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Journal of Assisted Reproduction and Genetics logoLink to Journal of Assisted Reproduction and Genetics
. 2019 Jun 28;36(7):1315–1328. doi: 10.1007/s10815-019-01473-2

Polymorphisms of methalenetetrahydrofolate reductase in recurrent pregnancy loss: an overview of systematic reviews and meta-analyses

Boran Du 1, Xiangjun Shi 2, Chenghong Yin 1,✉, Xin Feng 1,✉
PMCID: PMC6642244  PMID: 31254142

Abstract

Purpose

The aim is to summarize and evaluate current systematic reviews and meta-analyses on MTHFR polymorphisms in recurrent pregnancy loss (RPL).

Methods

We searched Pubmed and Embase databases and selected in form of PICOS (participants, interventions, comparisons, outcomes, and study design). Our methodology was registered on PROSPERO (CRD42017042762). Systematic reviews and meta-analyses containing primary studies were extracted for meta-analyses, along with their OR and 95%CI. We assessed the quality of the included studies using AMSTAR and OQAQ criteria.

Results

Eleven systematic reviews and meta-analyses were identified. C677T was significantly related to RPL overall in Allele (OR, 95%CI 1.43, 1.29–1.60), Recessive (OR, 95%CI 1.66, 1.42–1.95), and Homozygous (OR, 95%CI 2.08, 1.66–2.61). There was no correlation observed between A1298C and RPL, except for in Heterozygous (OR, 95%CI 1.62, 1.17–2.25).

Conclusions

We identified a difference in the association between MTHFR C677T polymorphism and RPL, especially in Asian population. No significant correlation was found between A1298C and RPL.

Keywords: Recurrent pregnancy loss (RPL), Methalenetetrahydrofolate reductase (MTHFR), Polymorphism, C677T, A1298C, Systematic review and meta-analysis

Introduction

Recurrent pregnancy loss (RPL) is defined as two or more consecutive spontaneous miscarriages before 20 weeks of gestation. RPL affects at least 2% of women in reproductive age. Despite anatomical abnormalities, autoimmune diseases, and environmental factors, the causes of RPL in 50% of cases are still unknown [1]. Methylenetetrahydrofolate reductase (MTHFR) plays a critical role in the folate pathway, which is widely believed to play a key role in pregnancy outcome. MTHFR gene is located on chromosome 1p36.6 and features 11 exons. Polymorphisms of MTHFR, especially C677T (rs1801133) and A1298C (rs1801131), are believed to be associated with RPL [2]. These mutations cause a reduction in the activity of MTHFR, a vital enzyme which catalyzes 5,10-methylenetetrahydrofolate to 5-methylenetetrahydrofolate (5-MTHF). Then, the reduction of 5-MTHF, which participates in the conversion of homocysteine to methionine, results in elevated levels of total homocysteine (tHcy) in both blood and urine [2]. Subsequently, hyperhomocysteinemia, or homocystinuria, can induce inherited thrombophilia, a significant risk factor for RPL [3]. Accumulation of homocysteine is also associated with arteriosclerosis, pre-eclampsia, and neural tube defects [4].

A few systematic reviews and meta-analyses have investigated the correlation between MTHFR polymorphisms and RPL; however, the outcome on this work remains controversial as results have been inconsistent. The reasons for such discrepancies may be related to differences in ethnicities and the selection of primary case-control studies. The accumulation of primary case-control studies may also contribute to variation in views of different authors in term of MTHFR polymorphisms and RPL [4].

Therefore, in the present study, we conducted an overview of systematic reviews and meta-analyses in a comprehensive manner, including different ethnicities and genotype models, in order to assess the association between MTHFR genotype variants and the risk of RPL. A citation matrix was created to analyze the influence of selection in primary studies by different authors. Cumulative meta-analyses were also performed to investigate the tendency of results in genotype models with possible risk for RPL. We also evaluated systematic reviews with Assessment of Multiple Systematic Reviews (AMSTAR) and Overview Quality Assessment Questionnaire (OQAQ) scales to assess the quality of the systematic reviews and meta-analyses which have already been published on this topic.

Materials and methods

Search strategy

We identified potential articles by searching Medline and Embase databases with the following key terms: “recurrent pregnancy loss”; “recurrent miscarriage”; “spontaneous abortion”; “methylenetetrahydrofolate reductase”; “MTHFR”; “homocysteine”; “polymorphisms”; “single nucleotide polymorphisms”; and “SNP”. Only systematic reviews and meta-analyses that had been published in English were included.

Eligibility criteria and study selection

According to methodology registered on PROSPERO (CRD42017042762), two researchers independently screened and selected potential articles in accordance with the following eligibility criteria.

