RU2611358C1 - Method of predicting high risk of reproductive loss in first trimester of pregnancy - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to field of medicine, in particular to obstetrics and gynecology, and is intended for prediction of high risk of reproductive loss in first trimester of pregnancy. From peripheral venous blood of women DNA is extracted with the following amplification of polymorphous variants of A66G of gene MTRR, -31C-T (rs1143627) IL-1β, -174G-C (rs1800795) IL-6 and C677T (rs1801133) MTHFR. In case of detection of one of four genotypes: -31CT IL-1β / -174GG IL-6 / 677CT MTHFR / 66GG MTRR; -31CT IL-1β / -174CC IL-6 / 677CC MTHFR / 66AG MTRR; -31CC IL-1β / -174CC IL-6 / 677CC MTHFR/ 66AG MTRR; -31CC IL-1β / -174GC IL-6 / 677CT MTHFR/ 66AG MTRR, high risk of reproductive loss is predicted in first trimester of pregnancy.

EFFECT: invention makes it possible to form among women groups of risk of reproductive loss in first trimester of pregnancy and timely realise necessary thethapeutic-preventive measures in these groups.

1 dwg, 1 tbl, 7 ex

Description

FIELD OF THE INVENTION

The present invention relates to the field of molecular biology and genetics and can be used in obstetrics and gynecology as genetic markers in the diagnosis of reproductive pathologies of the first trimester of pregnancy both at the stage of pregnancy planning and at the preclinical stage.

State of the art

Reproductive loss in humans is about 50% in relation to the total number of conceptions. The rate of miscarriage remains high, despite the successes achieved in the prevention and treatment of human reproductive disorders. Currently, various types of spontaneous abortion are considered as multifactorial diseases, the development of which can be triggered by a combination of several factors. The individual contribution of each factor may be insignificant, and only their sum leads to the development of the disease. The vast majority of work aimed at studying the genetic causes of miscarriage is associated with the study of allelic variants of genes of one functional gene network, for example, folate cycle genes, xenobiotic detoxification genes, genes encoding coagulation factors of the blood system. At the same time, it is known that the early stages of embryonic development are based on the mechanisms underlying the functioning of several functional groups of proteins and genes encoding them.

From the existing level of technology for assessing the risk of reproductive losses due to chorionic and placental abruption in the first trimester of pregnancy, a method is known which consists in the amplification of allelic variants - 675 5G / 4G gene of type 1 plasminogen activator inhibitor using the PNR method (RU 2494400, publ. 27.09.09 .2013) / 1 /. The probability of developing chorionic and placental abruption in women with a high risk of spontaneous reproductive loss with the 4G / 4G genotype is predicted as 50%, with the 4G / 5G genotype as 35.3%. However, this method takes into account the influence of only one gene, while the pathogenesis of early embryonic losses is complex and multicomponent. In addition, the method is characterized by a limited contingent for possible use, since the invention determines the possibility of predicting the development of chorionic and placental detachment only in women with a high risk of spontaneous reproductive loss.

A known method of assessing a predisposition to the development of habitual miscarriage (RU 2330071 C1, publ. 27.07.2008) / 2 /. The method involves the analysis of a combination of C677T polymorphisms of the MTHFR gene and R353Q gene of coagulation factor VII gene and drawing a conclusion about the risk of developing habitual miscarriage. The RRTT genotype qualifies as extremely unfavorable. The limitation of this method is to take into account the influence of only two genes that increase the risk, primarily thrombotic complications, and do not take into account the role of immunological factors in the development of pregnancy.

Based on a combination of cytokine genes, a method is known for predicting the risk of severe preeclampsia (RU 2568892 C1, publ. 11/20/2015) / 3 /, which involves the extraction of DNA from the peripheral blood of women and the analysis of polymorphisms -308G / A TNFα, + 36A / G TNFR1 , -801G / A SDF1, C / G MCP-1. An increased risk of severe preeclampsia is predicted with a combination of the genetic variants -308A TNFα, + 36GG TNFR1 and -801A SDF1. The limitation of this method is the prediction of pregnancy complications only from the second trimester.

