Introduction:

Early recurrent pregnancy loss (further RPL), defined as three or more losses up to 12 weeks of pregnancy, is a heterogeneous disorder, affecting up to 3% of couples who are in the reproductive period^1,2.

The etiological causes of RPL are very diverse: genetic factors (chromosomal abnormalities in parents), which amount to 2-5%, presence of chromosomal disorders in embryo, anatomical factors (uterine abnormalities) -10-15%, endocrine diseases (untreated hypothyroidism, uncontrolled diabetes mellitus and etc.) -17-20%, autoimmune - 20% and infectious causes - 0.5-5%. 40-50% of RPL have not established etiology and belong to idiopathic RPL (further iRPL)^1,3-5.

Published systematic scientific reviews^6,7 of studies of SNP associations polymorphisms with iRPL, such as genes of hereditary thrombophilia, pro-inflammatory cytokine genes, angiogenesis genes, and placental function genes, have confirmed their significant genetic contribution to the development of iRPL in not all populations.

Genome-wide association studies (GWAS) RPL were conducted in heterogeneous ethnic populations, in small sample sizes, and did not find significantly significant predisposition genes for RPL that were confirmed in independent replicative studies of other populations^7-9. GWAS studies to identify genetic variants of iRPL development risk have not been conducted, due to the heterogeneity of the disease and lack of clear definitions of iRPL, the complexity of recruiting and small size of samples; the insufficient number of replicative studies in ethnically homogeneous populations^10,11.

According to literature data, thrombophilia makes a significant contribution to susceptibility to RPL by increasing platelet aggregation, the level of activity of coagulation factors and an excess of fibrinolytic inhibitors¹¹. At iRPL, these associations may not be observed, which requires further study of the genetic contribution of new predisposing factors, including the SNP of other genes of cardiovascular system¹².

The most studied genetic associations with RPL and iRPL are the Leiden mutation FV (A506G, rs6025), the mutation in prothrombin gene FII (G20210A, rs1799963), the unfavorable genotypes of folate metabolism genes: MTHFR (C677T, rs1801133, and A1298C, rs1801131)^6,7,10,11. Despite the contradictory results, the Practical Committee of the American Society of Reproductive Medicine² recommends that G20210A FII, A506G FV, C677T, and A1298C MTHFR gene be included in the mandatory screening study of couples with RPL.

Thus, associations studies of gene polymorphisms of coagulation and cardiovascular systems with the risk of iRPL development were few and showed inconsistent results in subsequent replicative studies of other ethnic populations^13-15. This is due to the high heterogeneity of the disease and lack of clear definitions of iRPL, the complexity of recruiting and small size of samples; ethnic differences and different research methodologies.

Aim of the study: to conduct GWAS independent replicative genotyping of statistically significant polymorphisms of coagulation and cardiovascular system genes associated with iRPL in an ethnically homogeneous population of Kazakhs. The gene polymorphisms were studied: F5 (A506G), F2 (G20210A), FGB (G455A), ITGB3 (Leu33Pro), GPIa (C807T), MTHFR (C677T, A1298C), MTR (A2756G), MTRR (A66G), ACE (I/D), AGTR (A1166C), eNOS3 (Glu298Asp).

Materials and Methods:

The study was conducted by prospective "case-control" method in the outpatient department of Scientific Center of Obstetrics, Gynecology, and Perinatology (SCOGP), the medical center "Center of Molecular Medicine." All study participants gave informed consent to the use of their blood samples and anamnestic data, the permission of the Ethical Committee of SCOGP to hold these studies is available.

RPL is classically defined as three or more spontaneous miscarriages up to 20 weeks’ gestation^1,2. The American Society of Reproductive Medicine (ASRM) believes that two consecutive pregnancy losses are sufficient for the diagnosis of RPL, since the recurrence rate and risk factors are similar to those observed after three losses².

The main group with iRPL consisted of 302 women of Kazakh nationality; age 18-45 years; who have two or more miscarriages before 12 weeks of gestation.

The control group is represented by 300 female Kazakh women with normal reproductive function without indication of the presence of spontaneous miscarriages with at least one child.

Recruitment criteria included: belonging to the Kazakh nationality by maternal and paternal grandparents; age 18-45 years; the presence of 2 or earlier spontaneous miscarriages, the presence of pregnancies was confirmed by ultrasound data and/or pregnancy hormones.