  • Inclusion criteria: (1) concerning the association between MTHFR C677T or A1298C polymorphisms and recurrent pregnancy loss; (2) human studies; (3) systematic reviews and meta-analyses

  • Exclusion criteria: (1) reviews, comments, editorials; (2) animal studies; (3) without the number of pregnancy losses ≥ 2; (4) controls without having at least one successful live birth

In form of PICOS (participants, interventions, comparisons, outcomes, and study design), the study was described as follows [5]:

  • P: women with two or more pregnancy losses;

  • I: N/A;

  • C: women with at least one successful live birth;

  • O: MTHFR polymorphisms including C677T or A198C;

  • S: systematic reviews and meta-analysis; case-control studies in the systematic reviews included

Data extraction and quality assessment

Two reviewers (Boran-Du, Xiangjun-Shi) screened titles and abstracts according to the eligibility criteria. Studies which met the inclusion criteria were evaluated with full text. For the articles finally included, two reviewers independently extracted the following information: first author, year of the publication, ethnicity, and frequencies of MTHFR in cases and controls. Any disagreement was resolved through discussion. If necessary, a third reviewer (Xin-Feng) made the final decision with regard to discrepancies.

We assessed the methodological quality of systematic reviews and meta-analyses with Assessment of Multiple Systematic Reviews (AMSTAR) [6], which consists of 11 items relating to search bias, selection, and the process of data synthesis. The quality of the systematic reviews and meta-analyses included was also evaluated using Overview Quality Assessment Questionnaire (OQAQ) scale [7], which consists of 9 items and a final total score item. In this research study, we omitted the last item in comparison to AMSTAR scale.

Data synthesis

We described and synthesized data from all selected publications. The odds ratio (OR) with 95% confidence interval (95%CI) was used to compare differing results from systematic reviews relating to associations between MTHFR polymorphisms and RPL. Data synthesis was performed with Stata 12.0 (Stata Corporation, College Station, TX, USA). A two-sided p value < 0.5 was considered to be statistically significant. Heterogeneity was identified if I2 > 50%, and a random-effect model was used to synthesize data. In order to explore the optimal genotype model of MTHFR and its relationship with RPL, we analyzed a range of different genetic models including Allele model, Dominant model, Recessive model, Homozygous model, and Heterozygous model (Table 1).

Table 1.

Genotype contrast model of MTHFR polymorphisms and RPL(C677T and A1298C)

Contrast model C677T A1298C
Allele T/C C/A.
Dominant CT+TT/CC AC+CC/AA
Recessive TT/CT+CC CC/AC+AA
Homozygous TT/CC CC/AA
Heterozygous CT/CC AC/AA.
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Results

Literature searches and study selection

Our initial search generated 29 articles, of which 16 were excluded due to the duplication of results (n = 8) [8–15], non-relevance (n = 2) [16, 17], or the fact that they were not systematic reviews and meta-analyses (n = 6) [18–23] (Fig. 1). After full-text screening, 2 studies were removed according to the eligibility criteria (without the number of pregnancy losses ≥ 2 [24], which did not concern MTHFR polymorphisms [25]). Finally, 11 reviews and meta-analyses remained; all of these were published in English between 2000 and 2016.

Fig. 1.

Fig. 1

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Flowchart of selection of systematic reviews published

Characteristics of the systematic reviews and meta-analyses included in this study

The characteristics of the 11 systematic reviews and meta-analyses included in our final analysis are summarized in Table 2. These publications all focused on the correlation between RPL and MTHFR polymorphisms, of which there were 9 reviews related to C677T and 5 reviews related to A1298C (Table 2). The number of primary studies included in each systematic review ranged from 6 to 53. There were ten systematic reviews which involved the analysis of ethnicity and MTHFR polymorphisms; one of these was especially focused on Asian population while another focused on Chinese population. Two reviews were partial systematic reviews and meta-analyses. The researchers (Nair, 2013 [10]; Parveen, 2013 [27]) of these two reviews conducted a case-control study in their own country and compared with results of the systematic reviews and meta-analyses they executed.

Table 2.

