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Association between Thrombophilic Genes Polymorphisms and Recurrent Pregnancy Loss Susceptibility in the Iranian Population: a Systematic Review and Meta-Analysis

Studies have indicated that thrombophilic genes polymorphisms are associated with recurrent pregnancy loss (RPL) in the Iranian population. We aimed to evaluate the precise association between thrombophilic genes polymorphisms (MTHFR C677T, MTHFR A1298C, Prothrombin G20210A, FVL G1691A, and PAI-1 4G/5G) and RPL risk in the Iranian population. PubMed, Web of Science, Google Scholar, and ISC were searched for eligible articles published up to April 1, 2017. In total, 37 case-control studies in 18 relevant publications were selected: 1,199, 1,194, 630, 830, and 955 RPL cases and 1,079, 1079, 594, 794, and 499 controls for MTHFR C677T, MTHFR A1298C,Prothrombin G20210A, FVL G1691A, and PAI-1 4G/5G, respectively. The results indicated a significant increased risk of RPL in all genetic models in the population. Also, Prothrombin G20210A and FVL G1691A as well as PAI-1 4G/5G polymorphisms were associated with RPL risk in the Iranian population. Hence, thrombophilic genes polymorphisms are associated with an increased RPL risk in the Iranian population.

INTRODUCTION he miscarriage of three or more consecutive pregnancies in the first or early second trimester is termed as recurrent pregnancy loss (RPL) [1] . Several etiological factors, including endocrinologic problems, uterine structural, chromosomal anomalies, and antiphospholipid antibody syndrome can be the causes of some RPL cases. However, in many cases, the pathogenesis of RPL remains unknown [2,3] . For more than two decades, researchers have focused on certain inherited thrombophilic factors that may be the risk of arterial and/or venous thromboses and their possible association with pregnancy complications such as early pregnancy loss [4] . It is estimated that the thrombophilia is a common cause of RPL and is found in 40-50% of cases [5] .

Search strategy
To identify eligible studies for this meta-analysis, we searched the PubMed, Web of Science, Google Scholar databases, ISC, and EMBASE. In the search, we considered all eligible articles published up to April 1, 2017 that examined the association between the MTHFR C677T, MTHFR A1298C, Prothrombin G20210A, FVL G1691A, and PAI-1 4G/5G polymorphisms and RPL risk in the Iranian population. The following key terms were included in our search: "recurrent pregnancy loss", ''recurrent miscarriage'', ''habitual abortion'', "RPL", "thrombophilic gene", "MTHFR C677T'', "MTHFR A1298C'', "Prothrombin G20210A'', "Factor V Leiden G1691A'', "PAI-1 4G/5G'', "polymorphism", "variant", "gene", "genotype", "SNP", and "allele". The extracted publications were limited to Persian and English languages and conducted only on human subjects. We retrieved those publications matching the keywords without no restriction, and then the studies were evaluated by reading the title and abstract. We have also screened the reference lists of the retrieved articles for original papers. If there were multiple reports of the same study or overlapping data, only the study with the largest sample sizes or the most recent one was selected in our meta-analysis, and the others were excluded.

Inclusion and exclusion criteria
The included studies to the meta-analysis had to be consistent with the following criteria: (1) published in full-text, (2) be case-control or cohort design, (3) evaluate the association between MTHFR C677T, MTHFR A1298C, Prothrombin G20210A, FVL G1691A, and PAI-1 4G/5G polymorphisms and the risk of RPL in the Iranian populations, (4) offered the size of the sample and sufficient data (genotype distributions of both cases and controls were available) for estimating OR with 95% CI, and (5) written in English or Persian. The exclusion criteria were as follows: (1) abstracts, case reports, letter to the editor, and reviews, (2) studies with only case group (no control population), (3) studies on other polymorphisms of thrombophilic genes, (4) studies without detail genotype frequencies in which calculation of OR is impossible, and (5) duplicate publications of data from the same study.