Criteria for exclusion from the project, according to -disorders of luteal phase in the results of endometrium biopsy, uterine anatomical abnormalities diagnosed by hysterosalpingography, hysteroscopy or sonohysteroscopy, carriers of balanced chromosomal abnormalities by karyotyping of both spouses, presence of antiphospholipid syndrome, confirmed by the analysis of anti-beta2-glycoproteinI (IgGorIgM) antibodies, anti-cardiolipin (IgGorIgM) antibodies, lupus anticoagulant; multiple pregnancies, confirmed by ultrasound, presence of sexually transmitted infections, confirmed by two different analyses of various biological materials (IgG or IgM; PCR, smear, PCR real-time), dysfunction of thyroid gland according to analyses of TSH and thyroid antibodies.

DNA isolation was performed by separating M-PVA magnetic particles on a Prepitto automatic analyzer (PerkinElmer) to isolate ChemagicPrepito nucleic acids (Wallac, Finland) using the PrepitoDNACytoPure reagent kit.

Molecular genetic studies were performed by TaqMan using a single site-specific amplification and real-time genotyping (Real-Time PCR) on a StepOnePlus instrument (AppliedBiosystems, USA) using test systems (TestGene, Russia).

Table 1 shows polymorphisms of the studied genes with an identifier (SNP Identifier), location of polymorphism on the chromosome - the physical distance in paired bases (base-pair position - bp), name of polymorphism.

Table 1. Description of studied polymorphisms of coagulation and cardiovascular system genes.

Statistical data processing was performed using the PLINK program. Comparative analysis of allelic and genotypic frequencies was carried out in the main group with iRLP and the group with normal reproduction using the Pearson χ2 test and/or t - Fisher test. The odds ratios (OR) and 95% confidence interval (95% CI) were calculated using unconditional logistic regression analysis.

Statistical analysis included the calculation of associations based on various models - genotypic, additive, allelic, dominant and recessive.

Obtained Results:

Due to the lack of GWAS studies of iRPL, we used the results of the GWAS meta-analysis of the search for candidate RPL genes. In a systematic review, 428 case-finding studies (1990–2015) were analyzed, which differed significantly in the definition of RPL, the clinical evaluation of patients and the choice of control group^{6,7,9,12,16,17}.

Association with iRPL, defined as two or more spontaneous abortions without apparent etiology, was found for 13 gene polymorphisms of coagulation and cardiovascular system, immune response, angiogenesis, chromosomal segregation, and placental function.

This article presents the results of studying associations of coagulation and cardiovascular system genes with iRPL.

Analysis of allelic and genotypic frequencies in the main group with iRPL and the control group with normal reproduction is presented in Table 2. As presented in Table 2, significant differences in allelic and genotypic frequencies of polymorphisms of blood coagulation and cardiovascular system genes are MTHFR (C677T, A1298C), MTR (A2756G), MTRR (A66G), F5 (A506G), F2 (G20210A), FGB (G455A), ITGB3 (Leu33Pro), AGTR1 (A1166C), ACE (I/D), GPIa (C807T), PLANH1 (5G/4G), eNOS (Glu298Asp) in the compared group of patients with iRPL and control, not detected (p>0.05).

Table 2. Allele and genotype frequencies in the main group with iRPL and control group.

The absence of statistically significant differences in the compared groups by coagulation and cardiovascular system genes: MTHFR (C677T, A1298C), MTR (А2756G), MTRR (A66G), F5 (А506G), F2 (G20210А), FGB (G455A), ITGB3 (Leu33Pro), AGTR1 (A1166C), АСЕ (I/D), GPIa (C807T), PLANH1 (5G/4G), eNOS (Glu298Asp) due to the fact that presence of thrombophilia is an antiphospholipid syndrome, confirmed by analysis anti-beta2-glycoproteinI (IgGorIgM) antibodies, anti-cardiolipin (IgGorIgM) antibodies, lupus anticoagulant; were criteria for exclusion from recruitment of women with iRPL. The obtained results do not confirm the possible contribution of coagulation system genes and indicate the presence of another etiopathogenetic factor in the development of iRPL.

Association calculations of studied polymorphisms using various models.

Statistical analysis using PLINK includes the calculation of associations based on various models. The allelic model is based on strength evaluation of allelic frequencies association is the simplest test, but it does not take into account the general genotype of two chromosomes; therefore, we used more accurate models of genotypic tests. The genotypic test for SNP association polymorphisms with the risk of iRPL is based on the use of frequencies of three possible genotypes in the main and control groups. The unit of calculation is not allele, but three possible genotypes with df =2. The additive (trend) model suggests that the presence of two copies of the minor allele in homozygous unfavorable AA genotype is two times more associated with iRPL than single allele in heterozygous genotype. The basis of the additive mathematical model is that the more copies of the minor allele are in the study group, the greater the adverse effect on the risk of iRPL development is heterozygotes having phenotypes lying between two homozygotes. This test has 1 df and is known as the "Cochran-Armitage Trend Test".