Characteristics of systematic reviews included

Author Year of publication C677T A1298C
Number of studies Case number Control number Conclusion Number of studies Case number Control number Conclusion
Nelen [11] 2000 6 Pooled estimate OR of C677T TT genotype was 1.4 (1.0–2.0). The role of C677T TT genotype was not clear in RPL.
Wiwanitkit [26] 2005 8 53.1% subjects with T allele have RPL; on the other hand, 55.3% subjects without T allele have RPL. C677T might not be a useful marker for RPL. There was no association between C677T and ethnicities.
Ren [13] 2006 26 2120 2949 C667T was not a genetic risk factor for RPL, except in a Chinese population.
Wu [14] 2012 27 2427 3118 C667T was associated with increased risk of RPL, except for Caucasians.
Cao [8] 2012 37 3559 5097 Significant association between C677T and RPL in East Asian and mixed subgroup. 8 1163 1061 No significance between A1298C and RPL.
Nair [10] 2013 5 1080 709 A1298C was a genetic risk factor for RPL.
Parveen [27] 2013 12 1725 2392 Estimated OR of C677T was 1.383 (1.045–1.830). Moderate risk of C677T in RPL.
Rai [12] 2014 17 2338 2588 A1298C was not associated with RPL
Chen [24] 2015 16 1420 453 Significant association between C677T and RPL in Chinese population. 5 1408 376 No association between A1298C and increased risk of RPL.
Yang [15] 2015 53 6078 9441 C677T was associated with RPL. 16 2924 4759 A1298C was associated with RPL.
Rai [28] 2016 29 3725 4105 Strong association between C677T and RPL in Asian population.
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The conclusions from 11 of these reviews clearly differed. Seven reviews reached the conclusion that C677T was associated with RPL in Asian population, while two reviews held the opinion that C677T has no association with RPL. In the case of A1298C, two studies claimed this mutation as a genetic factor for RPL, while three other systematic reviews reached the opposite conclusion.

Primary study characteristics

The primary studies included in each systematic review were extracted and compared, including citation matrixes (Tables 3 and 4). With regard to C677T, six studies presented only the number of Homozygous type (TT) and total numbers of patients in RPL and control group; the other 72 provided a complete set of data for case-control studies. Of these 78 studies, 45 were conducted in Asian populations and 33 were conducted in Caucasian populations (Table 3). In term of A1298C, 31 case-control studies provided a complete data set; of these, 15 were based on Asian populations and 16 on Caucasian populations (Table 4).

Table 3.

