[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.

[Investigation of cytokeratin and vimentin intermediate filaments in polycystic ovaries (PCOS) -- presence and specific structure of Balbiani body in primordial follicles].

Despite of the importance of cytoskeletal proteins for ovarian function and pathology, very few studies have addressed the presence and distribution of these proteins in polycystic ovaries. We investigated sections of human polycystic ovarian tissue for vimentin and a set of cytokeratins by epifluorescence. The studied proteins showed strong colocalization. Positive reaction was detected in two main ovarian compartments: with weak intensity in follicular cells and very strong in perinuclear position in oocytes of primordial follicles. Epifluorescent study of the oocytes from primordial follicles allowed us to identify the immunopositive structure in oocytes as Balbiani body, a transient organelle responsible for establishing oocyte polarity and ooplasm gradients in nonmammalian vertebrates. Our results suggest functional interaction of different types of cytoplasmic intermediate filament proteins in polycystic ovaries and a possible importance of the Balbiani body for human oogenesis in norm and pathology.

[Role of oocyte cytoplasmic factors in human IVF failure].

Premature chromosome condensation (PCC) of the sperm chromatin and abnormal dispersion of oocyte chromosomes are independent indirect indicators for cytoplasmic immaturity and cytoskeletal anomalies in the oocyte affecting the normal zygote and embryo formation in human IVF-ET practice. In a group of 66 human unfertilized oocytes, two types of cytoplasmic factors were registered: PCC in 49 (74.24%) and cytoskeletal anomalies in 17 oocytes (25.76%). These data were analyzed according to the main stimulation regimen and female age as well as to the familial factor of infertility. Our results displayed a higher proportion of PCC if the stimulation regimen included gonadotrophins alone (77.55%) compared with the combined GnRH agonist and gonadotrophins administration (22.45%) and in the female age group of 36-40 years (63.33%). Cytoskeletal defects were predominant in the female age group of 31-35 years (46.06%). Remarkable was the significance of cytoplasmic factors in cases of unknown infertility factor--oocytes from these patients included 34.69% of all cells with PCC and 52.94% of all cells with cytoskeletal defects. Hence, these anomalies were probable causes for IVF failure in cases of unexplained infertility Conclusions: 1. The main regimen for hormonal stimulation and the female age influence the cytological status of studied oocytes (cytoplasmic immaturity causing PCC and polyspermy). 2. Cytoplasmic factors (immaturity and cytoskeletal anomalies) are a prognostic factor for the IVF success in cases of unknown infertility problem in the family.

[Sperm parameters in male idiopathic infertility after treatment with prelox].

UNLABELLED: The diagnosis of male infertility is determinate after assessment of sperm quality and clinical study. In nearly 30% of the cases nevertheless detailed clinical and laboratory study it can't be discovered the cause and on the bases of exclusion criteria set the diagnosis idiopathic infertility. The object of our study was investigation of the group patients (n=50) with idiopathic infertility treated with Prelox and to be studied its effects on spermatozoa parameters. MATERIAL AND METHODS: The study design was double-blind, placebo-controlled, cross-over, randomized study, including introduction period (1 month), two therapeutic periods (each one of 1 month) separated with 1 month wash out period and concluding period of 1 month. There was applied a new method for treatment with mechanism of action stimulation the production cGMP of spermatozoa endothelial nitric oxide synthase (eNOS). This is not surprising achieving results show improvement of sperm quality. The methods of the study were: 1. Assessment of the conventional semen analysis (according the criteria of WHO, 1999). 2. Spermatozoa function tests. 3. Spermatozoa-cervical mucus penetration tests. RESULTS: The obtained results showed improvement of sperm quality, in the middle-aged men the therapeutic answers was better than in younger. In conclusion the therapy with Prelox improve sperm parameters in men with idiopathic infertility. Pycnogenol (one of the constituents of Prelox) has powerful antioxidative influence ameliorating spermatozoa function.

[Selecting function of female genital tract in relation with spermatozoa interaction].

The present study includes 50 infertile couples followed sperm parameters: morphology and others indices for ability of penetration in ovulatory cervical mucus during 12 months. It is discussed the possibility of which spermatozoa can participate in the fertilization and the impotence and place of the female genital tract for the selection of morphologically abnormal spermatozoa before reaching the place of fertilization.

The European study of assisted reproduction families: a comparison of family functioning and child development between Eastern and Western Europe.

