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A major factor affecting male fertility is excessive death of germ cells, both immature germ cells and mature spermatozoa. It can be due to various factors causing testicular and/or post-testicular damage, such as infections, obstructive conditions, toxins, oxidative stress, hormonal imbalance, hyperthermia, and anti-sperm antibodies. Massive death of spermatozoa leads to a high proportion of dead sperm cells in the ejaculate (necrozoospermia or necrospermia) while death of immature germ cells can lead to low sperm count (oligozoospermia or oligospermia). Cell death can occur both by necrosis and by apoptosis; in recent decades, it has been found that apoptosis of mature spermatozoa is not only possible but quite common, and can contribute to infertility. Treatment approaches are primarily directed to the underlying condition, i.e. removing the cause(s) of sperm cell death whenever possible, but include also attempts to bypass the cell death event by intracytoplasmic sperm injection with testicular spermatozoa.

The potential of viruses as appropriate vectors for the development of new therapeutic strategies, as well as for the design of molecular (DNA, RNA, and/or protein) vaccines via substitution of nucleotide sequences, has been proven. Among the most appropriate DNA and/or RNA fragments, members belonging to families Parvoviridae (particularly adeno-associated virus, AAV) and Poxviridae have frequently been suggested for this purpose. In previous studies, the vaccine avipoxvirus strains FK (fowl) and Dessau (pigeon) have been proven able to infect mammalian cells (as well as avian cells), and to replicate productively in a small number of them; thus, we may be able to adapt them using incubation, and in these conditions. Additionally, we have previously proved, based on AAV recombinant DNA vectors, that it is possible to transfer appropriate genes of interest via mouse embryonic stem cells (mESCs). In the current study, we develop methods for the application of the same vaccine avipoxviral strains, based on the AAV DNA genome recombinant constructs, to be used for gene transfer in cells, for the transfer of DNA and/or RNA fragments (for the suppression of unwanted viral and/or cellular genes), and for the production of molecular (DNA, RNA, and/or protein) anti-cancer and anti-viral vaccines. To this end, sub-populations of embryonic mammalian cells infected with the two forms of both vaccine avipoxviral strains were frozen in the presence of cryo-protector dimethylsulfoxide (DMSO), subsequently thawed, and re-incubated. In most cases, the titers of the intra-cellular forms of the two strains were higher than those of their extra-cellular forms. These data were explained by the probable existence of the intra-cellular forms as different sub-forms, including those integrated in the cellular genome proviruses at a given stage of the cellular infection, and suggest the possibility of transferring nucleotide (DNA and/or RNA) fragments between cellular and viral genomes; this is due to the influence of activated fusion processes on DMSO, as well as drastic temperature variations.

Natural killer (NK) cells can discriminate between normal and cancer cells and are known to directly recognize and kill malignant cells or induce apoptosis. Thus, activation of NK cells is considered as a promising strategy for cancer treatment. However, clinical application has been somewhat limited because of difficulties in the preparation of sufficient number of highly cytotoxic/activated NK cells in vitro . We used cytokine stimulation to provide a suitable environment (activating receptor-ligand interactions) for the expansion of NK cells. This method potently expanded NK cells, and the final product was composed of highly proliferating NK cells. The expanded NK cells showed significant upregulation of various activation receptors such as CD69 and NKG2D. The latter is a particularly important receptor for triggering NK cell responses toward tumor cells.

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.

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.

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 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.

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.

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.