical appliance.

[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 incidence of sperm antibodies in patients included in a program of assisted reproduction]. / Chestota na spermoantitelata u patsienti, vkliucheni v programa za asistirana reproduktsiia.

The aim of the present investigation was to establish the sperm antibody incidence among patients included in a program for assisted reproduction by the means of the classical and new methods for sperm antibody detection; to analyze the correlation between the results demonstrated by the different techniques; to evaluate the obtained data in the context of the application of new assisted reproduction technologies (ART). In the Laboratory of Reproductive Immunology, Department of Biology, Medical University of Sofia, 73 sera from patients (35 couples and 3 men) with primary and secondary infertility, aged 23-46 years, grouped according to the diagnosis and included in the program for assisted reproduction "Technobioassistance" were tested. Our results demonstrated the highest incidence of sperm antibodies amongst patients with primary unexplained infertility. The percentage of positive reacting couples for at least one of the applied tests was 31%. The highest relative share of those reacting positively, was observed with ELISA and the tray agglutination test (TAT) of Friberg. For ELISA 31.48% of the sera reacted positively, while clinically relevant liters of sperm antibodies were found with TAT in 21.92% of the tested sera. We also found that 17.46% and 13.7% of the tested sera were positive, respectively in the sperm immobilization test (SIT) of Isojima and the gelatin agglutination test (GAT) of Kibrick. The high degree of correlation (P < 0.0001) between the tests of Kibrick and Friberg showed the appropriateness of their application for patients in an ART program. At the other hand, the lack of correlation between the other applied tests (the Isojima test and ELISA) confirmed the assumption that immunity against spermatozoa should be sought with at least two diagnostic tools. Five of the tested families with clinically relevant TAT titers were included in our ART program for "in vitro" fertilization and embryo transfer, after preliminary absorption of the seminal fluid with autologous semen, or underwent the ICSI technique. For these patients, one ICSI pregnancy finished with the successful birth of a healthy child and one "in vitro" pregnancy is developing at the moment.

Heterogeneity in the presence of CD4-like molecules on human spermatozoa.

The object of the present study was to study if there are differences in the presence of CD4-like molecules on human ejaculated spermatozoa in fertile donors and infertile patients (with globozoospermia). Indirect and absorption enzyme-linked immunosorbent assay and indirect immunofluorescence were applied. The enzyme-linked immunosorbent assay data showed that monoclonal anti-human CD4-antibody recognizes an epitope common to the human spermatozoa with normal morphology and round-headed spermatozoa. Localization of the antigenic determinants, identified by anti-human CD4-monoclonal antibody, in the acrosomal region, including equatorial segment, postnuclear cap and tail was determined in normozoospermic samples. A positive reaction was found on the sperm head both in the acrosomal and postacrosomal region of some round-headed spermatozoa in the samples with globozoospermia. The tails of the normozoospermic spermatozoa and of some round-headed spermatozoa were weakly immunopositive. The results of the experiments carried out are evidence of heterogeneity in the presence of CD4-like antigen determinants on human spermatozoa. These data increased the information about the CD4-antigen characteristic of the spermatozoa from fertile donors and infertile patients.

Characterization of anti-sperm antibodies and their coding cDNA sequences by Epstein-Barr virus transformed B cell lines from lymphocytes of infertile women possessing anti-sperm antibodies.

Epstein-Barr virus (EBV)-transformed B cell lines that produce human antisperm antibodies were established using peripheral blood lymphocytes (PBLs) from infertile women with sperm immobilizing antibodies in their sera. We obtained three stable cell populations (designated B1, B2, D5) of transformed PBLs originating from three different patients. They produced IgM sperm-reacting antibodies directed against the tail of live, methanol-fixed and NaIO4-treated human spermatozoa. The established antisperm antibodies recognized noncarbohydrate sperm membrane antigens with different specificity and distribution in the male reproductive system. Antisperm antibody-B2 corresponding antigen appears to be specific for the male reproductive system. This antigen is excreted from the epithelial cells of the ductus epididymidis and bound to the spermatozoa in the lumen of the ductus. Antisperm antibodies B1 and D5 corresponding antigens were expressed on the spermatozoa in the seminiferous tubules and were common to the secretions of the ductus epididymidis, prostate and some other somatic organs. The cDNA of the immunoglobulin heavy chain genes were analyzed by reverse transcription polymerase chain reaction (RT-PCR) using RNA extracted from these clones. The immunoglobulin heavy chain cDNA sequences of these antisperm antibodies showed extremely high homology to previously reported immunoglobulin germline DNA sequences, implying that these antisperm antibodies might be natural autoantibodies rather than antibodies stimulated by external antigen.