Influence of genetic markers on the immune cells in women with miscarriage.

Author: Loginova Yuliia Vladimirovna

Co-author: Abramovskikh Olga Sergeevna

Research Institute of Immunology, South Ural State Medical University of the Ministry of Health of Russia

Russia, Chelyabinsk

Miscarriage is the spontaneous termination of pregnancy between conception and 37 weeks, counting from the first day of a woman’s menstrual cycle. Termination of pregnancy from conception to 22 weeks refers to spontaneous miscarriage (the frequency of occurrence is 15-20%). Three or more consecutive pregnancy losses are regarded as recurrent pregnancy loss (RPL) [1]. The etiology of miscarriage is diverse, where immunogenetic factors play an important role, especially in cases of miscarriage of unknown origin [2]. It is known that under the control of «individual characteristics of the genotype» (single nucleotide polymorphisms of cytokine genes, for example) are the mechanisms of immunological tolerance that are formed and implemented in relation to the allogeneic fetus [3]. 

Aim of the study. Study of the effect of the polymorphic variant of the IL6 gene (rs1800795, C174G) on the immune cells in women with miscarriage living in the city of Chelyabinsk and the Chelyabinsk region. 

The study involved 174 women of reproductive age, of which 63 patients (36,2%) were diagnosed with miscarriage, 39 patients (22,4%) were diagnosed with RPL and 72 conditionally healthy women (41,4%) having 2 or more realized pregnancies in a pair with the same partner, without a burdened obstetric and gynecological history (control group). The peripheral blood of patients was studied in the first phase of the menstrual cycle. The material for genotyping was DNA samples obtained from peripheral venous blood leukocytes of the examined women using a commercial kit of reagents «K-Sorb-100» (CJSC «Synthol», Russia). To analyze the SNP of the IL6 gene (rs1800795, C174G), the real-time polymerase chain reaction method was used on a Rotor-Gene amplifier (Corbett Research Pty Ltd., Australia) using primers and probes manufactured by CJSC Sintol (Russia) according to the manufacturer’s instructions. In order to quantify the content of cellular immunity factors, hematological parameters were determined using an automatic hematological analyzer MEDONIC M16 (Boule Diagnostics AB, Sweden) and the content of lymphocytes and their subpopulation composition by flow cytometry on a Navios™ apparatus (Beckman Coulter, USA) using monoclonal antibody conjugates : CD45-PC7, CD3-PE, CD4-FITC, CD-8-PC5.5, CD-3-FITC/CD19-PE, CD3-FITC/CD(16+56)-PE, CD-25-PE, CD3-FITC/HLA-DR-PE (Beckman Coulter, USA), CD183-PerCP/Cy5.5, CD194-APC, CD196-PE/Cy7 (BioLegend, USA). Statistical processing of the obtained data was carried out using StatTech v. 2.8.8 (developer — Stattech LLC, Russia) and PAST v. 4.05. Quantitative indicators were evaluated for compliance with the normal distribution. Comparison of groups in terms of a quantitative indicator having a normal distribution, provided that the variances were equal, was performed using Student’s t-test. Comparison of groups according to a quantitative indicator, the distribution of which differed from the normal one, was performed using the Mann-Whitney U-test. Comparison of percentages in the analysis of multifield contingency tables was performed using Pearson’s chi-square test. In case of low saturation of the tables (n<5), statistical significance was assessed using the Monte Carlo randomization technique (n = 99,999). In order to determine the cells of the contingency tables that ensured the statistical significance of the revealed effects, the Freeman-Tukey FTdev deviations were calculated and their statistical significance was evaluated. Differences were considered statistically significant at p ≤ 0,05, insignificant at p > 0,10; in intermediate cases (0,05 < p ≤ 0,10), trends towards differences were discussed. 

It was established that the frequency of occurrence of the CC genotype of the polymorphic variant of the IL6 gene (rs1800795, C174G) in women of the control group is higher than in women with miscarriage and RPL (p<0,05). The CG rs1800795 genotype of the IL6 gene in the group of women with RPL was statistically significantly less common than in patients with miscarriage and apparently healthy women (p<0,05). No statistically significant differences were found in the analysis of the distribution frequencies of the GG rs1800795 genotype of the IL6 gene. Analysis of the association of genotypes SNP IL6 (rs1800795, С174G) with immunity parameters in the studied groups showed that women with miscarriage are carriers of CC genotype is accompanied by a decrease in the content of B-lymphocytes (p<0,05) and a trend towards higher numbers of T-lymphocytes with markers of early activation and Th2-lymphocytes, and in the group of women with RPL, there is a lower number of B-lymphocytes, an increase in the content of T-lymphocytes with markers of early activation (p<0,05) and a tendency to an increased content of NK-T-lymphocytes in comparison with similar indicators in the control group. In the group of women with RPL, the carriage of the CG genotype is associated with a higher content of T-lymphocytes in the peripheral blood with markers of early activation and a tendency to decrease in Th2-lymphocytes, and the carriage of the GG genotype is associated only with a tendency to increase the relative number of NK cells in the peripheral blood of the examined patients compared with the control group. 

According to the results of the study, it was found that the C allele and the CC genotype of the IL6 gene (rs1800795, C174G) have a probable protective effect on the risk of developing RPL. In women with RPL, whose genotypes contain the G allele of the polymorphic variant of the IL6 gene (rs1800795, C174G), there is an increase in the amount and reactivity of cellular immunity factors, as well as a shift in the Th1/Th2 dichotomous axis towards Th1-lymphocytes. 

References: 

1. Sidelnikova V.M., Sukhikh G.T. Miscarriage: A guide for practitioners. M.: LLC «Medical Information Agency»; 2010. 

2. Grimstad F., Krieg S. Immunogenetic contributions to recurrent pregnancy loss. J Assist Reprod Genet 2016; 33:833–847 

3. Ma J., Zhang X., He G. et al. Association between TNF, IL1B, IL6, IL10 and IFNG polymorphisms and recurrent miscarriage: a case control study. Reprod Biol Endocrinol 2017; 15:83