Characteristics of primary studies included on C677T

Author Year of publication Ethnicity Case Control Nelen 2000 Wiwanitkit 2005 Ren 2006 Wu 2012 Cao 2013 Parveen 2013 Chen 2015 Yang 2015 Rai 2016
CC CT TT CC CT TT
1 Nelen 1997 Caucasian 77 79 29 48 59 6 √ √ √ √
2 Grandone 1998 Caucasian 35 42 17 45 77 28 √ √ √ √ √
3 Quere 1998 Caucasian 28 52 20 32 54 14 √ √ √ √
4 Brenner 1999 Asian 54 8 14 86 9 11 √ √ √ √ √
5 Holmes 1999 Caucasian 102 57 14 31 30 6 √ √ √ √ √
6 Kutteh 1999 Caucasian 50 4 50 2 √ √ √
7 Lissak 1999 Caucasian 17 20 4 7 7 4 √ √ √ √ √
8 Foka 2000 Caucasian 80 6 100 15 √ √
9 Murphy 2000 Caucasian 24 3 0 527 13 0 √ √ √ √
10 Wramsby 2000 Caucasian 17 17 2 27 35 7 √ √ √
11 Pihusch 2001 Caucasian 41 47 14 55 61 12 √ √ √ √ √
12 Carp 2002 Caucasian 108 14 82 7 √ √
13 Unfried 2002 Caucasian 64 46 23 46 24 4 √ √
14 Dilley 2002 Asian 27 26 7 39 45 8 √ √ √
15 Wang 2002 Asian 13 33 16 43 53 23 √ √ √ √ √ √ √
16 Pauer 2003 Caucasian 28 32 9 56 51 15 √ √ √ √
17 Kumar 2003 Asian 18 6 0 22 2 0 √ √ √ √ √ √
18 Hohlagschwandtner 2003 Caucasian 72 52 21 53 41 7 √ √ √ √ √
19 Wang 2004 Asian 49 78 20 43 34 5 √ √ √ √
20 Dossenbach-Glaninger 2004 Caucasian 49 8 48 5 √
21 Li 2004 Asian 16 32 9 25 20 5 √ √ √ √ √ √
22 Makino 2004 Caucasian 56 55 14 29 32 15 √ √ √ √ √
23 Wang 2004 Asian 14 17 8 43 34 5 √ √ √
24 Couto 2005 Caucasian 29 47 12 53 26 9 √ √ √ √
25 Gerhardt 2005 Caucasian 104 14 277 28 √
26 Guan 2005 Asian 13 59 55 19 73 25 √ √ √ √ √ √ √
27 Kobashi 2005 Asian 34 40 9 67 82 25 √ √ √ √ √
28 Song 2005 Asian 36 2 12 40 12 4 √ √ √ √ √ √
29 Parle-McDermott 2005 Caucasian 55 55 14 271 270 73 √ √ √
30 Mtiraoui 2006 Caucasian 92 47 61 156 30 14 √ √ √ √ √
31 Dong 2006 Asian 2 12 20 11 26 18 √ √
32 Wang 2006 Asian 36 2 12 89 27 9 √ √ √
33 Jivraj 2006 Caucasian 136 32 714 58 √
34 Sotiriadis 2006 Caucasian 24 61 12 32 57 13 √ √
35 Wan 2006 Asian 6 46 28 19 33 8 √
36 Ren 2007 Asian 9 40 22 29 38 26 √
37 Xu 2007 Asian 21 48 43 32 50 18 √
38 D’Uva 2007 Caucasian 0 5 15 8 9 3 √
39 Callejon 2007 Caucasian 10 233 99 195 170 70 √ √
40 Vettriselvi 2008 Asian 86 15 3 98 19 3 √ √ √ √ √
41 Makino 2008 Asian 33 42 10 29 32 15 √
42 Toth 2008 Caucasian 71 68 12 68 70 19 √ √
43 Ma 2008 Asian 12 32 16 19 34 7 √ √ √
44 Cardona 2008 Caucasian 38 43 12 93 83 30 √
45 Zhong 2008 Asian 72 50 16 116 43 3 √
46 Morales-Machin 2009 Caucasian 10 18 2 19 29 2 √
47 Govindaiah 2009 Caucasian 111 25 4 112 28 0 √ √ √ √
48 Mukhopadhyay 2009 Asian 75 6 3 78 2 0 √ √ √
49 Ciacci 2009 Caucasian 47 0 31 85 0 59 √ √
50 Bae 2009 Asian 82 104 36 45 63 14 √ √
51 Rodriguez-Guillen 2009 Caucasian 6 7 10 16 39 19 √ √
52 Zhang 2009 Asian 12 25 19 20 22 8 √ √ √
53 Abu-Asab 2010 Asian 145 151 33 182 177 43 √ √
54 Kim 2010 Asian 26 19 12 63 60 32 √ √
55 Zhong 2010 Asian 72 53 16 114 43 3 √ √ √
56 Settin 2011 Caucasian 40 26 4 67 68 1 √ √ √ √
57 Nair 2011 Asian 75 26 5 118 21 1 √ √ √ √
58 Wang 2011 Asian 18 82 59 28 78 21 √ √
59 Jeddi-Tehrani 2011 Asian 43 42 15 66 25 9 √
60 Park 2011 Asian 14 16 9 17 26 7 √
61 Ozdemir 2011 Asian 231 239 73 76 30 0 √ √
62 Parveen 2012 Asian 110 70 20 196 90 14 √ √ √
63 Han 2012 Asian 10 35 26 25 15 18 √ √
64 Dissanayke 2012 Asian 158 39 3 142 27 2 √
65 Torabi 2012 Asian 43 42 15 66 25 9 √
66 Zonouzi 2012 Asian 53 30 9 27 22 1 √ √
67 Creus 2012 Caucasian 23 26 11 13 13 4 √
68 Puri 2012 Asian 86 16 5 263 69 11 √
69 Yin 2012 Asian 13 25 15 33 18 12 √
70 Kaur 2013 Asian 86 16 5 463 109 21 √
71 Cao 2013 Asian 29 43 10 53 83 30 √ √
72 Chen 2013 Asian 30 20 9 50 36 1 √
73 Hu 2014 Asian 29 14 9 11 4 1 √
74 Luo 2014 Asian 40 70 15 60 65 10 √
75 Yousefian 2014 Asian 96 90 18 63 43 10 √ √
76 Farahmand 2015 Asian 180 114 36 230 85 35 √
77 Vanill 2015 Asian 13 2 0 13 2 0 √
78 Hubacek 2015 Caucasian 208 214 42 1068 1116 302 √
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Table 4.