There is a method of determining a hereditary predisposition to the development of habitual miscarriage (RU 2532367 C2, publ. 10.11.2014) / 4 /, carried out by isolating genomic DNA from blood samples of the subject, followed by genotyping of polymorphic variants 34V / L of the FXIII, M235T gene of the AGT gene, 4a / b of the eNOS gene, PLA1 / A2 of the GpIIIa gene and 353R / Q of the FVII gene. In this method, after the establishment of genotypes for all 5 genes, the effect of the identified alleles on the formation of a predisposition to habitual miscarriage is determined by calculating the total score. A total score of less than 0.16 is the basis for classifying the subject as a low-risk group, a value of 0.16 to 0.4 points is a group with an average risk, and a value of more than 0.4 points is a group with a high risk of formation habitual miscarriage. This method takes into account the effect of only polymorphisms of hemostasis system genes, which does not allow an objective assessment of the risk of miscarriage.

The closest to implementation of the claimed invention is a method for predicting chorionic detachment in the first trimester of pregnancy (RU 2566729 C1, publ. 10.27.2015) / 5 /, taken as a prototype in which DNA is extracted from the patient’s blood with subsequent amplification of polymorphic Arg353Gln variants ( G10976A) of the coagulation factor VII gene and Ile22Met (A66G) of the MTRR reductase methionine synthase gene using the PCR method. If a combination of the GA heterozygosity of the polymorphic variant Arg353Gln (G10976A) of the coagulation factor VII gene with the homozygote GG of the polymorphic variant Ile22Met (A66G) of the MTRR reductase methionine synthase gene is predicted, the risk of chorion detachment in the first trimester of pregnancy is revealed.

The limitation of the prototype method is that chorion detachment is only one of the possible complications of the gestational period of the first trimester, moreover, chorion detachment in the first trimester of pregnancy is possible in the absence of a combination of polymorphisms of the coagulation factor VII gene and the MTRR reductase methionine synthase gene. In the prototype method, the role of only factors affecting the coagulation processes is taken into account, while the early stages of ontogenesis are determined by the interaction of factors of several functional groups, which does not allow an objective assessment of the risk of reproductive losses of the first trimester.

Disclosure of invention

The technical result of the present invention is to increase the reliability of predicting a high risk of reproductive loss in the first trimester of pregnancy by using a new combination of genetic variants 677CT MTHFR, 66AG MTRR, -31CT IL-1β and -174GC IL-6, i.e. genotype for 4 allelic variants of genes of two functional groups - pro-inflammatory cytokines and folate cycle.

The specified technical result is achieved in that a method for predicting a high risk of reproductive loss in the first trimester of pregnancy involves the extraction of DNA from the peripheral venous blood of women, followed by amplification of the polymorphic variant A66G of the methionine synthase reductase gene MTRR.

According to the invention, allelic variants of the pro-inflammatory cytokine -31C-T (rs1143627) IL-1β, -174G-C (rs1800795) IL-6 gene and folate cycle enzymes C677T (rs1801133) MTHFR are amplified and one of the four genotypes is identified:

-31CT IL-1β / -174GG IL-6 / 677CT MTHFR / 66GG MTRR;

-31CT IL-1β / -174CC IL-6 / 677CC MTHFR / 66AG MTRR;

-31CC IL-1β / -174CC IL-6 / 677CC MTHFR / 66AG MTRR;

-3ICC IL-1β / -174GC IL-6 / 677CT MTHFR / 66AG MTRR

predict a high risk of reproductive loss in the first trimester of pregnancy.

