Keywords
infertility - thrombophilia - MTHFR C677T mutation
Palavras-chave
infertilidade - trombofilia - mutação MTHFR C677T
Introduction
The C677T mutation in the methylenetetrahydrofolate reductase (MTHFR) gene has been investigated for the past 20 years. However, the importance of this mutation as a risk factor for the occurrence of thrombosis remains controversial. Some authors consider it an inherited thrombophilic factor.[1]
In women who have difficulty conceiving, the possible causes of embryo implantation failure have been investigated, both in spontaneous cycles and in induced cycles in assisted reproduction. Among the various factors that may interfere with the implantation process, thrombophilic factors are considered to hamper fertility.[2]
A study conducted with a Brazilian population found a greater incidence of homozygous MTHFR mutations in Caucasian individuals (10%) compared with Blacks (1.45%) and Brazilian Native Indians (1.2%).[3] An analysis of the association between this mutation and hyperhomocysteinemia showed that among the Brazilian population, folic acid levels are the greatest determinants of plasma homocysteine.[4]
Over the past ten years, thrombophilias have been identified with increasing frequency in women submitted to repeat treatment cycles of in vitro fertilization (IVF) with failed embryo implantation when compared with fertile women. Azem et al [5] conducted a case-control study analyzing hereditary thrombophilia factors in infertile, fertile and women with failed implantation. That study reported a high frequency of thrombophilia in the subgroup of women with implantation failure. Those authors suggested a negative effect of a state of hypercoagulability on embryo implantation. Similar results were reported by Grandone et al.[6]
Nevertheless, the relationship between thrombophilia, including the MTHFR C677T mutation, and infertility cannot be considered proven based on the level of scientific evidence from case-control studies conducted with limited sample sizes.
Studies were then conducted to investigate the presence of the MTHFR C677T mutation in infertile women and in women with failed implantation; however, the results are controversial. Some authors failed to identify inherited thrombophilia, including the MTHFR C677T mutation, as a risk factor for infertility,[7]
[8] while others reported conflicting findings, particularly in women in whom embryo implantation had failed.[9]
In view of these data, we deemed pertinent to conduct the present study to clarify the importance of investigating the MTHFR C677T mutation in infertile women. The study was aimed at evaluating whether the MTHFR C677T mutation is more common in infertile women, and whether it constitutes a risk factor for the occurrence of infertility in the Brazilian population.
Methods
A case-control study was conducted with a sample of 390 women divided into 2 groups: a group of fertile women (n = 260) and a group of infertile women (n = 130).The frequency of the MTHFR C677T mutation was then determined for the two groups.
The infertile women were selected by reviewing the files of patients attending a private clinic between March 2003 and March 2005.[10] All of the women were Caucasian and aged over 18 years, with primary and unexplained infertility. Male or tubal factors were excluded.
The control group was composed by healthy fertile women who were selected at the family planning clinic of our institution between April 2012 and March 2013. The inclusion criteria were: being Caucasian, having had at least 1 previous term delivery, age ≥ 18 years old, and having no previous obstetric concerns, no history of liver or hematological diseases, nor infection.
The sample size was calculated using different sources based on the difference in the prevalence of thrombophilic factors between the infertile and fertile women.[11]
[12] Considering a significance level of 5%, a proportion of 2 fertile women for every infertile woman and a power of the test of 80%, based on the formula developed by Pocock[13] for the Chi-squared test, a sample size of 390 women was calculated: 130 infertile women and 260 fertile women.
The Chi-squared and Fisher exact tests were used in the statistical analysis.
MTHFR Mutation Detection
A venous blood sample (5 mL) was retrieved from each patient and quickly placed into sterile Vacutainer tubes (Becton, Dickinson and Company [BD], Franklin Lakes, NJ, US) containing ethylenediamine tetra acetic acid and then divided into sterile Eppendorf tubes (Eppendorf, Hamburg, Germany) and stored at -80°C until it was time to perform the molecular study of the MTHFR polymorphisms. We extracted DNA from nucleated blood cells using a phenol chloroform method.[14] The MTHFR polymorphisms were detected by polymerase chain reaction–rapid fragment length polymorphism (PCR-RFLP) using the HinfI restriction analysis of a 198-bp PCR-amplified fragment in the gene for MTHFR.[15] The products of the HinfI digestion were electrophoresed on 3% agarose gels. The normal allele with cytosine at position 677 (C677) formed an undigested fragment of 198 bp, while the mutant allele with thymine in position 677 (T677) formed fragments of 175 and 23 bp.
The Ethical Committee of another institution approved the study protocol under reference number 153 on July 13, 2009. All patients signed an informed consent form, and the ethical aspects were observed in accordance with the Declaration of Helsinki.