Characteristics of primary studies included on A1298C

Author Year of publication Ethnicity Case Control Nair 2012 Cao 2013 Rai 2014 Chen 2015 Yang 2016
AA AC CC AA AC CC
1 Hohlagschwandtner 2003 Caucasian 63 67 15 35 50 16 √ √
2 Li 2004 Asian 33 21 3 29 18 3 √
4 Mtiraoui 2006 Caucasian 108 65 27 130 62 8 √ √ √ √
5 Sotiriadis 2006 Caucasian 44 37 7 45 39 6 √ √
6 Wang 2006 Asian 103 35 10 60 20 2 √ √ √ √
8 Callejon 2007 Caucasian 209 89 44 248 149 37 √ √ √
9 Ren 2007 Asian 0 17 54 1 6 86 √
10 Bae 2009 Asian 144 68 9 75 43 4 √ √
11 Ciacci 2009 Caucasian 22 0 56 52 0 92 √ √ √
12 Rodriguez-Guillen 2009 Caucasian 18 5 0 60 14 0 √
13 Kim 2011 Asian 34 21 2 113 38 4 √ √ √
14 Jeddi-Tehrani 2011 Asian 69 27 4 94 6 0 √
15 Klai 2011 Asian 47 14 2 93 7 0 √
16 Ozdemir 2011 Caucasian 201 257 85 71 35 0 √ √ √
17 Settin 2011 Caucasian 15 49 6 36 97 3 √ √ √
18 Dissanayke 2012 Asian 74 78 43 72 79 46 √
19 Nair 2012 Asian 48 68 13 116 80 6 √ √
20 Torabi 2012 Asian 69 27 4 94 6 66 √
21 Zonouzi 2012 Caucasian 35 46 8 13 34 3 √ √
22 Chen 2013 Asian 24 29 6 38 44 5 √
23 Herodez 2013 Caucasian 36 48 16 43 47 18 √
24 Parveen 2013 Caucasian 88 92 20 157 127 16 √ √
25 Cao 2014 Asian 49 31 2 132 31 3 √
26 Hefler 2014 Caucasian 49 38 7 43 40 11 √
27 Hu 2014 Asian 33 12 7 12 3 1 √
28 Lino 2014 Caucasian 71 32 9 52 43 3 √
29 Luo 2014 Asian 82 40 3 78 54 3 √
30 Yousefian 2014 Asian 98 81 25 68 39 9 √
31 Hubacek 2015 Caucasian 209 212 43 1145 1066 275 √
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The citation matrix showed the differences of selection for each systematic review, along with a meta-analysis which identified similarities and controversies in the published conclusions (Tables 3 and 4).

A summarized meta-analysis combining primary studies reported in 11 systematic reviews and meta-analyses (overall analysis and sub-group analyses by ethnicity)

According to the citation matrixes and the primary studies included in 11 systematic reviews and meta-analyses, we re-calculated the OR and 95%CI of different genotype models (Table 5). Due to high heterogeneity, the OR and 95%CI were removed in a random-effect model. The OR and 95%CI of C677T in Recessive model were not heavily influenced by the 6 primary studies in which only the number of TT genotypes and the total number of cases and controls were provided.

Table 5.

Re-meta-analysis of primary studies on MTHFR polymorphisms

Allele Dominant Recessive Recessive (6 included) Homozygous Heterozygous
OR, 95%CI I 2 OR, 95%CI I 2 OR, 95%CI I 2 OR, 95%CI I 2 OR, 95%CI I 2 OR, 95%CI I 2
C677T
  Asian 1.54 (1.38, 1.72) 62.30% 1.61 (1.39, 1.86) 57.90% 1.85 (1.53, 2.25) 46.70% 1.85 (1.53, 2.25) 46.70% 2.32 (1.84, 2.92) 53.20% 1.44 (1.24, 1.67) 53.80%
  Caucasian 1.28 (1.03, 1.58) 87.20% 1.33 (1.01, 1.76) 84.70% 1.42 (1.05, 1.91) 70.60% 1.43 (1.10, 1.86) 68.80% 1.66 (1.07, 2.58) 83.60% 1.27 (0.96, 1.67) 81.90%
  Total 1.43 (1.29, 1.60) 78.50% 1.50 (1.30, 1.73) 75.20% 1.68 (1.42, 1.99) 60.30% 1.66 (1.42, 1.95) 59.60% 2.08 (1.66, 2.61) 72.90% 1.37 (1.18, 1.57) 70.80%
A1298C
  Asian 1.25 (0.90, 1.75) 85.50% 1.44 (1.05, 1.98) 73.70% 1.15 (0.62, 2.14) 73% 1.47 (0.79, 2.75) 67.40% 1.62 (1.17, 2.25) 71.50%
  Caucasian 1.16 (0.95, 1.41) 76.60% 1.11 (0.88, 1.41) 72% 1.44 (1.00, 2.08) 63.50% 1.43 (0.97, 2.12) 65.70% 1.02 (0.81, 1.28) 65.30%
  Total 1.19 (1.00, 1.42) 81.60% 1.25 (1.03, 1.50) 72.60% 1.29 (0.93, 2.12) 68% 1.43 (1.03, 1.98) 64.20% 1.25 (1.03, 1.53) 70.60%
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In term of the MTHFR C677T polymorphism and RPL, there were 78 primary studies, including 8907 cases and 13,636 controls. The OR and 95%CI of C677T in Allele model were 1.54 (1.38, 1.72) in Asian population, 1.28 (1.03, 1.58) in Caucasian population, and 1.43 (1.29, 1.60) overall. The most significant genotype model for C677T was Homozygous model, in which OR and 95%CI were 2.32 (1.84, 2.92) in Asian population compared with 1.66 (1.07, 2.58) in Caucasian population and 2.08 (1.66, 2.61) overall.

