Expression of the NUP153 and YWHAB genes from their canonical promoters and alternative promoters of the LINE-1 retrotransposon in the placenta of the first trimester of pregnancy.

The placenta has a unique hypomethylated genome. Due to this feature of the placenta, there is a potential possibility of using regulatory elements derived from retroviruses and retrotransposons, which are suppressed by DNA methylation in the adult body. In addition, there is an abnormal increase in the level of methylation of the LINE-1 retrotransposon in the chorionic trophoblast in spontaneous abortions with both normal karyotype and aneuploidy on different chromosomes, which may be associated with impaired gene transcription using LINE-1 regulatory elements. To date, 988 genes that can be expressed from alternative LINE-1 promoters have been identified. Using the STRING tool, genes (NUP153 and YWHAB) were selected, the products of which have significant functional relationships with proteins highly expressed in the placenta and involved in trophoblast differentiation. This study aimed to analyze the expression of the NUP153 and YWHAB genes, highly active in the placenta, from canonical and alternative LINE-1 promoters in the germinal part of the placenta of spontaneous and induced abortions. Gene expression analysis was performed using real-time PCR in chorionic villi and extraembryonic mesoderm of induced abortions (n = 10), adult lymphocytes (n = 10), spontaneous abortions with normal karyotype (n = 10), and with the most frequent aneuploidies in the first trimester of pregnancy (trisomy 16 (n = 8) and monosomy X (n = 6)). The LINE-1 methylation index was assessed in the chorionic villi of spontaneous abortions using targeted bisulfite massive parallel sequencing. The level of expression of both genes from canonical promoters was higher in blood lymphocytes than in placental tissues (p < 0.05). However, the expression level of the NUP153 gene from the alternative LINE-1 promoter was 17 times higher in chorionic villi and 23 times higher in extraembryonic mesoderm than in lymphocytes (p < 0.05). The expression level of NUP153 and YWHAB from canonical promoters was higher in the group of spontaneous abortions with monosomy X compared to all other groups (p <0.05). The LINE-1 methylation index negatively correlated with the level of gene expression from both canonical (NUP153 - R = -0.59, YWHAB - R = -0.52, p < 0.05) and alternative LINE-1 promoters (NUP153 - R = -0.46, YWHAB - R = -0.66, p < 0.05). Thus, the observed increase in the LINE-1 methylation index in the placenta of spontaneous abortions is associated with the level of expression of the NUP153 and YWHAB genes not only from alternative but also from canonical promoters, which can subsequently lead to negative consequences for normal embryogenesis.

IMMUNOPHENOTYPICAL CHARACTERISTICS OF CELLULAR COMPOSITION IN BREAST MILK.

The morphology and immunophenotype of female colostrum adherent cells with the help of CD3, CD31, CD34, CD45, CD68, vimentin, and osteocalcin antibodies panel was studied in short-term (6­7 days) culture in vitro. Approximately equal (1 : 1) ratio of fibroblast-like and rounded cells was observed in 20 % of cultural flasks. The cells with regular shape mixed with single fibroblasts were noted in 80 % of cultural flasks. The diameter of spreaded cells varied within 10­100 mm. All cells adhered to plastics did not express CD3 and interacted slightly (sl) with antibodies to CD31, CD34, and CD45. At the same time, adherent cells with intensive CD68, vimentin and osteocalcin staining have been revealed. Literature data allows to interpret CD68+CD3­CD31slCD34slCD45sl immunophenotype of significant part of mother colostrum adherent cells as belonging to monocyte-macrophage lineage. Marked expression of stromal antigens (vimentin, osteocalcin) in 40­45 % adherent cells in cultural medium without osteogenic supplements (beta-glycerophosphate, ascorbic acid, dexamethasone) proposes an existence of osteoblasts fraction differentiated in colostrum from mesenchymal stem cells under an action of breast milk humoral factors.

[THE CHARACTERISTICS OF SYSTEMIC AND LOCAL IMMUNE REACTIONS UNDER HABITUAL NON-CARRYING OF PREGNANCY].

The study was carried out to evaluate clinical informativeness ofstudies of immunological parameters of structural and functional elements of endometrium in complex with profile of embritropic autoimmune antibodies under non-carrying of pregnancy The sampling consisted of 19 women of reproductive age with episodes of non-carrying in first semester. They were examined for inherited thrombophilia and profiles of immune reactivity of natural regulatory auto-antibodies (ELI-P-Complex). The biopsy material of endometrium was subjected to histological and immune histochemical (CD16, CD56, CD68, CB138, estrogen and progesterone receptors) analyses. In women with non-carrying of pregnancy polyclonal immunosuppression, increased level of embriotoxic auto antibodies to Fc- fragment of immunoglobulin, insulin and thyroglobulin are detected. The immune histochemical analysis established expressed decreasing of amount of progesterone receptors of stromal elements of endometrium after spontaneous abortion. The activation of killer activity of decidual lymphocytes under habitual non-carrying of pregnancy was detected too. The positive dependency was established between: expression of progesterone receptors in glandular cells of endometrium and level of anti-spermatic antibodies in blood serum; expression of progesterone receptors in stroma and level of antibodies to human chorial gonadotropin; expression of receptors of estrogens in stroma and level of antibodies to Fc-fragment of immmunoglobulines. The study data permits affirming that evaluation of level of regulatory auto-antibodies is informative at examination of woman after episode of non-carrying of pregnancy. The production of antibodies has direct relationship with pathological processes in endometrium and it can be one of criteria of completeness of pre-conceptional preparation.