[New screening method for aneuploidies based on analysis of cell-free DNA in the maternal blood].

UNLABELLED: The national program of screening for aneuploidies in Bulgaria is based on first trimester combined test, second trimester biochemical test and/or the combination between first and second trimester integrated test. OBJECTIVE: To review the literature for studies analyzing cell-free (cf) DNA in the maternal blood and to report the clinical implementation and validation of the method in the clinical practice. Literature search and study selection extracted studies since 2011 when the first article was published. The data source included searches from PubMed and Medline. The reported results for detection rates (DR) and false positive rates (FPR) in singleton pregnancies were about 99.0% and 0.08% respectively, for trisomy 21, 96.8% and 0.15% for trisomy 18, 92.1% and 0.20% for trisomy 13, 88.6% and 0.12% for monosomy X. For twin pregnancies, the DR was 94.4% and FPR was 0% for trisomy 21. CONCLUSION: Analysis of cell-free DNA in the maternal blood is an effective method of screening for aneuploidies.

[Increased absolute numbers of NK cell in peripheral blood correlate to up-regulated expression of HLA-DR and enhanced secretion Il-12 in infertile women].

It is well known that the increased numbers of NK cells with phenotype CD3-CD16+CD56+ can execute cytotoxic effect against trophoblast cells and might be responsible at least for a part the immune mediated reproductive failures. The aim of this study is to follow up the correlations between the increased absolute numbers of NK cells, their activation status and secretion of some cytokines in infertile patients. The results obtained clearly show that in patients with increased numbers of NK cells the percentages of HLA-DR positive NK cells were significantly higher compared to the patients with normal number of NK cells. A similar correlation was revealed when the data of blood plasma where the concentrations of IL-12 were significantly higher in patients from the first group (with increased NK cells numbers). The results from our study demonstrate that the increased numbers on CD3-CD16+CD56+ NK cells is accompanied by enhanced activation of these cells and secretion of cytokines.

Characterization of clonogenic stromal cells isolated from human endometrium.

Human endometrium is an object of extensive restructuring and remodeling during the female reproductive life and it is quite tempting to assume that these periodic changes happen with the participation of cells that should have the basic characteristics of multipotent cells. The aim of this study was to search for the presence of cells with plastic adherence, clonogenicity, and differentiation in human endometrium. To this end, human endometrial stromal cells were cultured in vitro for more than 15 passages. Flow cytometry analysis of the cultured cells showed that they were positive for CD29, CD73 and CD90, which are considered to be the markers of cells with mesenchymal origin. The cells were negative for the hematopoietic cell markers (CD45, CD34, CD14, CD3, CD19, CD16/56, and HLA-DR). Further, it was shown that the cultured cells had 15% clonogenic efficiency and could be induced to differentiate into adipogenic cells containing typical lipid-rich vacuoles. These results demonstrate that the human endometrium contains a low number of cells with the characteristics of endometrial stromal stem/progenitor cells, which seem to belong to the family of the mesenchymal stem cells. It can be speculated that these cells are engaged into the monthly restructuring and remodeling of human endometrium.

[Gynalgin--one more opportunity in the treatment of bacterial vaginosis].

The data of 30 nonpregnant women in reproductive age BV treated with Gynalgin were presented. On clinical and microbiological indicator Gynalgin showed a good effect on BV-associated microorganisms (80%). Only one of the cases of BV was not affected (4%). With nixed infection of BV and Candida we established clinical improvement and recovery of Lactobacillus spp. In 66.6%, but in all cases on Candida is not effect. We consider that Gynalgin is still one more easily applicable and clinically effective opportunity for treatment of BV.

[Assesment of the woman's age as a main predictive factor of assisted reproductive technology treatment outcome].