In term of A1298C polymorphism and RPL, 31 primary studies were analyzed, including 4211 cases and 6208 controls. The OR and 95%CI of A1298C in Allele model were 1.25 (0.90, 1.75) in Asian group, 1.16 (0.95, 1.41) in Caucasian group, and 1.19 (1.00, 1.42) overall. Analysis showed no association between A1298C polymorphism and RPL, except that the OR and 95%CI of A1298C in Heterozygous model were 1.62 (1.17, 2.25) in Asians, 1.02 (0.81, 1.28) in Caucasians, and 1.25 (1.03, 1.53) overall, which might indicate moderate RPL risk for the heterozygous genotype AC of MTHFR.

Cumulative meta-analyses of MTHFR polymorphisms and RPL (overall analysis and sub-group analyses by ethnicity)

Cumulative studies were performed on MTHFR polymorphisms and RPL risk sorted by publication year. Differences of tendency across ethnicities were also evident in subgroups.

Trends of C677T and RPL risk in Allele model are shown in Fig. 2 a and b. The first statistical change appeared in the 16th study in 2003, which remained stable in the next 56 primary studies over 12 years (Fig. 2a). Compared with wagging trends for Caucasians in Allele model, C677T mutation of Asians in the Allele model remained unchanged except for the first 3 studies (Fig. 2b).

Fig. 2.

Fig. 2

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Cumulative meta-analysis of a C677T in Allele model, b C677T in Allele model of different ethnicities, c C677T in Recessive model, d C677T in Recessive model of different ethnicities, e C677T in Homogeneous model, f C677T in Homogeneous model of different ethnicities

With 6 primary studies only providing TT and total number, the first statistical association of C677T in Recessive model was achieved in the 14th study in 2002 (Fig. 2c). In the recessive model, C677T showed a similar tendency to Allele model for Asian population (Fig. 2d). Following the publication of 4 studies showing uncertainty, the association did not change in Asian population but remained uncertain in 33 primary studies involving Caucasians.

In Homozygous model, C677T mutation in Asian group did not change after the 3rd study and the width of the 95%CI became more narrow and more stable (Fig. 2e, f).

In all three models, the OR and 95%CI changed in a swinging trend across 31 primary studies (Fig. 3a, b). Heterozygous genotype showed only a moderate risk between A1298C and RPL (Fig. 3c, d). However, a statistical change appeared only in the last 6 studies, published between 2012 and 2014.

Fig. 3.

Fig. 3

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Cumulative meta-analysis of a A1298C in Allele model, b A1298C in Allele model of different ethnicities, c A1298C in Heterogeneous model, d A1298C in Heterogeneous model of different ethnicities

Publication bias and sensitivity analysis

Begg’s test and Egger’s test were performed for checking the publication bias of the primary case-control studies (Table 6). On C677T of MTHFR polymorphism, publication bias appeared in Dominant model of overall analysis and Caucasian subgroup, Homogeneous model of overall analysis and Asian subgroup, and Recessive model of overall analysis and Asian subgroup. Other primary studies on C677T did not have publication bias. On A1298C of MTHFR polymorphism, publication bias did not exist.

Table 6.

Begg’s test and Egger’s test on publication bias of primary case-control studies

Begg’s test
Allele Dominant Recessive Recessive (6 included) Homogeneous Heterogeneous
z p z p z p z p z p z p
  C677T
    Asian 1.12 0.265 1.66 0.096 2.82 0.005* 2.82 0.005* 3.02 0.002* 0.39 0.696
    Caucasian 1.13 0.26 2.17 0.03* 1.63 0.103 1.31 0.189 1.81 0.071 1.9 0.058
    Total 1.91 0.057 2.38 0.017* 3.17 0.002* 2.86 0.004* 3.4 0.001* 1.34 0.182
  A1298C
    Asian 0.79 0.428 0.89 0.373 0.99 0.322 0.79 0.428 1.63 0.102
    Caucasian 0.11 0.913 0 1 1.53 0.127 1.4 0.161 0.55 0.583
    Total 0.54 0.586 1.03 0.302 1.36 0.173 1.36 0.173 1.34 0.179
Egger’s test
Allele Dominant Recessive Recessive (6 included) Homogeneous Heterogeneous
t p t p t p t p t p t p
  C677T
    Asian 2.02 0.05 1.81 0.078 3.16 0.003* 3.16 0.003* 3.99 0.001* 0.32 0.754
    Caucasian 0.35 0.73 1.35 0.189 1.02 0.32 0.62 0.54 1.2 0.24 1.14 0.264
    Total 1.74 0.087 2.53 0.014* 3.09 0.003* 2.58 0.012* 3.48 0.001* 1.42 0.161
  A1298C
    Asian 0.92 0.374 1.48 0.161 0.92 0.374 1.14 0.273 2.04 0.062
    Caucasian 0.54 0.596 0 0.999 2.09 0.061 1.74 0.11 −0.43 0.678
    Total 0.98 0.337 1.19 0.243 1.48 0.15 1.82 0.081 1.58 0.126
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*In Begg’s test or Egger’s test, p value < 0.05