Data extraction
Two investigators independently extracted the data using a pre-designed form. Based on the inclusion and exclusion criteria, we extracted the following data from each study: the first author, year of publication, number of RPL patients and controls, genotype and allele frequency, minor allele frequencies (MAFs) in control subjects, and Hardy-Weinberg equilibrium (HWE) test in control subjects. For conflicting evaluation, these two investigators carried out discussions until a consensus was reached.

Statistical analysis
All the statistical analyses were performed by comprehensive meta-analysis (CMA) version 2.0 software (Biostat, USA). All P values were two-tailed with a significant level at 0.05. The strength of associations was assessed by using ORs and 95% CIs, and the significance of pooled ORs was examined by Z test . We performed a meta-analysis of the association between MTHFR C677T polymorphism and RPL under the allelic model (T vs. C), the homozygote model (TT vs. CC), the heterozygote model (CT vs. Heterogeneity assumption was checked by a chisquare-based Q-test, and I 2 statistics was calculated to quantify the proportion of the total variation across studies due to heterogeneity [26] . The heterogeneity was considered significant if either the Q statistics had p < 0.1 or I 2 > 50%. An I 2 value of 0% represents no heterogeneity, and with the values of 25%, 50%, 75%, or more, it represents low, moderate, high, and extreme heterogeneity, respectively. A P value greater than 0.10 indicated the lack of heterogeneity among studies; therefore, the fixed-effects model (Mantel-Haenszel method) was used to calculate pooled OR [27] . Otherwise, the fixed-effects model (Mantel-Haenszel approach) was used. HWEs were calculated with goodness-of-fit tests (i.e., chi-square or Fisher's exact tests). A value of p < 0.01 signified a departure from HWE [28] . One-way sensitivity analyses were carried out by consecutively omitting one study at a time to assess the power of the meta-analysis findings [29] . Visual inspection of the asymmetry of funnel plots was carried out to assess potential publication bias. Begg's funnel plot, a scatter plot of effect against a measure of study size, was generated as a visual aid to detect bias or systematic heterogeneity [30] . Publication bias was assessed by Egger's test; p < 0.05 was considered statistically significant [31] . Sensitivity analysis was performed to evaluate the stability of the results by removing the studies, but not in HWE.

Characteristics of included studies
Based on the search criteria, 53 individual literatures were found. After screening the titles and abstracts, 35 publications that did not meet the criteria were excluded. These studies were reviews, short reports, case reports, and other polymorphisms of MTHFR, Prothrombin, FVL, and PAI-1 genes. As summarized in Tables 1 and 2, a total of 37 case-control studies in 18 publications [8][9][10][11][12][13][14][15][16][17][18][19][20][21][22][23][24][25] were selected in the final metaanalysis, including 1,199 RPL cases and 1,079 controls for MTHFR C677T (from ten studies), 1,194 RPL cases and 1079 controls for MTHFR A1298C (from ten studies), 630 RPL cases and 594 controls for Prothrombin G20210A (from five studies), 830 RPL cases and 794 controls for FVL G1691A (from seven studies), and 955 RPL cases and 499 controls for PAI-1 4G/5G (from five studies). Genotype distributions in the controls of ten case-control studies were not in agreement with HWE. Table 3 Table 3 summarizes the ORs with corresponding 95% CIs for association of Prothrombin G20210A, FVL G1691A, and PAI-1 4G/5G polymorphisms with RPL risk in the Iranian population. Among the eligible studies were pooled to the meta-analysis of Prothrombin G20210A polymorphism, only allelic and dominant model was applicable because they provided the genotypes of AA + GA vs. GG. Therefore, the pooled analyses showed a significant association between Prothrombin G20210A polymorphism and RPL in the Iranian population in the allelic model (A vs. G: OR = 1.979, 95% CI = 1.128-3.472, p = 0.017,

Sensitivity analysis
To evaluate the influence of individual studies on the risk of RPL, the studies were sequentially deleted from this meta-analysis, and the pooled ORs were performed. However, the results did not change exactly, which verify that no individual studies significantly affected the pooled ORs. Additionally, sensitivity analysis was performed after excluding HWE-violating studies, and the corresponding pooled ORs were not materially altered, indicating that our results are statistically robust (not shown).

