To Study the Correlation of Methylenetetrahydrofolate Reductase C677T and MTR (A2756G) Gene Polymorphism in Endometriosis in Women of Reproductive Age Group

Endometriosis is the proliferation of endometrial glands and stroma outside the uterus. The cause of endometriosis is still a mystery; however, recent research suggests a genetic predisposition to the condition. Endometriosis is a complex gynecologic disorder diagnosed in approximately 10% of women of childbearing age and is characterized by the presence and growth of endometrial-like tissue outside the uterus.[ 1 2 ] Endometriosis causes disabling dysmenorrhea, pelvic pain, and inflammation in women of reproductive age and is responsible for 30%–50% of their infertility.[ 2 3 ] Risk factors for endometriosis include abnormalities in estrogen production and metabolism, immunologic abnormalities, exposure to environmental pollution and toxins, and a genetic background.[ 4 5 6 7 ] However, the etiopathogenesis and pathophysiology of endometriosis associated with inadequate fertility are still poorly understood.[ 2 ] Methylenetetrahydrofolate reductase (MTHFR) plays a key role in the enzymatic process of folate metabolism and its main function is to convert 5,10-methylenetetrahydrofolate to 5-methylenetetrahydrofolate with biological functions, which is then involved in DNA synthesis, modification and methylation.[ 8 ] The MTHFR gene consists of 11 exons and is located on the short arm of chromosome 1 (1p36.3).[ 9 ] One of the most common mutations is that at position 677 in exon 4, the cytosine (C) has been replaced by the thymine (T).[ 10 ] The 222 nd amino acid in the protein changes from alanine to valine, eventually leading to a decrease in MTHFR activity, an increase in homocysteine (Hcy) levels, and a decrease in DNA synthesis and methylation.[ 11 ] The MTHFR gene regulates MTHFR, while the MTR gene regulates methionine synthase reductase (MTRR) activity; these are the two major enzymes responsible for the remethylation of Hcy to methionine in the one-carbon pathway.[ 9 10 11 ] MTHFR converts 5,10-methylenetetrahydrofolate (THF) to 5-methyl-THF, the predominant form of folate in the bloodstream. The 5-methyl-THF product donates a methyl group to Hcy in the formation of S-adenosylmethionine, the major source of methyl groups required for DNA methylation. MTRR regenerates a functional methionine synthase (MS) through reductive methylation associated with the transfer of Hcy to methionine.[ 9 10 11 ] MTHFR and MTRR due to MTR and MTHFR gene polymorphisms lead to an increase in inflammatory cytokines involved in the etiopathogenesis of endometriosis. The two major MTHFR single nucleotide polymorphism variants, c.677C>T, also called C677T (Ala222Val), and c.1298A>C, also called A1298C (Glu429Ala), decrease the activity of the enzyme, affecting these two metabolic cycles and leading to adverse effects on methylation processes and an increase in circulating Hcy.[ 12 ] The formation of 5-methyl-tetrahydrofolate (5-MTHF), the active substrate for MS, may be impaired by up to 70% in the presence of the homozygous isoform c.677C>T. In c.1298A>C homozygotes, the enzymatic activity of MS may reach only 68% of normal. Alteration of MTHFR activity is not observed in wild-type (WT) patients: They do not carry either of these isoforms. A heterozygous compound mutation can also significantly affect the gene activity and regeneration of Hcy via the 1-CC, thus affecting the methylation process.[ 13 14 15 ] The aim of the present study was to investigate the association between MTR (A2756G) and MTHFR (C677T) gene polymorphisms and endometriosis in women of reproductive age. We also investigated the association between MTR (A2756G) and MTHFR (C677T) gene polymorphisms with the severity of endometriosis.