The prior art does not know the possibility of predicting the occurrence of reproductive losses in the first trimester of pregnancy by the presence of combinations of genetic variants of polymorphisms -31C-T IL-1β (rs1143627), -174G-C IL-6 (rs1800795), C677T MTHFR (rs1801133), A66G MTRR (rs1801394). The use of genetic markers of two functional groups increases the reliability of the diagnosis of reproductive pathologies of the first trimester of pregnancy both at the stage of pregnancy planning or at the preclinical stage of reproductive pathology.

The drawing shows an electrophoregram of the amplification results of the allelic variants -31C-T (rs1143627) of the IL-1β gene.

The table shows the prevalence of combinations of genetic variants of cytokine genes and folate cycle enzymes among women in the control group and women with reproductive losses in the first trimester of pregnancy.

The implementation of the invention

DNA is isolated from samples of peripheral venous blood of women by the thermocoagulation method using the commercial DNA-Express-blood reagent kit (Litech, Russia) according to the manufacturer's instructions.

1000 μl of women's whole blood is added to an Eppendorf tube, the sample is centrifuged at a speed of 3000 rpm for 5 minutes. Gently remove the plasma. Close the tube and freeze it at -20 ° C until the shaped elements are completely frozen for at least 1 hour. Thaw the contents of the tube at room temperature. Add the DNA-express-blood reagent into the test tube with a volume equal to the volume of the formed elements remaining in the test tube. The contents of the tubes are thoroughly mixed on a vortex. Install the tube in a thermostat preheated to 99 ° C and incubate for 15 minutes. At the end, cool to 70 ° C. Centrifuge the tube at 12,000 rpm at room temperature for 60 seconds. The resulting supernatant is used as a test sample of DNA.

The isolated DNA is then subjected to polymerase chain reaction using SNP-express reagent kits (Litech, Russia) according to the manufacturer's instructions:

1. Prepare numbered tubes for amplification with a capacity of 0.6 ml in accordance with the number of analyzed samples plus a negative control. For each sample, two test tubes are prepared: “norm” and “polymorphism”.

2. From the components of the SNP-express kit, working mixtures of reagents are prepared for amplification per 1 sample: 17.5 μl of diluent, 2.5 μl of the reaction mixture, 0.2 μl of Taq polymerase. Two working mixtures are prepared: with the norm reaction mixture and the polymorphism reaction mixture.

3. Add 20 μl of the appropriate working amplification mixture to all tubes prepared for amplification.

4. Add one drop (25 μl) of mineral oil to all tubes.

5. In a test tube with a working amplification mixture “norm” and in a test tube with a working mixture “polymorphism”, 5 μl of a DNA sample is added.

6. Sealed tubes are rapidly vortexed.

7. Transfer the tubes to a programmable thermostat heated to 94 ° C and amplify in the following mode: 94 ° C - pause; 93 ° C - 1 minute; 93 ° C - 10 s, 64 ° C - 10 s, 72 ° C - 20 s, a total of 35 cycles and 72 ° C - 1 minute at the end of the process.

The detection (separation) of amplification products of allelic variants of the pro-inflammatory cytokines -31C-T IL-1β (rs1143627), -174G-C IL-6 (rs1800795) and folate cycle enzymes C677T MTHFR (rs1801133), A66G MTRR (rs1801394phore) is carried out in a 3% agarose gel and fix the electrophoregram on a GelDoc transilluminator (BioRad (see drawing). As follows from the electrophoregram, in amplification wells No. 1, 3, 5, 7, 9 of the first lane, the amplification mixture with primers was added to the “normal” allele of the test gene to wells No. 2, 4, 6, 8, 10 - amplification mixture with primers on limorphic version of the gene. DNA samples of the studied women were added to wells No. 1-2, 3-4, 5-6, 7-8, 9-10. DNA of one was added to paired wells (for example: 1-2, or 3-4) the same person. In the wells No. 1, 3, 5, 7, 9, the reaction went through - in these individuals a “normal” allele of the studied gene was detected. In wells No. 2, 4, 6, 10 no amplification products were detected, which indicates the absence polymorphic variant of the gene In well 8, a PCR product corresponding to the polymorphic variant of the gene was detected.