Developments in reproductive technology over the past 20 years have given infertile couples new opportunities to have children. Although assisted reproduction does not appear to lead to negative consequences with respect to parent-child relationships or the socioemotional development of the child, the outcome for children and their parents may well differ according to the social and cultural environment. The aim of the present study was to compare family functioning and the social and emotional development of children in families created as a result of assisted reproductive techniques in Western Europe with assisted reproduction families in an Eastern European country (Bulgaria) where there is a history of specific pronatalist interventions. The findings indicate greater difficulties in parental adjustment and child behavior in assisted conception families in Eastern Europe. In addition, Eastern European parents were more uncertain than Western European parents about whether they would tell the child, and Eastern European parents were much more secretive with respect to telling others. Thus the outcomes of assisted reproduction for family functioning and child development appear to be independent, to some extent at least, on the social context in which these techniques are carried out.

Significance of chromosomal aberrations for the unsuccessful procedures of assisted reproduction.

Lymphocyte cultures from patients who had previously undergone at least three unsuccessful procedures of assisted reproduction were analysed for cytogenetic abnormalities. A total of 12,657 metaphases from 33 individuals (15 patients and 18 healthy controls with two normal offsprings) were studied. A significantly higher incidence (P<0.001) of chromosome aberrations was found in the patients (6.79+/-0.68%; x+/-SD) as compared to the controls (1.72+/-0.3%; P<0.001). Chromosomal breakages, particularly at the centromere region, were also observed with significantly increased frequency in the patients than in the controls (6.18+/-0.65 vs. 1.42+/-0.27%, respectively; P<0.001). It is possible that the high rate of centromere breakages in the ART patients (3.18+/-0.47 vs. 0.26+/-0.12%, P<0.001) may predispose to meiotic chromosomal abnormalities. A single cell aberration was demonstrated in 0.61+/-0.21% of ART patients' lymphocytes versus 0.3+/-0.12% in the controls (P<0.01). Structural rearrangements and chromosomal breaks predominantly affected the bands containing genes for the immune response and the cell cycle. Mosaic karyotypes were found in six patients. One of them had a karyotype 46,XX/46,XX,r(14) and the others had sex chromosomal mosaicisms. The prenatal diagnosis could be essential in these cases. It is concluded that chromosomal aberrations could play a role in the repeated failure of ART procedures.

Asymmetric vimentin distribution in human spermatozoa.

The subject of the study was vimentin distribution in spermatozoa from human ejaculates by immunofluorescence and immunogold electron microscopy. In accordance with earlier reports, vimentin was found in the sperm head and was localized mainly in the equatorial segment region. However, electron microscopic observations revealed an additional intriguing detail: vimentin-associated gold granules showed asymmetric distribution. This asymmetry tended to be more pronounced in heads with defects such as cytoplasmic droplets. Abnormal cells also had positive reaction for vimentin in the neck and the proximal midpiece, but in these domains gold granule distribution was apparently uniform. These findings seem to support the hypothesis that the surfaces of the mammalian sperm head are functionally non-equivalent, although morphological basis for such a phenomenon is evident only in rodents. It is also interesting that asymmetry in vimentin distribution correlates with distinct types of sperm structural defects.

Supernumerary human preembryos provide potential for preimplantation genetic diagnosis.

Preimplantation genetic diagnosis is an alternative to the classical prenatal diagnosis for couples undergoing in vitro fertilization. It allows very early embryo selection--before the intrauterine embryo transfer. Prior to clinical application of preimplantation diagnosis in the Infertility Treatment Centre "Technobioassistance", Sofia Medical Faculty, we have developed preimplantation diagnosis models of human spermatozoa and untransferred 2-8-cell human embryos obtained in vitro. Directly fluorescein isothiocyanate-labelled probes specific for the centromeric regions of chromosomes 1, 5, 19 and X (Boehringer Mannheim) were used. Eighty-six point three percent of fixed blastomeres with normal size and shape had unfragmented nuclei with dispersed interphase chromatin or mitotic chromosomes and all of them demonstrated successful hybridization. In cases with more than 75% of embryo cells suitable for analysis we were able to estimate the presence of mosaicism among the blastomeres.

[Our experience in the in vitro fertilization of human ovocytes]. / Nashiiat opit v oplozhdane in vitro na choveshki ovotsiti.

The authors describe the international experience in vitro fertilization. The programme, realized in the People's Republic of Bulgaria, is divided into three periods: First period--taking eggs by laparoscopy; Second period--during which eggs have been taken by laparoscopy as well as transvaginally by ultrasound, when two pregnancies have been successfully realized; Third period--again eggs are taken only by laparoscopy as 1.46 eggs are obtained on the average per a patient. The authors describe the difficulties and problems, which stand before them for solving. Taking into consideration their own and foreign experience, the authors think that the age of women, individual stimulation, monitoring and timely performance of the operation for taking eggs are of special significance.

[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.