The comparison of frequencies of different age, religion, literacy, socioeconomic status, and mean body mass index (BMI) was not significantly different in between cases and controls. Unmarried women were significantly more in cases. The frequencies of P0, P1, P2, and P3 parity were 37.25%, 29.41%, 15.69%, and 1.96% in cases and 0.00%, 0.00%, 72.55%, and 27.45% in control, respectively. Moreover, the frequencies of P0, and P1 parity were significantly more in cases as compared to controls [ Table 2 ]. Comparison of baseline characteristics in between cases and controls *Significant. BMI=Body mass index, SD=Standard deviation CA 125 level 30 (19 [38.00%]) in cases. Statistically, a significant difference was observed in CA 125 level among patients [ Table 3 ]. Distribution according to serum CA 125 in study population *CA125 levels has been based on a normal value of 35 U/mL. SD=Standard deviation The genotype and allele frequencies of the MTHFR C677T and MTR A2756G gene polymorphisms in cases and controls are shown in Table 4 . We compared the distribution of genotype and allele at each locus between cases and controls by using the Chi-squared test. The frequencies of CC, CT, and TT genotypes of MTHFR C677T were 17.65%, 54.90%, and 27.45% in cases and 64.71%, 15.69%, and 19.61% in controls, respectively. In addition, the frequency of C and T allele was 46.92% and 53.08% in cases; 72.55% and 27.45% in controls, respectively. The T-allele and CT and TT genotype of MTHFR C677T gene polymorphism was significantly associated with cases. The frequencies of AA, AG, and GG genotypes of MTR A2756G were 41.18%, 35.29%, and 23.53% in cases and 62.75%, 19.61%, and 17.65% in controls, respectively. In addition, the frequency of A and G allele was 58.82%, 41.18% in cases and 72.55%, 27.45% in controls, respectively. MTR A2756G gene polymorphism was not significantly associated with cases patients [ Table 4 ]. Comparison of genotype and allele frequency of methylenetetrahydrofolate reductase C677T and MTR A2756G gene polymorphisms in cases and controls OR=Odds ratio, CI=Confidence interval, MTHFR=Methylenetetrahydrofolate reductase The CT heterozygous and TT homozygous mutant of MTHFR C677T gene polymorphism was significantly more in Stages 1, II, and III as compared to Stage I. Whereas the C and T allele of MTHFR C677T and genotype and allele frequency of MTR A2756G gene polymorphisms were not significantly associated with different stage of endometriosis [ Table 5 ]. Comparison of genotype and allele frequency of methylenetetrahydrofolate reductase C677T and MTR A2756G gene polymorphisms in different stage (rASRM) of endometriosis *Significance. χ 2 =Chi square test. MTHFR=Methylenetetrahydrofolate reductase The clinical presentation of endometriosis showed that chronic pelvic pain[ 16 ] was the most common in endometriosis, followed by dysmenorrhea,[ 17 ] menorrhagia,[ 18 ] and primary infertility.[ 18 ] Dysmenorrhea was most common in Stage IV,[ 8 ] followed by III,[ 5 ] II,[ 4 ] and Stage I.[ 3 ] Dyparenuia,[ 4 ] chronic pelvic pain,[ 7 ] menorrhagia,[ 7 ] primary infertility,[ 11 ] secondary infertility,[ 2 ] pain or lump at the former scar site,[ 2 ] gastrointestinal symptoms,[ 9 ] urinary symptoms,[ 1 ] and asymptomatic symptoms[ 3 ] were all the most common at Stage IV [ Table 6 ]. Association of clinical presentation of endometriosis with staging N/A=Not available, OR=Odds ratio

We concluded that the P0 and P1 parity was significantly higher in cases. The average CA 125 level was significantly higher in the patients. The T allele and the CT and TT genotypes of the MTHFR C677T gene polymorphism were significantly associated with the cases. In addition, the CT heterozygous and TT homozygous mutants of the MTHFR C677T gene polymorphism were significantly more frequent in Stages II and III compared with Stage I. In contrast, the MTR A2756G gene polymorphism was not significantly associated with cases. There are no conflicts of interest.

In our study, most subjects were between 20 and 30 years old, of whom 66.6% were affected cases and 74.5% were controlled cases. The mean age of the 51 women in our study was 28 ± 5.6 years, which could be attributed to the diagnosis related to infertility and the lower awareness of young people to visit a medical facility. The distribution by religion was 62% and 68% among Hindus, 21% and 17% among Muslims, and 15% and 13% among Christians among women with and without endometriosis, respectively. A previous study reported that the mean age of patients with infertility was 29 ± 4.3 years, which is consistent with our study.[ 19 ] Another study found that the mean age at the diagnosis of endometriosis was 39.5 ± 9.7 years in 2863 women with endometriosis.[ 20 ] The mean age was higher than in our study. In our study, the mean BMI was 24.4 versus 23.7 in the case and control groups ( P = 0.51), which was not statistically significant. Our results show that 45% of the cases belonged to middle to lower socioeconomic class followed by middle 18 (35.29%). Similarly, BMI in women with and without endometriosis (median [interquartile range] = 23.6 [21.1–27.0] vs. 24.2 [21.5–28.1]).[ 21 ] In addition, the number of endometriosis cases was found to increase with increasing socioeconomic status. They found that the incidence was lower among women living in the most deprived and rural areas between 2009 and 2017. Our diagnosis showed that most women were Stage III (39.22%), followed by Stage II (25.49%), Stage IV (27.45%), and Stage I (7.84%). In our study, the percentage of endometriosis was Stage I–II (33.33%) and Stage III–IV (66.67%). In contrast, one study showed that the proportion of women in their study had Stage I–II (63%) and Stage III–IV (36%) endometriosis, which was not consistent with our study.