Evaluation of association between methylenetetrahydrofolate reductase and azoospermia: A meta-analysis.

Conceptualization: Guang-Xing Tan, Lin Jiang, Gang-Qin Li, Kuan Bai. Data curation: Guang-Xing Tan, Lin Jiang, Gang-Qin Li, Kuan Bai. Formal analysis: Guang-Xing Tan, Lin Jiang, Gang-Qin Li, Kuan Bai. Writing – original draft: Guang-Xing Tan, Lin Jiang. Writing – review & editing: Guang-Xing Tan, Gang-Qin Li, Kuan Bai.

The electronic search yielded a total number of 1281 articles. After thorough reading, 850 papers met the preliminary criteria. After further screening, 837 articles were excluded due to study design, insufficient data, and type of the articles. Finally, 13 papers [ 11 – 23 ] were selected for analysis and all the articles were randomized controlled trial. Figure 1 is a flowchart of identification, inclusion and exclusion, reflecting the search process, and the reasons for exclusion. In the process, we followed preferred reporting items for systematic reviews and meta-analyse Checklist. [ 24 ] Flow diagram of the study selection. Detailed characteristics of the included studies are presented in Table 1 . All these studies were published from 2000 to 2019. The sample size ranged from 216 to 685. Totally 2738 patients were in the fertile group, and 2726 patients were in the infertile group. Characteristics of studies included in the meta-analysis. The Cochrane bias risk tool was used to evaluate the risk of bias in the 13 trials in which 1 trial showed selection bias, 1 showed performance bias, and 1 trial showed attrition bias. The detailed results of the quality assessment are listed in Figs. 2 and 3 . Assessment of the quality of the included studies: low risk of bias (green hexagons), unclear risk of bias (white hexagons), and high risk of bias (red hexagons). Quality assessment of the included studies. To analyze the association between C677T polymorphism and azoospermia, we performed a meta-analysis to calculate the overall RRs using the random effects model in Caucasian populations and Mongolian populations based on heterogeneity analysis. The risk of azoospermia associated with the C allele was 0.94-fold that of the T allele (Fig. 4 , RR = 0.94 [0.90, 0.99], I 2  = 60.9%, P  = .002). Forest plots of association between C677T polymorphism and azoospermia. Similarly, a meta-analysis of association between A1298C polymorphism and azoospermia was performed. Both Caucasian and Mongolian populations were analyzed by the random effects model. The risk of azoospermia in A Allele was 0.98-fold that of the C allele (Fig. 5 , RR = 0.98 [0.94, 1.02], I 2  = 56.3%, P  = .011). Forest plots of association between A1298C polymorphism and azoospermia. The genetic heterogeneity of C677T polymorphism was assessed based on additive, dominant, and recessive models. The heterogeneity results of these 3 models are shown in Table 2 . Significant heterogeneity was observed in these studies. In the additive model (C vs T), the dominant model (CC vs CT + TT), and the recessive model (TT vs CT + CC), heterogeneity was observed in 13 selected studies (additive Model: P  = .002 and I 2  = 60.9%; dominant model: P  = .025, I 2  = 48.6%; recessive model: P  = .066, I 2  = 40.1%). A1298C polymorphism was also assessed by additive, dominant, and recessive models as well as additive models (A vs C), dominant models (CC vs CT + TT), and recessive models (TT vs CT/CT). Genetic heterogeneity. CC, heterogeneity was observed (additive model: P  = .011, I 2  = 56.3%; dominant model: P  = .009, I 2  = 57.5%; recessive model: P  = .286, I 2  = 16.5%). Additive, dominant, and recessive models of C677T and A1298C polymorphism. In view of significant heterogeneity and to seek for its potential sources, we performed a panel of subgroup analyses on ethnicities in Fig. 6 . When studies were stratified for ethnicity, significant risks of C677T were found among Caucasians (RR = 0.95 [0.90, 1.01], P  = .005, I 2  = 65.3%) while no difference was observed in the Mongolian population. Similarly, A1298C was analyzed according to ethnicity, and significant difference was present in the Caucasian population (RR = 1.01 [0.94, 1.09], P  = .008; I 2  = 65.5%) while no difference was present in the Mongolian population. Forest plots of subgroup about C677T and A1294C in different ethnicities. Sensitivity analysis was performed to assess the robustness of the meta-analysis results. Pooled RRs from different populations were not affected by the removal of one study in the C677T association analysis, which supported the stability of the meta-analysis (Fig. 7 ). Sensitivity analysis of forest plots of association between C677T polymorphism and azoospermia. A funnel plot was performed to assess publication bias (Fig. 8 ). In general, the funnel plots for both C677T and A1298C were symmetrically inverted funnels. These results indicated no significant publication bias in the meta-analysis. The above proves that our research conclusion is stable and reliable. Funnel plot of publication bias.