Results
The women selected for evaluation in the present study were all white, since this is the ethnic group in which the occurrence of thrombophilia is the highest. The mean age of the patients in the case group was 36 ± 4.5 years (± standard deviation [SD]) compared with 34.2 ± 8.1 years for the fertile women. As for parity, 38% of the women in the fertile group had had 2 previous term deliveries, and 25% had had 3 or more children. Among the non-hormonal contraceptive methods used by the women in the control group, 71% used the TCu 380A intrauterine device (IUD). Regarding marital status, most of the fertile women (82%) and all the women in the infertile group were in a stable union. In the group of infertile women, the mean time of infertility was 5 ± 3.3 years.
The MTHFR C677T mutation was found in 49.2% of the fertile women, and in 58.5% of the infertile women, a difference that was not statistically significant. The mutation was homozygous in 10% of the women in the case group (13 of the infertile women) and in 8.8% of the controls (23 of the fertile women). These results are shown in [Table 1].
Table 1
MTHFR mutation in cases (infertile women) and controls (fertile women)
|
Cases
|
Controls
|
p
|
|
Absence of MTHFR mutation
|
54 (41.5%)
|
132 (50.8%)
|
0.08
|
|
Presence of MTHFR mutation
|
76 (58.5%)
|
128 (49.2%)
|
|
|
Heterozygous MTHFR mutation
|
63 (48.5%)
|
105 (40.4%)
|
0.22
|
|
Homozygous MTHFR mutation
|
13 (10%)
|
23 (8.8%)
|
|
Abbreviation: MTHFR, methylenetetrahydrofolate reductase.
Out of the 260 fertile women evaluated, 37 had a history of a previous spontaneous abortion. However, when the presence of the MTHFR mutation was investigated in this subgroup and in the subgroup of women who had suffered no previous pregnancy loss, no statistically significant difference was found ([Table 2]).
Table 2
Fertile women with and without a history of pregnancy loss
|
MTHFR mutation
|
No previous
pregnancy loss
|
Previous
pregnancy loss
|
p
|
|
Present
|
111 (49.8%)
|
17 (45.9%)
|
0.66
|
|
Absent
|
112 (50.2%)
|
20 (54.1%)
|
|
Abbreviation: MTHFR, methylenetetrahydrofolate reductase.
Discussion
The results of the present study show a slightly higher frequency of the MTHFR C677T mutation in infertile women; however, this difference was not statistically significant. These findings are corroborated by the results of previous studies that also failed to find a greater frequency of inherited thrombophilia in infertile women.
In some case-control studies analyzed, which compared women with infertility of no apparent cause and fertile women, inherited thrombophilia was investigated. Those results are in agreement with the findings of the present study in relation to the MTHFR C677T gene mutation.[8]
[16]
Moreover, in 2010, Sharif and Ghunaim[17] conducted a prospective cohort study in which 273 cases of implantation failure were analyzed. That study emphasizes the importance of micro-thrombosis at the implantation site as a factor that prevents trophoblastic invasion and consequently embryo implantation. Since there was no subgroup with implantation failure in the present study, it is impossible to make any affirmations regarding the association between this mutation and women with implantation failure.
In 2016, Patounakis et al[18] conducted a prospective cohort to determine if thrombophilic single nucleotide polymorphisms (SNPs) affect outcomes in IVF treatments, and investigated the MTHFR (C677T and A1298C) mutations. They did not find any association between the MTHFR mutation and the IVF outcomes. They did not recommend this investigation for initial screening. Those results are in agreement with our findings in relation to the MTHFR C677T gene mutation.
The MTHFR C677T mutation has been associated with adverse obstetric events and with the risk of recurrent pregnancy loss.[1]
[19]
[20] In our study, this mutation was present in 49.2% of the fertile women, and it was homozygous in 8.8% of them. This significant frequency in fertile and healthy women suggests that this mutation is probably not responsible for causing adverse effects in pregnancy. Nevertheless, that was not the objective of this study; furthermore, the mutation was homozygous in only a small proportion of the sample evaluated (in 8.8% of the controls). In 2006, Ren and Wang[21] conducted a meta-analysis and found no association between the MTHFR mutation and recurrent pregnancy loss. Some studies suggested the role of the MTHFR C677T polymorphism in certain diseases (vascular diseases, cancers, neurologic diseases, diabetes, psoriasis, etc.). However, an interpretation of such results is compromised by the heterogeneity of the population sample and the small case series.[22]
Recently, Boas et al[23] evaluated the polymorphisms in genes involved in folate metabolism and their association with recurrent pregnancy loss in the Brazilian population. In the present study, no evidence was found of any association between the MTHFR C677T mutation and recurrent pregnancy loss.
The MTHFR mutation appears to be common in the general population, but its clinical implication does not appear evident in the reviewed literature. Therefore, MTHFR screening is not indicated for the asymptomatic general population, and its clinical expression is uncertain.[24]
Based on these findings and on the literature evaluated, it is our opinion that the MTHFR C677T mutation is not associated with infertility, and we would not recommend its investigation in this population. The investigation in infertile women is considered unnecessary.