Then associations of combinations of genetic variants with early reproductive losses by the bioinformatics method are revealed - Multifactor Dimensionality Reduction (MDR program, v. 1.1.0 www.epistasis.org/mdr.html) for modeling high-order genomic interactions when studying the nature of intergenic interactions for population-genetic studies of multifactorial polygenic diseases using relatively small sample sizes of patients and healthy. The risk of developing reproductive losses was judged by the odds ratio (OR). OR is indicated with a 95% confidence interval (CI) (see table). Differences were considered statistically significant at a significance level of p <0.05.

Identified variants of genotypes:

-31CT IL-1β / -174GG IL-6 / 677CT MTHFR / 66GG MTRR;

-31CT IL-1β / -174CC IL-6 / 677CC MTHFR / 66AG MTRR;

-31CC IL-1β / -174CC IL-6 / 677CC MTHFR / 66AG MTRR;

-31CC IL-1β / -174GC IL-6 / 677CT MTHFR / 66AG MTRR

indicate a high risk of reproductive loss.

The possibility of using the proposed method for predicting a high risk of early embryonic losses is confirmed by the results of observations of 141 patients with miscarriage of the first trimester and 120 women of the control group. The average age of women was 29 years. The control group included only those women who had no previous history of miscarriage.

The collection of anamnestic data and the formation of the studied groups of women was carried out by employees of the Department of Obstetrics and Gynecology of Rostov State Medical University, as well as obstetrician-gynecologists of the city hospital No. 8 of Rostov-on-Don and the maternity hospital No. 5.

Laboratory studies were conducted at the Laboratory of Human and Animal Genetics of the Southern Federal University. The study was approved by the Bioethics Committee of the Southern Federal University.

Allelic variants of the 31C-T genes IL-1β (rs1143627), -174G-C IL-6 (rs1800795) C677T MTHFR (rs1801133), A66G MTRR (rs1801394) were selected based on the results of scientific publications taking into account their pathogenetic contribution to the formation of early reproductive losses , as well as the results of our own research on the genotyping of women with reproductive losses in the first trimester.

A significant contribution to the pathogenesis of early embryonic losses is made by immune and autoimmune factors (Kerchelaeva SB, 2003) / 6 /. Immunological disorders can be due to the characteristics of the genotype, including the cytokine genes, which are endogenous mediators of intercellular interactions. The early stages of embryogenesis depend on the ratio of the functional activity of pro- and anti-inflammatory cytokines, mediating the maternal organism’s immune response to the developing fetus, controlling the processes of placenta formation and angiogenesis (T. Amchislavsky et al., 2003) / 7 /. The activity of immunological reactions, inflammation reactions can be determined by the features of the functioning of the folate cycle.

Cytokines represent a group of polypeptides whose main function is to provide interactions between cells. Cytokines are involved in the regulation of various physiological processes; enter the control system for cell survival, activate or inhibit their growth, differentiation. Cytokine-dependent processes are the stages of egg maturation, fertilization, adhesion, implantation, and the formation of the placenta. All these processes involve active intercellular interaction involving several families of cytokines. The most important of them are the cytokines of the family of IL-6, TGF-β, IL-1, as well as chemokines (Dimitriadis E. et al., 2005) / 8 /.

Violation of the intensity of inflammatory reactions, including due to the characteristics of the genotype for cytokine genes, may be a risk factor for pregnancy (Daher S., 2012) / 9 /. For example, excessive synthesis of IL-1β can lead to significant intensification of inflammatory reactions. Pro-inflammatory cytokines can act as an important link between inflammation and hypercoagulation (Yoshida N., 2009) / 10 /. Inflammation leads to an imbalance between pro- and anticoagulation reactions. Activation and binding of platelets to endothelial cells and leukocytes in the area of inflammation contributes to a procoagulant state, as it activates blood coagulation factors and enhances the formation of thrombin (Levi M. et al., 2005) / 11 /.

