ROLE OF GENE POLYMORPHISM OF IL-4 AND IL-17 IN RECURRENT MISCARRIAGE , CAME IN IVF CYCLES

Most researchers suggest that more than 50% of pregnant women after use of assisted reproductive technologies (ART) are faced with the problem of miscarriage, especially in the first trimester. The aim of the study was to investigate the frequency of genotypes and allelic variants of the IL-4 (S589T and S33T), IL-4R (Q576R), IL-17A (G197A) and IL-17F (488T/C) genes, depending on the reproductive status and assessment of their association with recurrent miscarriage, which occurred in ART cycles. Peripheral blood of 240 patients – the eastern Slav with habitual miscarriage, came in ART cycles, and 100 apparently healthy fertile women in the control group with a history of at least one term delivery and lack of spontaneous abortion episodes was studied. The groups were representative of age. DNA for molecular genetic studies of nuclei isolated from peripheral blood lymphocytes. The method is based on the destruction of lymphocytes using lysis buffer without affecting the integrity of the membranes of lymphocytes nuclei. For typing single nucleotide polymorphisms of IL-4 immune response, IL-4R, IL-17A and IL-17F using polymerase chain reaction product with the melting reaction in the presence of “adjacent” oligonucleotides. It was found that the genotype CT-33 (OR 3.99; 95% CI 2,42–6,57) and CT-589 (OR 3.99; 95% CI 2,42–6,57) gene IL-4; genotype GG576 (OR 5.95; 95% CI 1,39–25,58) gene IL-4R and TT488 genotype (OR 2.33; 95% CI 1,39–3,91) and CC488 (OR 2.27; 95% CI 0,76–6,84) IL-17F gene were associated with a significant increase in the risk of miscarriage. On the other hand, no significant differences were found between the control group and the basic frequency of polymorphisms IL-17A-G197A (rs227593). The results of the molecular-genetic typing it possible to identify in the Eastern Slavic population of residents of Odesa region of Ukraine immunogenetic markers of susceptibility/ resistance to the development of recurrent miscarriage. It is shown that cytokine gene typing can be used as a method of early diagnosis and pregravid prognosis of immune forms of reproductive loss in women.


INTRODUCTION
Recurrent miscarriage (RM) is a genetically heterogeneous condition as a result of the coexistence of the two governing factors: maternal and embryonic [1,2].The embryo expresses antigens inherited from both parents, and survival of hemiallotransplant fetus to term is one of the most challenging processes associated with pregnancy.Mother's immune system plays an important role in a successful pregnancy by controlling the fertilization, implantation, development and maintenance of the pregnancy itself [3][4][5].

ANALYSIS OF PUBLISHED DATA AND RESEARCH TASKS
Successful maintenance of pregnancy is dependent on the delicate balance between Th-1, Th-2, Th-17 and Treg-cells of the immune system as well as between specific cytokines inherent [6,7].Cytokines are cellular signaling proteins, mainly secreted by immune cells and mediate cell-cell communication [8].It was revealed that the cytokine polymorphisms may be associated with RM.It is known that one of the major cytokines involved in the gestation are IL-4 and IL-17.Human cells Th17, produce IL-17, may play a role in the rejection conceptus antigens and consequently may be harmful to the maintenance of pregnancy [9].It is shown that IL-17-decidual, producing by CD4+T-cells, are not harmful to human pregnancy, as they also produce .Therefore, a special interest is the study of the characteristics of polymorphisms of IL-4 and IL-17 in women with RM.
IL-4 regulates the growth and differentiation of B-lymphocytes, as well as processes of biosyn-thesis and secretion of antibodies.It is produced by activated CD4+T-lymphocytes (Th2), mast cells and eosinophils, generally -by activated Th2 cells.IL-4 stimulates the differentiation of Th0 to Th2, inhibits the generation of cytotoxic lymphocytes, natural killer cells and the production of interferon-γ and antitumor activity of macrophages, reduces the production of proinflammatory cytokines IL-1 and TNFα [11,12].In the presence of genetically determined disorders of the immune system excessive synthesis of pro-inflammatory interleukins occurs, resulting in the rapid development of inflammation in the tissues even in the absence of pathogens in the hearth.Some evidence suggests that Th2-cytokine IL-4 and its receptor may be of particular interest for controlling a Th17-induced inflammation.In humans, decreased response to IL-4 is believed to contribute to autoimmune inflammation.
In humans, the gene, coding for IL-4, is located on chromosome 5 in the 5q31-q33, which is a socalled cytokine cluster, where they were found and other genes that are important for the immune system, such as IL-5, IL-9, IL-12, IL-13 and granulocyte-macrophage colony stimulating factor.IL-4 gene contains four exons and exon 2 is the shortest (48 bp encoding 16 AA); exon 3 is the longest.The full length protein IL-4, which is encoded all four exons, is an option that is commonly referred to simplest IL-4, and it is certainly the most studied isoform structurally and functionally [12].