[ 18 ] In our study, a significant association was found between CA-125 and endometriosis, which means that CA-125 was 54.58 U/mL in the study. Similarly, the mean serum level CA-125 was 49.93 ± 4.30 U/mL.[ 17 ] There was a significant relationship between the stage of endometriosis and the level of CA-125, but the study of CA-125 is of greatest use in the diagnosis of endometriosis, especially in moderate-to-severe endometriosis. A normal CA-125 value in women with endometriosis neither proves the absence of endometriosis nor predicts its presence, as it is not specific. It has been reported that a patient with endometriosis has an abnormally elevated serum level CA-125 during menstruation. This is an intriguing laboratory discovery. Consistent with previous studies, we found that the average CA-125 level was significantly higher in affected than in control subjects. In our study, the T allele and CT and TT genotype of MTHFR C677T gene polymorphism was significantly associated with endometriosis. Moreover, the CT heterozygous and TT homozygous mutant of MTHFR C677T gene polymorphism were significantly more in Stages I, II, and III as compared to Stage I endometriosis. Whereas the MTR A2756G gene polymorphism was not significantly associated with endometriosis. An important enzyme for folate metabolism is MTHFR. 677C>T and 1298A>C are the two common polymorphisms in the MTHFR gene that have been associated with enzyme activity.[ 13 16 ] Decreased enzyme activity is associated with the 677TT genotype. Compared with individuals with genotypes 677CC or 677CT, who have intracellular levels of mainly 5-methyltetrahydrofolate, individuals with genotype 677TT have greater intracellular concentrations of 5,10-methylenetetrahydrofolate.[ 16 ] According to some studies, these polymorphisms may affect the relationship between folate consumption and cancer risk,[ 22 23 24 ] by altering the balance of folate.[ 16 ] The only two previous studies that examined these polymorphisms and endometrial cancer risk reported either no association or no higher risk with the 677T allele.[ 25 ] Xu et al . found no evidence of a significant association between these MTHFR polymorphisms and endometrial cancer risk, consistent with the findings of the nurses’ health study.[ 25 ] In the endometriosis group, the homozygous c.677C>T isoform is twice as frequent as in the nonendometriosis group: 21.5% versus 10.2% ( P > 0.01). In the nonendometriosis group, the proportion of patients with MTHFR WT decreased symmetrically by half (8.2%–17.2%) in the endometriosis group.[ 12 ] In a meta-analysis, no significant association was found between MTHFR polymorphisms C677T and A1298C and increased risk of endometrial cancer. To better understand how these MTHFR polymorphisms relate to susceptibility to endometrial cancer, larger, well-designed studies need to be conducted.[ 26 ] The polymorphisms MTHFR C677T and A1298C are present in women with idiopathic recurrent pregnancy loss and altered Hcy levels.[ 27 ] The endometriosis patients had a higher prevalence of MTHFR homozygotes (.677C>T), which affected their fertility.[ 28 ] The most common mutation in the MTR gene is 2756A>G, which leads to substitution of glycine by aspartic acid and fluctuations in plasma Hcy levels. In our study, the case has a genotype frequency of (GG + AG) of 58%, compared with 37.25% in the control group. Statistically, a nonsignificant difference in genotype and allele frequency was found between patients. The abnormalities of folate metabolism in infertile endometriosis patients, it was found that patients with endometriosis more frequently had the MTR-G allele and the GG genotype.[ 12 ] In the Brazilian population, they also discovered lower Hcy levels and more clinical pregnancies. In heterozygous genotype CT or TT, the MTHFR-C677T polymorphism has been associated with an increased risk of neural tube defects (NTDs) in fetuses and neonates.[ 29 ] Another study that found a significant difference in the frequency of heterozygous MTHFR-C677T CT versus CC and a 1.8-fold increased risk of NTDs in heterozygous CT made a similar observation.[ 30 ] Moreover, the risk was 1.9-fold higher in combined dominant models than in CC WT homozygotes. In addition, the risk for the T-allele was 1.9-fold higher than the risk for the C-allele. For the MTR-A2756G polymorphism, the risk of developing NTD was 1.6-fold higher in heterozygotes AG than in homozygotes AA. However, there was no discernible difference in the frequencies of model GG versus AA and model AG + GG versus AA. Similar results were found with a 1.4-fold higher risk of NTDs for the G allele compared to the A allele.[ 31 ] The combination polymorphism of MTHFR C677T and MTR A2756G was investigated to determine whether two genes interact and whether they might be associated with NTDs. The frequency of individuals heterozygous CT for MTHFR C677T and AA homozygous for MTR A2756G compared with individuals homozygous CC and AA genotype was similar in both groups. Compared with individuals with WT AA and CC homozygous genotypes, patients with both AG and CT heterozygous genotypes had a threefold higher risk of developing NTDs. In addition, there were no differences in the frequencies of distribution of the combined CC and GG homozygous genotypes compared with the reference genotypes.