We conducted sensitivity study to test the origin of heterogeneity. The results showed no individual case-control study had marked effect on the meta-analysis of primary studies of C677T or A1298C.

Quality of systematic reviews and meta-analyses

Table 6 and Table 7 show the quality assessments of 11 systematic reviews and meta-analyses executed with AMSTAR and OQAQ scales. The AMSTAR assessment showed one review of high methodology quality (≥ 8), 2 of low quality (≤ 3), and 8 of moderate quality with scores ranging from 5 to7 (Table 7). The OQAQ scale showed similar results for the quality of 11 systematic reviews, of which there were 2 of low quality (≤ 3) and 9 of moderate quality with scores between 5 and 7 (Table 8).

Table 7.

AMSTAR scores of 11 systematic reviews included

Author Year 1. A priori design 2. Duplicate selection 3. Literature search 4. Status of publication 5. List of studies 6. Characteristics of included 7. Quality of included 8. Scientific quality used 9. Appropriate methods 10. Likelihood of bias 11. Conflict of interest
Nelen 2000 + + + + – – – – + – +/−
Wiwanitkit 2005 + – – + – + – – – – +/−
Ren 2006 + + + + – + – – + + +/−
Wu 2012 + + + + – + – – + + +/−
Cao 2012 + + + + + + – – + + +/−
Nair 2013 + +/− + + – + – – + + +/−
Parveen 2013 + +/− – + – – – – +/− + +/−
Rai 2014 + – + + – + – – + + +/−
Chen 2015 + + + + – + – – + + +/−
Yang 2015 + + + + – + – – + + +/−
Rai 2016 + – + + – + – – + + +/−
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+: yes; −: no; +/–: partially answered or unclear

Table 8.

OQAQ scores of 11 systematic reviews included

Author Year 1. Search methods stated 2. Search comprehensive 3. Inclusion criteria reported 4. Selection bias avoided 5. Validity criteria reported 6. Validity assessed appropriately 7. Combining methods reported 8. Finding combined appropriately 9. Conclusions supported by the data
Nelen 2000 + + + + – – + + +
Wiwanitkit 2005 – – – – – – – – +
Ren 2006 + + + + – – + + +
Wu 2012 + + + + – – + + +
Cao 2012 + + + + – – + + +
Nair 2013 + + + + – – + + +
Parveen 2013 – +/− – +/− – – +/− + +
Rai 2014 + + – – – – + + +
Chen 2015 + + + + – – + + +
Yang 2015 + + + + – – + + +
Rai 2016 + + + – – – + + +
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+: yes; −: no; +/–: partially answered or unclear

Discussion

MTHFR plays a key role in catalyzing synthetic folic acid to 5-MTHF, also absorbed as natural folate from food [29], which participates in the conversion of homocysteine to methionine (Fig. 4). The transformation of homocysteine causes a reduction in the level of homocysteine in the plasma and promotes the methylation of substances such as DNA, protein, and lipids involving S-adenosylmethionine to S-adenosylhomocysteine [30].

Fig. 4.

Fig. 4

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Mechanism of MTHFR in recurrent pregnancy loss (RPL)

DNA methylation plays a crucial role in trophoblast development, affecting imprinted or non-imprinted genes after global demethylation on morula stage (third day postconception) [31]. Epigenetic modification in the placenta leads to intrauterine growth restriction, through methylation on gene promotors [32]. Recent research has reported such placental epigenetics are regulated with imprinted genes such as IGF2/H19, PEG10 on paternal chromosome and PHLDA2, CDKN1C on maternal chromosome [33].

MTHFR polymorphisms induce structural changes of MTHFR protein. For instance, C677T causes alanine to be substituted for valine and A1298C causes glutamate to be substituted for alanine. The thermolability and reduction of enzyme activity lead to elevated concentration of homocysteine [34]. Hcy concentration is also controlled through folate-independent pathways including CBS (cystathionine β-synthase) and BHMT (betaine-homocysteine methyltransferase) in liver and kidney [35]. The compensatory regulations fail to remove Hcy accumulated in pregnant women with MTHFR deficiency, as the peak demand cannot be reached [36, 37]. High level of homocysteine leads to RPL with pathway of Hcy toxicity such as homocysteinylation, oxidative stress induction, and biotoxicity itself [38].