Genetic factors associated with an increased risk of thrombosis include folate cycle genes (methylenetetrahydrofolate reductase (MTHFR), methionine synthase reductase (MTRR), methionine synthase (MTR)). In human cells, folates are necessary for the synthesis of nucleotides, amino acid metabolism, methylation of nucleic acids and proteins. Folate deficiency, regardless of the factors causing it, leads to hyperhomocysteinemia, which can cause the development of a number of pathological conditions due to increased expression of proinflammatory cytokines, induction of oxidative stress, activation of apoptosis, and disruption of methylation processes (Jacques P. et al., 2001) / 12 /. It was shown that a high concentration of homocysteine can induce the expression of IL-6 by monocytes (Su S. et al., 2005) / 13 /. An increased level of homocysteine during pregnancy can lead to circulatory disturbances in the placenta, implantation defects, a decrease in trophoblast invasion depth, and a disturbance in the normal development of fetoplacental circulation.

Causes of folate metabolism disorders may be due to the presence of polymorphic gene variants. The relationship of single nucleotide substitutions in the folate cycle enzyme genes with the development of pregnancy pathology has been shown (Wu X. et al., 2012) / 14 /. According to the literature, it is known that hyperhomocysteinemia creates conditions for the intensification of free-radical reactions, as well as reactions of the inflammatory response (Lazzerini P. et al., 2007) / 15 /.

Genetic studies of reproductive pathology have several components. Including the identification of DNA / RNA markers that have prognostic value for assessing the risk of miscarriage. Genotyping on one polymorphism does not allow to obtain a result with high prognostic significance. The prognostic significance of the study increases with the inclusion of allelic variants of several functional groups of genes that control key processes of early embryonic development. Polymorphisms of such genes form various combinations that enhance or weaken the effect of any particular genetic polymorphism. The inclusion of polymorphisms of the IL-1β, IL-6, MTHFR, MTRR genes in the program of genetic testing of women will increase the reliability of predicting a high risk of reproductive loss in the first trimester of pregnancy.

As follows from the table, two combinations of genetic variants

-31CT IL-1β / -174GG IL-6 / 677CT MTHFR / 66GG MTRR and

-31CT IL-1β / -174CC IL-6 / CC677 MTHFR / 66AG MTRR occur with the same frequency (6.4%) among women with reproductive losses in the first trimester of pregnancy, while in the control group two of these genotypes were not found. These combinations of allelic variants of cytokine genes and the folate cycle are risk factors for reproductive loss of the first trimester, since the odds ratio for these two genotypes was 17.3 (CI: 1.0-300.3, p = 0.01).

A combination of genetic variants (-31CC IL-1β / -174CC IL-6 / CC677 MTHFR / 66AG MTRR) occurs in 7.8% of women with reproductive losses in the first trimester of pregnancy, while this genotype was not detected in the control group. This combination of allelic variants of cytokine genes and the folate cycle is a risk factor for reproductive losses of the first trimester of pregnancy, since the odds ratio for this genotype was 21.2 (CI: 1.2-364.5, p = 0.005).

The combination of genetic variants (-31CC IL-1β / -174GC IL-6 / CT677 MTHFR / 66AG MTRR) occurs in 9.9% of women with reproductive losses in the first trimester of pregnancy, which is 3.9 times higher than the same indicator in the control group. This combination of allelic variants of cytokine genes and the folate cycle is a risk factor for reproductive losses of the first trimester of pregnancy, since the odds ratio for this genotype was 4.3 (CI: 1.2-15.3, p = 0.03).

