The effect of A1298c polymorphism of the MTHFR gene on anti‐Müllerian hormone levels: experimental and Web‐based analysis

Abstract Background The 5,10‐methylenetetrahydrofolate reductase (MTHFR) is an important enzyme of folate and methionine metabolism, which is expressed in human oocytes and preimplantation. Due to the involvement of MTHFR in female reproduction, we tend to evaluate the influence of MTHFR A1298C polymorphism on ovarian marker reserves such as serum anti‐Müllerian hormone (AMH) levels in women after in vitro fertilization (IVF)/intracytoplasmic sperm injection (ICSI). Methods A total of 100 women, who underwent ART treatment due to male factor infertility, were recruited into this study. MTHFR A1298C polymorphism was detected by polymerase chain reaction‐restriction fragment length polymorphism (PCR‐RFLP) technique, and serum AMH concentrations were measured by an ultrasensitive enzyme‐linked immunosorbent assay (ELISA). Results Women with the CC genotype had higher AMH levels (4.15 ± 1.67 ng/ml), albeit not significant, than carriers with other genotypes after ovarian stimulation. No significant differences existed in terms of miscarriage and live birth rates among different genotype groups. Conclusion The presence of the C mutant allele of the 1298 polymorphism in the MTHFR gene led to an increasing trend in serum AMH concentrations; however, the numbers of oocytes retrieved decreased in women with mutated genotypes. The influence of the MTHFR C677T polymorphism on embryo quality and pregnancy rate after ART cycles remains unclear.

could be a crucial step for the successful outcome of ART. 2 Despite significant improvements in infertility treatment, the individual ovarian response to The FSH is unrespectable and may depend on their genetic makeup. 3,4 Maybe, the patient genetic background better than the stimulation protocol determined the ovarian response.
The flavoenzyme 5,10-methylenetetrahydrofolate reductase (MTHFR) is a key regulatory enzyme in folate metabolism, which involved in the process of DNA synthesis and regulation of homocysteine levels. 5 It catalyzes the conversion of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate as a methyl group donor in homocysteine remethylation to methionine. 5 A common A>C mutation in the MTHFR gene at nucleotide position 1298 causes the substitution of glutamate to alanine in the MTHFR protein and a consequent reduction in enzyme activity, approximately 40% of the wild-type individuals. 6 Although decreased MTHFR activity due to A1298C polymorphism may lead to impaired DNA methylation, which is essential for normal embryo development, 7 only a few studies have evaluated the relationship between the MTHFR A1298C polymorphism and infertility, even with controversial results. [8][9][10][11] We have reported that women carrying homozygous (1298CC) genotype were less likely to achieve pregnancy and deliver a baby after IVF than those with the wild-type (1298AA) genotype. However, a recent study failed to find an association between A1298C polymorphism with embryo quality, ongoing pregnancy rate, and abortion rate. 9 The MTHFR gene expression was observed in human oocytes and preimplantation embryos. 9 It has been suggested that it is involved in many physiologic processes such as folliculogenesis and maintenance of genomic stability in oocytes. 12 During folliculogenesis, in which granulosa cells (GC) proliferate in response to FSH, the ovarian response of patients to FSH stimulation could be related to MTHFR polymorphisms. 13,14 Due to the important role of MTHFR in female reproduction, we tend to analyze the MTHFR A1298C polymorphisms in 100 women who underwent ART treatment to determine their relationship with an ovarian reserve and ovarian stimulation.

| Blood sampling
Fresh blood samples were collected during the 2nd or 3rd day of the menstrual cycle for AMH measurement and genomic DNA extraction. In order to measure the AMH levels, samples were centrifuged at 2,000 g for 10 min, and all the sera were stored at −80°C.
The AMH levels in serum were measured using an ultrasensitive enzyme-linked immunosorbent assay (ELISA) kit (cat. AL-105-i, Ansh Laboratories), with limits of detection set at 0.023 ng/mL.

| Genotyping
Genomic DNA was extracted from leukocytes by the QIAamp DNA Blood Mini Kit.

| Treatment protocol
Patients were treated with GnRH agonist and antagonist protocol. A detailed description of the stimulation protocols, follicle monitoring, hCG triggering, oocyte retrieval, and embryo transfer has been described. 15 Primary endpoints included the total number of retrieved oocytes and the determination of the effect of the MTHFR A1298C polymorphism on serum AMH levels. Clinical pregnancy, miscarriage, and live birth rates were considered as secondary endpoints. 16 A poor ovarian response was defined as ˂4 oocytes retrieved, a normal ovarian response as 4 to 15 oocytes retrieved, and a high ovarian response as ˃ 15 oocytes retrieved. 17

| Statistical analysis
All statistical analyses were carried out with SPSS software version 20 (IBM Corporation). Parametric variables were compared by one-way ANOVA test, and nonparametric variables, by the Kruskal-Wallis test, and data were presented as the mean ± standard deviation (SD) or number and percent. To test for associations between genotypes and categorical variables, the chi-squared test was used.
Distributions of the genotypes were checked with a Hardy-Weinberg equilibrium test. In order to predict efficacy factors oocyte retrieve, we used linear regression. Multiple logistic regression analyses were used to indicate the association between the dependent (pregnancy compared with no pregnancy) and independent variables. Adjusted odds ratios (aORs) and 95% confidence interval (CIs) were reported.
p-values of <0.05 were considered as statistically significant.

