The role of methylentetrahydrofolate reductase (MTHFR) gene polymorphism in intrauterine growth restriction development

Recently, studies on the role of folic acid in the pathogenesis of the gestation complications have been widely distributed [1]. Today, there is no doubt that folic acid is one of the most important micronutrients involved in the formation of the fetus tissues and organs during critical periods of pregnancy. Lack of folates during pregnancy is associated with such complications as malformations, delayed growth of the fetus, habitual abortion, antenatal fetal death, premature detachment of the placenta and premature birth [1–3].

According to modern studies, most people consume an insufficient amount of folate with food [3, 4]. Recommendations for pregnant women and puerperas include daily folate intake from 400 to 800 µg [1, 3]. However, the degree of absorption and utilization of folate and folic acid does not always depend on the nature of the food consumed. The cooking and the state of the gastrointestinal tract can directly affect the degree of folic acid absorption, as well as some drugs (anti-inflammatory drugs, folic acid antimetabolites, hypoglycemic, diuretic and sulfanilamide medicines) [2, 5, 6].

Some authors note the influence of genetic factors in the development of pathological conditions in the gestation period. Indeed, polymorphisms of tumor necrosis factor-α, interleukin-6, interleukin-10 genes are associated with a high risk of preeclampsia, fetal metabolic and respiratory distress syndromes. The development of placental insufficiency (PI) and intrauterine growth restriction (IUGR) are associated with the polymorphism of several genes, one of which is methylenetetrahydrofolate reductase (MTHFR) [7, 8].

The MTHFR enzyme plays a key role in folic acid metabolism and DNA synthesis, ensuring the conversion of the homocysteine amino acid to methionine. The decrease of the MTHFR enzyme activity leads to the accumulation of homocysteine in plasma and the deposition of homocysteine on the vascular endothelium [5, 9, 10]. According to current knowledge, the regulation of enzyme activity is due to a number of polymorphisms. The clinical manifestations of this dependence are expressed in some pathological states of the gestational process [11–14]. Of particular interest are studies on MTHFR gene polymorphism: 677 C>T (Ala222Val).

In recent years, a link has been established between folic acid deficiency and pathology of the placenta, and the need in this vitamin is selectively high for it. The risk of placental bleeding, placental detachment, premature birth, and growth retardation increases [11, 15]. At the same time, a number of studies have shown that supplemental folic acid intake favorably affects the pregnancy course and fetal mass and length [12, 15].

The objective of the study is to determine the role of the MTHFR gene polymorphism in the IUGR syndrome development.

Materials and methods

The study included 248 pregnant women (case-control) who were divided into 2 groups: pregnant women with IUGR were included in group I (n = 100), and pregnant women without signs of IUGR were included in group II (n = 148). At the second stage, MTHFR genes polymorphisms and their dependence with the IUGR development were studied. At the third stage, the impact of folic acid intake during the gestation period and the frequency of the IUGR development were assessed based on the anamnesis data retrospectively.

DNA isolation was carried out using the method of R. Higuchi (R. Higuchi, H. Erlich, 1989) with some modifications. 0.5 ml of blood, with EDTA as an anticoagulant, was mixed in 1.5 ml of Eppendorf-type microcentrifuge tubes with 0.5 ml of a lysing solution consisting of 0.32 M sucrose, 10 mM Tris-HCl pH 7.5, 5 mM MgCl, 1% Triton X-100, centrifuged for 1 min at 10.000 rpm, the supernatant was removed, and the cell nuclei were washed twice with the indicated buffer. The DNA concentration determined on a DNA minifluorimeter (Noahfer, USA) was on average 50–100 µg/ml . Estimation of single nucleotide substitutions was carried out with a modified method of “adjacent samples” (adjacent probes, kissing probes), using the original oligonucleotides. The genotype was determined by analyzing the melting curves. Polymerase chain reaction and determination of the melting point of oligonucleotide samples were carried out using a DT-96 detecting thermocycler (NPO DNK-Tekhnologiya LLC, Russia). The determination of polymorphic loci of the MTHFR gene was carried out: 677 C>T (Ala222Val).

All the results of observation and surveys were entered into a specially developed thematic file and MS Excel. Statistical processing of the results was carried out on a personal computer with the software package SPSS Statistics 17.0 for Windows. When assessing the reliability of the detected differences between the mean values of the samples and the reliability of the revealed correlation, the p-value was calculated. According to generally accepted terminology in analytical statistics, statements with p ≤ 0.05 are called significant; the statements with p ≤ 0.01 are very significant, and the statements with p ≤ 0.001 are the most significant. To determine the statistical significance of differences in the frequencies of alleles and genotypes in groups of patients, the χ2 test was used. Linkage disequilibrium analysis was performed using the free product Haploview software version 4.2. Data preparation for analysis was carried out using SNP_tools version 1.61, a free extension for MS Excel. Statistical data processing was carried out using the WINPEPI version 10.7, a freeware software product.