From the results of statistical processing of the obtained data, it follows that the genetic polymorphisms -31C-T (rs1143627) IL-1β, -174G-C (rs1800795) IL-6, C677T (rs1801133) MTHFR, A66G (rs1801394) MTRR play a significant role in the formation of genetic predisposition to a high risk of reproductive loss in the first trimester of pregnancy. In this case, a combination of polymorphic options:

-31CT IL-1β / -174GG IL-6 / 677CT MTHFR / 66GG MTRR;

-31CT IL-1β / -174CC IL-6 / 677CC MTHFR / 66AG MTRR;

-31CC IL-1β / -174CC IL-6 / 677CC MTHFR / 66AG MTRR;

-31CC IL-1β / -174GC IL-6 / 677CT MTHFR / 66AG MTRR

associated with a high risk of reproductive loss in the first trimester of pregnancy.

The presence of one of these genotypes confirms the high probability of reproductive loss in the first trimester of pregnancy with the following examples.

Example 1

Patient A., Russian, born in 1981. At the time of the study, the woman was diagnosed with the first pregnancy, which stopped developing for a period of 6 weeks. The patient received venous blood; genotyping of DNA markers revealed a combination of the studied polymorphisms of cytokine genes and folate cycle enzymes: -31CT IL-1β / -174GG IL-6 / 677CT MTHFR / 66GG MTRR, which confirms the high probability of reproductive loss in the first trimester.

Example 2

Patient S., Russian, born in 1992. He has a history of 1 birth, ending in the birth of a living child. At the time of the study, the woman had a spontaneous abortion at 7 weeks of gestation. The patient received venous blood; genotyping of DNA markers revealed a combination of the studied polymorphisms of cytokine genes and folate cycle enzymes: -31CT IL-1β / -174GG IL-6 / 677CT MTHFR / 66GG MTRR, which confirms the high probability of early reproductive losses.

Example 3

Patient U., Russian, born in 1983. He has a history of 1 birth, ending in the birth of a living child. At the time of the study, the woman had a spontaneous abortion at 7 weeks of gestation. The patient received venous blood; genotyping of DNA markers revealed a combination of the studied polymorphisms of cytokine genes and folate cycle enzymes: -31CT IL-1β / -174CC IL-6 / 677CC MTHFR / 66AG MTRR, which confirms the high probability of reproductive losses in the first trimester.

Example 4

Patient B., Russian, born in 1987. At the time of the study, the woman was diagnosed with the first pregnancy, which stopped developing for a period of 7 weeks. The patient received venous blood; genotyping of DNA markers revealed a combination of the studied polymorphisms of cytokine genes and folate cycle enzymes: -31CC IL-1β / -174CC IL-6 / 677CC MTHFR / 66AG MTRR. Thus, a genotype of an increased risk of early reproductive loss was identified.

Example 5

Patient I., Russian, born in 1984. In the history of women, childbirth is absent. At the time of the study, the woman was diagnosed with a second pregnancy, which stopped developing for 10 weeks. The patient received venous blood; genotyping of DNA markers revealed a combination of the studied polymorphisms of cytokine genes and folate cycle enzymes: -31CC IL-1β / -174CC IL-6 / 677CC MTHFR / 66AG MTRR. Thus, a genotype of an increased risk of reproductive loss in the first trimester was identified.

Example 6

Patient M., Russian, born in 1989. He has a history of 1 birth, ending in the birth of a living child. At the time of the study, the woman had a spontaneous abortion at 6 weeks of gestation. The patient received venous blood; genotyping of DNA markers revealed a combination of the studied polymorphisms of cytokine genes and folate cycle enzymes: -31 SS IL-1β / -174GC IL-6 / 677ST MTHFR / 66AG MTRR, which confirms the high probability of early reproductive losses.

Example 7

Patient O., Russian, born in 1978. He has a history of 1 birth, ending in the birth of a living child. At the time of the study, the woman was diagnosed with 4th pregnancy, which spontaneously ended at 9 weeks. The patient received venous blood; genotyping of DNA markers revealed a combination of the studied polymorphisms of cytokine genes and folate cycle enzymes: -31CC IL-1β / -174GC IL-6 / 677ST MTHFR / 66AG MTRR, which confirms the high probability of reproductive loss in the first trimester.