The case–control study was conducted on 51 patients with endometriosis who were registered at Department of Obstetrics and Gynaecology, King George’s Medical University and 51 healthy controls. Informed written consent was obtained from all subjects. Ethical clearance was obtained from the Institutional Ethical Committee. Clinical information was obtained through review of patients’ medical charts. Clinical data as well as stage of disease and history of comorbid conditions (diabetes, stroke, heart disease, kidney disease, liver disease, etc.) was extracted from the patients’ charts. All females between 18 and 24 years with menstrual abnormality, severe dysmenorrhea, premenstrual pain, chronic pelvic pain, dyspareunia, GI complaints, infertility and patient undergoing laparoscopy/laparotomy for pain, infertility, and pelvic lump were included in this study. Patients with a history of previous surgeries, acute fever, pelvic inflammatory disease, urinary tract infection, and chronic systemic illness (diabetes mellitus, tuberculosis, jaundice, and immuno-compromised) were excluded. Peripheral venous blood (2 mL) from each patient was collected in an EDTA tube. Genomic DNA was extracted from the blood samples using a DNA extraction kit and stored at –20°C for further use. The MTR (A2756G) and MTHFR (C677T) gene polymorphism was genotyped using a polymerase chain reaction (PCR)-restriction fragment length polymorphism method. PCR was carried out using the designed primer [ Table 1 ]. The 10-µL PCR reaction mix contained 30 ng genomic DNA, 1 × reaction PCR master mixture (TaKaRa, Inc.), and 200 nM of each primer. The initial denaturation was performed at 94°C for 3 min, followed by 30 cycles of 94°C for 30 s, 56°C for 30 s/61°C for 30, and 72°C for 30 s, and a final extension at 72°C for 7 min. The MTR (A2756G) PCR product was digested by 3 U of HaeIII at 37°C for 15 h. Digested PCR products were then loaded onto 3% agarose gel, stained with ethidium bromide, and visualized under ultraviolet illumination. The 919D allele resulted in a single band of 189 bp while the 919G allele resulted in two bands of 30 and 159 bp [ Figure 1 ]. The MTHFR (C677T) PCR product was digested by 3 U of HinfI (Thermo Fisher Scientific, USA) at 37°C for 15 h. Digestion of the 198 bp PCR product of the 677TT genotype results in two fragments of 175 and 23 bp, whereas the 677CC genotype results in one fragment of 198 bp [ Figure 2 ]. Further, the correlation of MTR (A2756G) and MTHFR (C677T) gene polymorphism with endometriosis in adolescents and young women was analyzed. Polymerase chain reaction (PCR)-restriction fragment length polymorphism analysis of MTR A2756G genotype. The fragments after digestion of PCR product with HaeIII (lanes 1: 100 bp ladder, lane 2: D, lane 5: G allele and lane 3, 4, 6: DG genotypes) Polymerase chain reaction (PCR)-restriction fragment length polymorphism analysis of MTHFR C677T genotype. The fragments after digestion of PCR product with Hinf (lanes 1: 100 bp ladder, lane 2, 5, 7: C allele, lane 4: T allele and lane 3, 6: CT genotypes) Primer sequences for MTR and methylenetetrahydrofolate reductase gene polymorphisms MTHFR=Methylenetetrahydrofolate reductase, SNP=Single-nucleotide polymorphism Data were entered into Microsoft Excel and analyzed using the statistical software SPSS (SPSS Inc., Chicago, IL, USA) for Windows program (26.0 version). The continuous variables were evaluated by mean (standard deviation) or range value when required. The dichotomous variables were presented in the number/frequency and were analyzed using the Chi-square or Fisher’s extract test. Chi-square-goodness-of-fit test was used to test the distribution of genotype frequencies for deviations from Hardy–Weinberg equilibrium between the patients and controls. Odds ratios and 95% confidence intervals would be calculated to measure the risk associated with variant genotypes by the unconditional logistic regression method. Age-adjusted risks were also be calculated using a multivariate logistic regression model. The differences would be considered significant if the P ≤ 0.05.