Ethics approval was waived because this study does not involve any human participants or animals. Multiple electronic databases including MEDLINE, EMBASE, Cochrane library, and China journal full-text database were searched from January 2000 to September 2019 using combinations of the following key terms: MTHFR and azoospermia. The Mesh words included methylenetetrahydrofolate reductase (NADPH2), azoospermia, and nonobstructive. There was no language restriction. Title and abstract were initially reviewed and the references were also examined (G-XT and LJ). Two authors assessed possibly related articles independently complying inclusion criteria and exclusion criteria. If there is disagreement between 2 researchers, a third author will help to solve it. Studies were included if: They were randomized trials or case-control studies. They analyzed the association between MTHFR and azoospermia. The details of MTHFR polymorphism were reported. Studies were excluded if: Patients did not have azoospermia. Data were limited or insufficient. They were duplicate publications. The full texts of the articles were read carefully and the characteristics from each study were extracted using a predetermined form. [ 6 ] Data about base number in C677T and A1298C among azoospermia patients were extracted as main outcome. The data extracted from these studies included the first author's name, year of publication, country, ethnicity, sample size (infertile/fertile subjects). Crude relative risks (RRs) with the corresponding 95% confidence intervals (CIs) were used to assess the strength of the association between MTHFR polymorphism and azoospermia. Cochran X 2 based Q -statistic and I 2 test were performed to assess heterogeneity in the combined studies. Generally, I 2 values 50% correspond to strong heterogeneity between studies. Random-effects and fixed-effect summary measures were calculated as inverse-variance-weighted average of the log odds ratio. The results of random-effects summary were reported in the text because it takes into account the variation between studies. Sources of heterogeneity were investigated through a hierarchical meta-analysis based on ethnicity and sample size. Ethnicity was defined as Mongolian and Caucasian. Publication bias is presented through a funnel chart. Sensitivity analysis was performed by removing a study from the population and then reanalyzing the remaining studies. Analysis was performed using STATA software version 12.0 (Stata Corp., College Station, TX). All P values were used for double-sided analysis and a P value of .05 was considered statistically significant.

In conclusion, the results showed that Caucasian populations had higher risk than Mongolian populations in MTHFR and azoospermia.

Infertility often has a great impact on families and society. Surveys show that the divorce rate of infertile couples is 2.2 times that of the normal population, which has become an important medical and social issue. [ 25 , 26 ] The causes of infertility are very complex and any factors affecting ovulation, fertilization, and implantation can cause disease. The causes of infertility vary widely around the world. It is reported that the proportion of unknown causes of female infertility in Asia, Africa, and developed countries is 31%, 16%, and 40% respectively, fallopian tube factors account for 39%, 85%, and 36%, respectively, ovulation factors account for 34%, 26%, and 33%, respective, and endometriosis accounts for 10%, 1%, and 6%, respectively. [ 27 – 29 ] Because the mutation of MTHFR gene-related site lead to a decrease of MTHFR activities and the increase of plasma Hcy concentration. [ 30 ] On one hand, for pregnant women, it can lead to blood hypercoagulability, risk of placental thrombosis, placental embolism, insufficient maternal and fetal circulation, which can lead to abortion, fetal growth restriction, and placental abruption. On the other hand, high concentration of Hcy can damage the structure and function of vascular endothelial cells, thus triggering pregnancy-induced hypertension. [ 31 , 32 ] In addition, mutations in MTHFR-related sites affect the metabolism, DNA methylation of pyrimidine, purine and nucleic acid, which lead to inadequate methylation of DNA and protein, and it is also one of the causes of abortion and restricted fetal growth. In the analysis of the association between C677T polymorphism and azoospermia, the results of this research showed that the risk of azoospermia associated with the C allele was 0.94-fold that of the T allele. Moreover, we found that Caucasian populations had higher risk than Mongolian populations. Liu reported that MTHFR C677T gene polymorphism is associated with male infertility. CT and TT genotypes of MTHFR C677T gene may be susceptible genes for male infertility, T allele may be a risk factor for male infertility, and T allele may be associated with malformed spermatozoa. The risk of azoospermia in A Allele was 0.98-fold that of the C allele. The relationship of A1298C polymorphism with azoospermia was statistically significant. Yang stated that MTHFR A1298C polymorphism may be a potential risk factor for male infertility. In the subgroup analysis, both in C677T and A1298C, the association between MTHFR and azoospermia was significant among Caucasian population while in the Mongolian population, the association was insignificant. The results in different ethnicities are consistent with Li report. [ 16 ] In conclusion, the results showed that Caucasian populations had higher risk than Mongolian populations in MTHFR and azoospermia. However, this article had some limitations. Firstly, the comparison in different age areas was not considered, which could be evaluated in further research. Secondly, more singlenucleotide polymorphisms (SNPs) were not included and diverse SNPs could be evaluated in the future. However, some limitations existed in this article. Firstly, the comparison in different age areas was not considered, which could be evaluated in the further research. Secondly, the more SNP was not included and diverse SNPs could be evaluated in the future.

At present, infertility affects childbearing age couples all over the world. Statistics show that infertile couples account for about 10% to 15% of married couples, including men and women. [ 1 , 2 ] Despite current improvement in clinical diagnostic techniques, the causes of infertility are unknown in about half of couples. Mutations in the gene and the level of metabolism are 2 influencing factors. [ 3 ] The methylenetetrahydrofolate reductase ( MTHFR ) gene is located in 1p36.32 and encodes a protein with 656 amino acids. MTHFR is an important enzyme in the metabolism of folic acid and homocysteine (Hcy), mediating the transformation of 5,10-methylenetetrahydrofolic acid into 5-methyltetrahydrofolic acid. [ 4 , 5 ] 5-Methyltetrahydrofolic acid provides materials for DNA methylation in cells. At the same time, it acts as a methyl donor and uses vitamin B12 as a coenzyme under the catalysis of methionine synthase to remethylate Hcy in blood to produce methionine (Met). Folic acid is an antioxidant. [ 6 , 7 ] If folic acid intake is reduced and there is no enough folic acid in the body to protect against oxidative stress caused by high Hcy, sperm DNA will be damaged and fertility will be affected. At present, common MTHFR polymorphisms are C677T (rs1801133) and A1298C (rs1801131). [ 8 – 10 ] There are several articles analyzing association between infertility and MTHFR including polymorphisms C667T and A1298C. [ 7 – 10 ] They are varied in research designs, recruitment methods, exclusion criteria, and methods. A meta-analysis is required to evaluate the association between infertility and MTHFR.