The application of the claimed method will allow to form risk groups among women at the stage of pregravid preparation and to timely implement the necessary preventive measures in these groups.

Information sources

1. RU 2494400, IPC G01N 33/50, publ. 09/27/2013.

2. RU2330071 C1 IPC C12Q 1/68, publ. 07/27/2008.

3. RU 2568892 C1, IPC G01N 33/52, C12Q 1/68, publ. 11/20/2015.

4. RU 2532367С2, IPC G01N 33/68, publ. 11/10/2014.

5. RU 2566729 C1, IPC G01N 33/68, publ. 10/27/2015.

6. Kerchelaeva S.B. The value of antibodies to phospholipids and phospholipid-binding proteins during an undeveloped pregnancy // Russian Bulletin of the Obstetrician-Gynecologist. 2003. - T. 4. - S. 11-16.

7. Amchislavsky E.I., Sokolov D.I., Starikova E.A. Cytokine control of the process of angiogenesis. Medical Immunology 2003, T. 5, No. 5-6, S. 493-506.

8. Dimitriadis E., White S., Jones R. Cytokines, chemokines and growth factors in endometrium related to implantation // Human Reproduction Update. - 2005. - V. 24. - P. 612-628.

9. Daher S., Mattar R., Gueuvoghlanian-Silva BY. Genetic polymorphisms and recurrent spontaneous abortions: an overview of current knowledge // Am J Reprod Immunol. - 2012. - V. 67. - P. 341-347.

10. Yoshida H., Granger D.N. Inflammatory bowel disease: a paradigm for the link between coagulation and inflammation // Inflamm Bowel Dis. - 2009. - V. 15. - P. 1245-1255.

11. Levi M., Van der Poll T. Two-way interactions between inflammation and coagulation // Trends Cardiovasc Med. - 2005. - V. 15. - P. 254-259.

12. Jacques P., Bostom A., Wilson P. Determinants of plasma total homocysteine concentration in the Framingham Offspring cohort // Am J Clin Nutr. - 2001. - V. 73. - P. 613-621.

13. Su S.J., Huang L.W., Pai L.S., Liu H.W. Homocysteine at pathophysiologic concentration activates human monocyte and induces cytokine expression and inhibits macrophage migration inhibitory factor expression // Nutrition. - 2005. - V. 21. - P. 994-1002.

14. Wu X., Zhao L., Zhu H. Association between the MTHFR C677T polymorphism and recurrent pregnancy loss: A Meta-analysis // Genet Test Mol Biomarkers. - 2012. - V. 7. - P. 806-811.

15. Lazzerini P.E. Hyperhomocysteinemia, inflammation and autoimmunity // Autoimmun Rev. - 2007.- V. 6 (7). - P. 503-509.

Claims (6)

A method for predicting a high risk of reproductive loss in the first trimester of pregnancy, including the extraction of DNA from the peripheral venous blood of women, followed by amplification of the polymorphic variant A66G of the MTRR gene for methionine synthase reductase, characterized in that the amplification of allelic variants of the pro-inflammatory cytokine -31C-T genes (rs1143627) IL- 1β, -174G-C (rs1800795) IL-6 and folate cycle enzymes C677T (rs1801133) MTHFR and when one of the four genotypes is detected: -31CT IL-1β / -174GG IL-6 / 677CT MTHFR / 66GG MTRR; -31CT IL-1β / -174CC IL-6 / 677CC MTHFR / 66AG MTRR; -31CC IL-1β / -174CC IL-6 / 677CC MTHFR / 66AG MTRR; -31CC IL-1β / -174GC IL-6 / 677CT MTHFR / 66AG MTRR predict a high risk of reproductive loss in the first trimester of pregnancy.

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