The dominant model suggests that disease manifests itself only if there is at least one copy of the adverse allele. All subjects are classified into two groups, depending on whether or not there is a minor allele, the dominant test has 1 df. The recessive model suggests that the effect on phenotype is manifested only if the subject has two copies of the minor allele, the number of freedom degrees =1. The most significant if we are not sure of a genetic model of the association between genotypes and phenotype is the additive model, which is less based on the principle of inheritance but is statistically less effective due to the additional degree of freedom.

Table 3 presents the results of a comparative associations’ analysis of studied polymorphisms with iRPL based on the use of several models at a threshold of significance at p=0.05 for multiple testing.

Table 3. Comparative analysis of studied associations polymorphisms of coagulation and cardiovascular system.

As can be seen from Table 3, statistically significant associations of iRPL, which imply a specific relationship between genotype and phenotype, include statistically highly significant differences in genotypic, additive (trend), allelic, general recessive and dominant models, were not detected for the studied polymorphisms (p˃0.05).

Low reliable associations of A66G polymorphism (rs1801394) of MTRR folate metabolism gene with iRPL were detected using a genotypic model (χ2 = 4.174; p = 0.041). The absence of similar results for other models suggests a random trend associated with a high POPULATION frequency of the adverse minor allele in this polymorphism, which was 0.43.

Discussion:

Despite numerous scientific studies of possible causes of RPL, such as fetal chromosomal abnormalities, infectious agents, adverse environmental factors, bad habits, anatomical defects, thrombophilic disorders, etc., etiology of RPL (up to 50% of cases) remains uncertain^1,5-10. These cases of RPL have no explainable etiology and effective therapy, require an in-depth study of their etiopathogenesis and are considered idiopathic RPL (hereinafter iRPL).

The lack of large-scale GWAS research on iRPL is due to several objective reasons: lack of clear definitions of iRPL, difficulty in recruiting and small sample size; lack of replicative studies in ethnically homogeneous populations^6,7,9,11,12.

Published systematic scientific reviews [8–17] of more than 80 studies of associations of genetic polymorphisms with iRPL, such as genes of hereditary thrombophilia, pro-inflammatory cytokine genes, angiogenesis genes, and placental function genes, did not confirm the unambiguous connection of studied polymorphisms and iRPL. The question of choice validity of these genetic polymorphisms, based on the modern understanding of the physiology of implantation processes, which is a long and complex process of balanced interaction between the mother and the fetus, mediated through the placenta^10-17 is discussed.

Disorders of this process at all stages can lead to abortion, which led to our choice of specific polymorphisms of the maternal genome responsible for disorders of decidualization and endometrial angiogenesis^18-20.

Due to the high frequency of iRPL, its significant contribution to reproduction and fertility rates, genetic causation and lack of reliable data on genetic markers that would predict the development of iRPL, a replicative study was conducted in an ethnically homogeneous population of Kazakhs with clear criteria for recruiting and choosing etiopathogenetic polymorphisms of iRPL.

The aim of the study was to evaluate the genetic contribution of 13 potentially significant polymorphisms of coagulation and cardiovascular system genes: MTHFR (C677T, A1298C), MTR (A2756G), MTRR (A66G), F5 (A506G), F2 (G20210A), FGB (G455A) ITGB3 (Leu33Pro), PLANH1 (5G/4G); GPIa (C807T), AGTR1 (A1166C), ACE (I/D), eNOS (Glu298Asp) in development of iRPL in ethnically homogeneous population of Kazakhs.

Conclusion:

The results of independent replicative TaqMan genotyping of 302 patients with iRPL and 300 women with normal reproduction did not reveal an association of studied polymorphisms of coagulation and cardiovascular system genes with the development of iRPL in the Kazakh population. The obtained results are consistent with the data of replicative genotyping in other ethnic populations^13,21, with published meta-analyzes^8,22-25.

The obtained contradictory results in different ethnic populations may reflect the methodological errors associated with an insufficient sample size, ethnic heterogeneity and the methodology of research conducted.

Our study on a sufficient sample in ethnically homogeneous groups, using international diagnostic criteria, will make a certain contribution to the search for genetic associations of iRPL in independent human populations.

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