К.P. GOLOVATYUK
PhD, assistant at the department of reconstructive and regenerative medicine with reproductology course, Odessa National Medical University, chief medical officer of LLC Medical Center of Reproductive Health Protection "Gamete", Odesa ORCID: 0000-0002-9033-3583 ers.It was installed large variability of occurrence of abnormal IL-4 alleles in different populations [13].Different ethnic groups may have especially immunopathogenesis that are both theoretical and practical interest [13].
A single nucleotide polymorphism (SNP), in IL-4R coding region, I50V [rs1805010] detects the presence of isoleucine (I) according to valine (V) at position 50 in the aminoacid sequences.This polymorphism in the IL-4R is functionally important, since it affects the signal force receptor and may be able to regulate the production of IL-17 involved in the gestation [14,15].At the same time, [15] found no significant difference in IL-4 VNTR genotype frequencies between RM and control (OR 0.91; 95% CI 0.58-1.45);[16] showed that IL-4R I50V [rs1805010] plays a causal role in the RM.The contradictory literature data requires further research.
T-helper 17 (Th17) -the third type of the newly discovered T helper cells.T helper 17 cell characterized by the expression of IL-17A, IL17F, IL-6, TNF-α and IL-22.Six ligands of family IL-17 (IL-17A-F) and five receptors (IL-17RA-RD and SEF) were identificated.IL-17A and IL-17F are members of the cytokine IL-17 responsible for pathogenic cell activity Th17; they induce multiple proinflammatory mediators including chemokines, cytokines and metalloproteinases of epithelial cells and fibroblasts [17].The genes IL-17A and IL-17F are located on chromosome 6P12.Development Th17-cells and cytokine secretion are reduced in vitro upon exposure IFN-γ and IL-4 produced by Th1 and Th2-cells, respectively.Polymorphisms IL-4R (I50V) may monitor the possibility of the immune systems control the amount of human IL-17 production.
The aim of the study was to study the incidence of genotypes and allelic variants of the IL-4 (S589T and S33T), IL-4R (Q576R), IL-17A (G197A) and IL-17F (488T/C) genes, depending on the reproductive status and evaluation of associative due to recurrent miscarriage in IVF cycles.

MATERIAL AND METHODS
Under supervision there were 240 patients of group R with habitual miscarriage, came in IVF cycles and 100 apparently healthy fertile women in the control group K with a history of at least one term delivery and lack of spontaneous abortion episodes.All the women were Eastern Slav.
Patients were taken peripheral whole blood into vacuum tube type plastic Vacuette volume of 4.0 ml added as anticoagulant disodium ethylenediaminetetraacetate salt in a final concentration of 2.0 mg/ml.
Molecular genetic studies carried out in the molecular genetics laboratory of "Diagnostic center 'Eugenics'" (Head Lab.PhD Makshaeva E.T.) in Odessa.DNA is isolated from nuclei of lymphocytes.The method is based on the destruction of lymphocytes using lysis buffer without affecting the integrity of the membranes of lymphocytes nuclei.For typing of gene SNPs immune response using PCR fusion reaction products in the presence of "adjacent" oligonucleotides (variant of the adjacent probes, kissing probes).
Statistical data processing carried out with the help of the electronic program Microsoft Office 2007 for Windows XP Professional, STATISTICA 6.0 (StatSoft Inc, US) with the definition of the differences were significant at a value of p <0.05.For comparison, the frequency for a normal distribution of feature t-test was used.To estimate the nonparametric, unrelated metrics using the Mann-Whitney test related -Wilcoxon test.Compliance with the observed distributions of genotype frequencies, theoretically expected by the Hardy-Weinberg equation, evaluated using χ 2 test.To assess the association calculated the relative risk (OR).For OR calculated confidence interval (CI) at 95% significance level.If OR is equal to 1, it is considered that there is no association if OR was greater than 2 was considered a positive association.It was assessed preventive and etiological fraction.

RESULTS AND ITS DISCUSSION
The groups were representative of age.The average age of women surveyed group N was 29.80 ± 0.30 years and in the group K -30.09 ± 0.30 (p> 0.05).Termination of pregnancy in Group H in the first trimester had 51.25% of the patients, in the second -34.17%, in the third -14.58%.
The presence of polymorphisms of IL-4, IL-4R, IL-17A and IL-17F, we have studied on the basis of the total (Table 1) and a multiplicative model of inheritance (Table 2).
As seen from Table 1, both the genotype CT -33 (OR 3.99; 95% .27;95% CI 0.76-6.84)IL-17F gene were associated with a significant increase in the risk of miscarriage.On the other hand we found no significant differences between the study and control group in frequency of polymorphisms IL-17A-G197A (rs227593).

CONCLUSIONS
1.The results of the molecular-genetic typing of IL-4, IL-4R, IL-17A and Il-17F possible to identify markers in the Eastern Slavic population immunogenetic susceptibility/resistance to the development of recurrent miscarriage.
2. Typing of cytokine genes can be used as a method of early diagnosis and prediction of Pregravidar immune forms of reproductive loss in women.