Several factors could be used as a predictors of in vitro fertilization treatment outcome: female age, aetiology of sterility, previous ovarian surgery, embryo morphology and cleavage rates and also the technique of embryo transfer. The aim of the present study is to evaluate the role of the age to be used as a prognostic factor for the sterility treatment outcome. The tasks we assigned were to compare the several parameters: duration of the stimulation, number of retrieved oocytes, fertilization rate, implantation rate and clinical pregnancy rate after IVF/ICSI in different age's groups. The study results clearly demonstrate that in spite of decreasing number of retrieved oocytes after the age of 30, the fertilization rate remains elevated till 40 years. Regardless of this fact, the implantation rate and the clinical pregnancy rate were significantly lower after the age of 35. Reduction of the success rate in these cases showed that ART, despite of its renewals, is not the strategy for getting over the age in human reproduction.

In vitro effects of inhibin on apoptosis and apoptosis related proteins in human ovarian granulosa cells.

OBJECTIVE: To clarify the in vitro effects of inhibin A (I) on apoptotic cell death and its mechanisms in ovarian granulosa cells the immunoexpression patterns of the apoptosis markers caspase-3 and pro- and anti-apoptotic proteins (Bcl-2, Bcl-xl, Bak) were evaluated in ovarian granulosa cells collected from women with different hormonal status. MATERIALS AND METHODS: Granulosa cells were isolated from follicles of women participating in an in vitro fertilization (IVF) program, normally cyclic (NC) and premenopausal women (PrM). The obtained cells were cultured for 72 h with inhibin A (Sigma, USA)--10 ng/ml. The concentration of estradiol in the culture medium was determined by radioimmunoassay using the Coat-A-Count kit (Nippon, Japan), whose intra- and interassay coefficients of variations were 6,8% and 6,2% respectively. The expression of caspase-3, Bak, Bcl-2, Bcl-xl was investigated immunohistochemically. The percentages of immunopositive cells were calculated and Student's t-test was used for statistical analysis. RESULTS: Addition of inhibin A (10 ng/ml) to granulosa cells cultures resulted in increased estradiol production. Maximal stimulation was observed in granulosa cells collected from women participating in IVF whereas minimal effect of inhibin treatment on estradiol production was demonstrated in premenopausal women. Inhibin A exposition enhanced the immunoexpression of prooncogenes (Bcl-2, Bcl-xl) and reduced the expression of caspase-3 and pro-apoptotic protein Bak in ovarian granulosa cells from the three experimental groups. CONCLUSIONS: Our findings suggest that inhibin A in vitro stimulates the estradiol secretion by granulosa cells dependently of the woman hormonal status, while it inhibits apoptotic process in granulosa cells independently of the hormonal status.

Potential role of nitric oxide in endothelin-1 provoked inhibition of progesterone secretion by isolated ovarian granulosa cells.

OBJECTIVE: Nitric oxide (NO) is involved in different cell functions including ovarian steroid production. Endothelin-1 (ET-1) was found to regulate the steroidogenesis in ovarian granulosa cells (GC). The present study was designed to receive more information about the mechanism of action of NO in the process of ET-1 induced progesterone (P) inhibition, using nicotine amide dinucleotide phosphate-diaphorase (NADPH-d) histochemistry as a cofactor of oxidoreductase enzymes (e. g. nitric oxide). METHODS: Granulosa cells were isolated from ovaries of: 1. young women with natural cycle or after in vitro fertilization (IVF), 2. premenopausal women. The obtained cells were cultured with endothelin-1 and the concentration of progesterone in conditioned media was determined by RIA. For the estimation of NADPH-d the histochemical reaction was used. RESULTS: The suppressive effect of ET-1 on P production in granulosa cells was more pronounced in young women with natural cycles, slightly weaker after IVF and the most ineffective in premenopausal patients. The number of NADPH-d positive GCs was higher in young non-hormonally stimulated women, slightly lower after IVF and small in premenopausal ones. CONCLUSIONS: The results indicate the possible role of NADPH-d or NOS in the mechanism of ET-1 provoked P suppression.