| RE SULTS
We screened 134 patients for eligibility, of which 18 were excluded since they failed to meet the inclusion criteria. The MTHFR A1298 genotyping analysis on the blood samples of the remaining 116 patients was performed. Of these, 16 patients failed to start treatment (the IVF/ICSI cycles) and PCR-RFLP results show that these patients had the subsequent MTHFR 1298 genotype: AA (n = 8), AC (n = 5), and CC (n = 3). Statistical analysis of the 100 patients who received controlled ovarian stimulation in the IVF/IESI cycle was performed, and the cycle outcomes were compared according to the patients' MTHFR 1298 genotypes.
The three genotype groups were compared with respect to basic characteristics, ovarian response, and cycle outcomes (Table 1). No significant differences were found among groups in terms of age, BMI (kg/m 2 ), basal FSH and LH levels, type of infertility, infertility duration, AFC, the total dose of gonadotropins used, duration of stimulation, and the total number of oocytes retrieved (Table 1). Women carrying the homozygous CC genotype showed higher levels of AMH than those carrying other genotypes; however, they failed to reach significance. No significant differences were shown among groups in terms of numbers of good-quality embryos, fertilization rates, clinical pregnancy rates, miscarriage, and live birth rates (Table 1).
Linear regression and scatter plot were used to evaluate the associations between AMH concentrations and the number of oocytes retrieved (NOR) separately in the two genotypes (normal and mutant) of MTHFR A1298C (Figure 1). The results showed significant correlations between serum AMH concentrations and NOR in two genotypes. According to the AMH concentrations, there was significantly lower NOR in mutant MTHFR individuals than those with the normal MTHFR genotype. As seen in Figure 1, the mean oocyte numbers for different MTHFR genotype individuals with AMH concentrations ranged from 1 to 5 ng/ml were as follows: 12.09 (normal) and 12.61 (mutant).  Table 2).
The multivariable logistic regression test performed in a backward manner was adjusted for age and BMI, then applied to determine the significant predictive variables for live birth in the studied population. All possible important variables such as type and duration of infertility, AFC, serum AMH level, and MTHFR genotype were entered in the model. Our result fails to show any significant predictive when the outcome was live birth (Table 3).

| Bioinformatics analysis
The analysis of the MTHFR protein sequence at the UniProt database showed that this protein was expressed as a dimer. Position 429 is in the α-helix structure; according to the study, this position is not in the domain. The molecular weight and acidic condition of wild-type protein are higher than mutated protein, but hydrophobicity and isoelectric point of mutated protein are higher. Protein charge in wild-type protein is −9 and in mutated protein −8. Furthermore, the instability index and aliphatic index of wild protein are less than mutated protein, and the grand average of hydropathicity (GRAVY) was −0.418 and −0.410 for wild and mutated protein, respectively. The difference between wild-type and mutant form is presented in Table 4.
Data obtained from SNAP2 database revealed that the Glu429Ala substitutions could affect protein function (prediction: non-natural; score = 3; expected accuracy: 53%). Moreover, the PolyPhen-2 server predicted the Glu429Ala substitutions as a benign mutation with a score of 0.021 (sensitivity: 0.95; specificity: 0.80). Data from PROVEAN server showed that this substitution has deleterious effects for MTHFR activity (PROVEAN prediction score: −3.09; SIFT prediction score: 0.100). The effect of A1298C mutation on RNA secondary structure was assessed by RNAsnp server. The data demonstrated substantial changes, a p-value of less than 0.2, in the secondary structure of mRNA with a distance of 0.3215 (Figure 2).

| DISCUSS ION
The present study, for the first time, evaluated the association between the A1298C polymorphism in the MTHFR gene and serum AMH concentrations in women with normal ovarian function. Our findings indicate that there is a trend increase in serum AMH concentrations, albeit not significant, in women with a mutated genotype of MTHFR 1298A>C compared with wild-type genotype. Interestingly, we found that increasing serum AMH levels in mutated patients is not accompanied by increasing the number of oocyte recovery in these patients. Our study was done in women with a normal ovarian function to exclude the influence of elevated AMH serum concentrations, as seen in polycystic ovary syndrome.
MTHFR is a crucial enzyme in the folate metabolism, which maintains a balance in the flux of folates between DNA synthesis and methylation reactions. 4 The expression of the folic acid transport protein and MTHFR enzyme in human oocytes and preimplantation embryos enforces the idea for the key role of folate metabolism on aspects of human reproduction. 9 During times of high folate requirements, such as folliculogenesis and embryogenesis, reduced MTHFR activity due to A1298C polymorphism may impair ovarian function, implantation, and the entire process of pregnancy. 13,14 Another consequence of reduced MTHFR activity is impaired methionine synthase and homocysteine accumulation, which may induce cytotoxic and oxidative stress and lead to impaired embryo development, and endothelial cells. 18,19 Despite the relationship between MTHFR polymorphisms and aspects of female reproduction, the effects of the MTHFR polymorphisms on clinical relevance and biochemical parameters are still controversial. 8,9 In this study, we failed to demonstrate an association between MTHFR genotypes and a number of oocytes retrieved in patients; however, we confirmed previous results that have reported a significant association between AMH levels and the numbers of oocytes retrieved after ovarian stimulation. Rosen  Further studies with large sample size across different ethnic groups are needed to clarify the effect of A1298C MTHFR polymorphisms on female reproduction.

ACK N OWLED G M ENTS
We would like to thank all the participants in this study.

CO N FLI C T S O F I NTE R E S T
The authors declare that they have no existing conflicts of interest associated with the manuscript.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.