While numerous primary studies and systematic reviews have investigated the correlation between MTHFR polymorphisms and RPL, the outcome of this research remains unclear, and even controversial when comparing across different authors [39]. When comparing OR and 95%CI in Allele model conducted in this current study with the results from previous systematic reviews, it was evident that the C677T polymorphism could represent a risk marker for RPL, especially in Asian population. Results from Homozygous and Recessive models further indicated that TT genotype could represent a significant risk marker for RPL. Despite the moderate level of risk shown in Heterozygous model, there may be no relationship between A1298C and RPL.

Paternal MTHFR gene influence may have contributed to discrepancies in A1298C, resulting aneuploidy in embryo [40]. Despite chromosome abnormality, paternal MTHFR polymorphisms could also lead to destruction in sperm nucleus DNA as well [41]. Cornet [42] reported that, in population included (18 homozygous, 77 heterozygous, 1405 control) with SDI (sperm nucleus decondensation index) above 20%, the homozygote group presented with 67% comparing to 23% in the control group and 30% in heterozygote group. The selection of primary studies from systematic reviews and meta-analyses most likely contributed to the discrepancies evident in the published conclusions from different authors; this was evident in the selection matrix.

AMSTAR and OQAQ scales showed moderate quality overall in 11 systematic reviews and low quality in 2 systematic reviews. A common deficiency of the 11 systematic reviews with moderate quality was the lack of validity criteria or the nature of the validity criteria used, which is commonly evaluated with the NOS (Newcastle-Ottawa Scale) [43] for case-control studies. The lack of qualification for primary studies may therefore have contributed to the heterogeneity and controversy evident in the published conclusions.

The tendency of stability was demonstrated by the cumulative meta-analysis of MTHFR polymorphisms in RPL. The meta-analyses relating to C677T, and based upon early primary studies, did not show any correlation with RPL; this was in accordance with the views of previous systematic reviews and meta-analyses which were published prior to 2005. However, as the number of primary studies increased, the OR and 95%CI of the C677T polymorphism changed to positive and became stable over the next few years in Allele, Recessive, and Homozygous models. The trend for the A1298C polymorphism in Allele model remains unclear; despite the positive change appearing in 2011, the data was mainly based on recent primary studies in Asian population.

The mild tendency of A1298C in Heterozygous model might attribute to composite C677T and A1298C. Xu [44] has found that compound 677/1298 heterozygous genotype is a risk factor in RPL. In 218 in RPL group and 264 in control group, no composite homozygote genotype appeared; however, patients with composite heterozygote (677CT-1298AC) presented with higher risk compared with the control group (OR, 95%CI 4.996, 1.65–15.129). Paternal effect may also have contributed to the compound MTHFR genotype in embryo [45].

There are some limitations in this present study which need to be considered. First of all, heterogeneity was observed in each genotype model for C677T and A1298C polymorphisms; thus, results based on primary studies should be interpreted with caution. Second, the NOS is not used to investigate the quality of primary studies and this might be the underlying factor responsible for the overall high heterogeneity. Finally, there are some factors which could have influenced our results but were not considered, such as age, body mass index (BMI), smoking, alcohol and drug abuse, and environmental factors [46].

In conclusion, MTHFR C677T polymorphism appears to represent a risk marker for RPL, especially in Asian population. The TT genotype of C677T appears more significant than the other genotypes, particularly in term of RPL. However, there is no significant correlation between A1298C and RPL, except for in Heterozygous model, which indicates moderate risk. We believe that differences in the selection of primary studies lead to the controversial and inconsistent conclusions made by different authors in the systematic reviews and meta-analyses considered in our present study. More comprehensive and rigorous systematic reviews and meta-analyses should now be performed, which incorporate quality-controlled primary case-control studies. Future investigations also need to consider different ethnicities, gene-gene interaction, and gene-environment interaction in order to fully investigate the influence of MTHFR polymorphisms on RPL.

Funding information

This research is supported by Beijing Obstetrics and Gynecology Hospital, Capital Medical University (FCYY201819). The authors are highly grateful to the support of Department of Pharmacy of Peking Union Medical College Hospital.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflicts of interest.

Footnotes

Publisher’s note

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Contributor Information

Chenghong Yin, Phone: +86-010-6313-8748, Email: modscn@126.com.

Xin Feng, Phone: +86-010-5227-7244, Email: fengxin1115@126.com.

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