Results and Discussion

In accordance with the objectives of the first stage of the study, we conducted an analysis of the obstetric and gynecological and somatic health of pregnant women who gave a birth at the National Medical Research Center for Obstetrics, Gynecology and Perinatology named after academician V.I. Kulakov, Moscow. The features of case history, initial clinical characteristics, the course of pregnancy, childbirth and the postpartum period in patients with PI were studied in detail, the features of the early neonatal period were analyzed. In the study of the early neonatal period, the mass-growth rates of the newborns, the Apgar score on the 1st and 5th minutes were considered.

The age of pregnant women included in the study ranged from 18 to 45 years and averaged 30.9 ± 0.9 and 29.0 ± 0.8 years, respectively. In patients of group I, mitral valve prolapse prevailed among somatic diseases – 19.0% (OR = 0.5; CI = 0.2–0.8), varicose disease - 23.0% (OR = 0.5; CI = 0.3–0.8), myopia with astigmatism – 20.0% (OR = 0.5; CI = 0.3–0.9), osteochondrosis – 31.0% (OR = 1.5; CI = 1.3–1.8), scoliosis – 49% (OR = 0.16; CI = 0.04–0.58). Ovarian dysfunction was more common among gynecological diseases in this group of patients – 7.0% (OR = 2.6; CI = 1.1–6.4).

The first trimester of current pregnancy in patients of group I was more often complicated by mild or moderate vomiting - 32.0% (OR = 13.5; CI = 18.2–24.5), threatening abortion - 48.0% (OR = 3.5; CI = 1.4–11.2) with the formation of a retrochorial hematoma – 7.0% (OR = 5.5; CI = 1.2–27.3). During the second trimester, group I patients more often had threatened abortions and threatened very early preterm births – 36.0% (OR = 4.8; CI = 2.6–8.9); in 4.0% of cases, surgical correction of isthmic-cervical insufficiency was performed. The third trimester of pregnancy in patients of this group was complicated by threatening preterm labor - 25.0% (OR = 3.3; CI = 1.6–6.4); fetal hypoxia - 26.0% (OR = 5.2; CI = 2.4–10.9). Children of group I mothers had significantly lower mass-growth rates and a lower Apgar score at birth.

At the second stage, MTHFR gene polymorphisms were studied in pregnant women of all groups and their dependence with the IUGR development was determined. When studying the MTHFR gene polymorphism (677 C > T), the frequency of the allele C occurrence was significantly lower in the IUGR than in the control group, and the C/C genotype was not detected in any patient. These molecular genetic studies prove the desirability of using folic acid in IUGR risk population.

At the third stage, a retrospective analysis of the data from case history was carried out with an assessment of the effect of folic acid supplementation during the gestation period and the IUGR frequency.

Of the 248 pregnant women included in the study, 150 women received drugs containing folic acid. In group I, 70% of pregnant women additionally received folic acid because other patients (30%) refused to take additional “unnecessary drugs.” In group II, 80% of women included folic acid in the daily diet.

Based on the obtained data, there was no significant relationship between the IUGR and folate intake in both groups. However, when taking into account the MTHFR gene polymorphism model (677 C > T) (according to a molecular genetic study) with folic acid supplementation, a correlation was established between folate withdrawal and the IUGR development.

Thrombophilic conditions (antiphospholipid syndrome, hyperhomocysteinemia, MTHFR, factor V and prothrombin gene mutations) are one of the important causes of miscarriage and IUGR risk. They cause impaired fibrin-mediated implantation of a fertilized ovum in the decidual tissue and are also associated with thrombus formation in the vessels of the placenta, leading to impaired vital activity of the implanted gestational sac and the fetus in the later stages of pregnancy [9, 14, 15].

Based on the study, significant PI clinical risk factors were identified: myopia with astigmatism, mitral valve prolapse, varicose vein disease, osteochondrosis, scoliosis. Threatening abortion in the first trimester with the formation of retrochorial hematoma and isthmic-cervical insufficiency should be attributed to predisposing factors of the PI development. Non-invasive PI predictors (MTHFR genes polymorphism), which should be determined in pregnant women of risk groups, have been established. In the presence of the MTHFR gene polymorphism, it is advisable to use folic acid for the IUGR prevention.

Today, folacin represented in the pharmaceutical market is produced by Jadran Galenski Laboratorij (Republic of Croatia) and has the optimal composition for use throughout pregnancy and lactation. Folacin is one of the modern, most balanced and easy-to-use multivitamin medicines that belong to group B vitamins.

The chemical composition of folic acid and folacin is identical, but the content of vitamin B9 is higher in the latter. Therefore, folic acid tablets should be taken three times a day, and folacin is prescribed for once daily administration, that can be indisputable advantage. Also, this medicine has the following advantages: good tolerability, the impossibility of overdosage and drug poisoning, the rapid therapeutic effect. Overdosing is almost impossible, because folic acid is absorbed in the required amount only, and the excess vitamin is excreted by the kidneys without any harm. But there are some side effects: in rare cases, women may experience nausea, lack of appetite, abdominal distension, belching, air itching, dizziness, but they disappear with the drug withdrawal.

Conclusion

Thus, the foregoing makes it possible to recommend the drug folacin for prophylactic purposes to pregnant women who are at risk of IUGR, carriers of the MTHFR gene polymorphism (677 C>T). Taking this medicine at the appropriate times in the surveyed patient population will reduce the IUGR incidence and improve perinatal outcomes.