Publication Bias
In this meta-analysis, Begg's funnel plot and Egger's test were used to assess the publication bias of included studies. The funnel plot revealed no obvious publication bias for MTHFR A1298C, Prothrombin G20210A, FVL G1691A, and PAI-1 4G/5G, and this result was confirmed by Begg's test and Egger's test (Fig. 2). However, the shapes of the funnel plots revealed obvious asymmetry for MTHFR C677T and MTHFR A1298C in the recessive model ( Fig. 2A), suggesting that there were obvious publication biases in these two genetic models. Moreover, the results of Egger's regression test provided sufficient evidence for publication bias for MTHFR A1298C in the recessive model (P Begg's = 0.348, P Egger's = 0.022), but not for MTHFR C677T (P Begg's = 0.020, P Egger's = 0.056). Therefore, we used Iran. Biomed. J. 22 (2): 78-89   the Duval and Tweedie non-parametric "trim-and-fill" method to adjust the results of publication bias for MTHFR A1298C polymorphism recessive model. However, the meta-analysis with and without ''trimand-fill'' method did not show different results, showing that the results of this meta-analysis are statistically robust.

DISCUSSION
It is known that folate is required for the proper development of fetus and placenta [32] . Aberrations in folate pathway such as maternal folate deficiency, maternal hyperhomocysteinemia, and either MTHFR C677T or A1298C polymorphisms were found to contribute to the etiology of RPL in different populations [33] . Although MTHFR A1298C polymorphism may not responsible for increased total homocysteine, it seems that this polymorphism contributes significantly to the increased homocysteine levels [34,35] . Previous studies have reported that MTHFR C677T polymorphism is significantly associated with the increased risk of RPL. For instance, Chen et al. [36] in a meta-analysis of 16 articles involving 1420 cases with RPL and 1408 controls reported that MTHFR C677T was significantly associated with RPL risk in the Chinese population under all genetics models. Similarly, Wu et al. [37] and Cao et al. [38] findings supported that the idea that MTHFR C677T polymorphism was associated with the increased risk of RPL among Asians, but not Caucasians. Based on these studies, the MTHFR 677TT polymorphism has a significant increased likelihood of RPL in Asians, which is in agreement with our conclusion. The data of a meta-analysis by Nair et al. [39] showed that MTHFR A1298C polymorphism was a genetic risk factor for RPL. However, Cao et al. [38] did not find any significant association between MTHFR A1298C polymorphism and RPL susceptibility. The combined data, based on previous studies, showed that both MTHFR C677T and MTHFR A1298C polymorphisms might be a risk factor for RPL.
As for the other two polymorphisms, we also find a significant association of polymorphisms in Prothrombin G20210A, FVL G1691A, and PAI-1 4G/5G with the risk of RPL, which were consistent with the majority but not all previous studies [40][41][42] . In a meta-analysis conducted by Gao et al. [41] , the Prothrombin G20210A variant was reported to increase the risk of RPL (fetal loss, primary RPL, or secondary RPL) [43] , particularly in Europeans and women older than 29 years. In another meta-analysis, Kovalevsky et al. [44] found that FVL G1691A or Prothrombin gene polymorphisms were associated with the increased risk of two or more miscarriages compared with women without these polymorphisms. In addition, in three meta-analyses, Chen et al. [45] , Su et al. [46] and Li et al. [47] suggested that PAI-1 4G/5G polymorphism might be associated with RPL development.
Between-study heterogeneity, a multifactorial phoneme in meta-analyses, is a potential problem when interpreting the results [48][49][50] . In addition to ethnicity and the source of controls, selection of controls, race variation, age, gender, and prevalence of lifestyle factors might also generate the heterogeneity [35,48,49] . In the present meta-analysis, between-study heterogeneity was observed in all polymorphisms, and thus a random-effect model was used for those genetic models.
There was an inevitable publication bias in our metaanalysis because we retrieved only published studies. Publication bias was assessed by funnel plots whose symmetries were further evaluated by Egger's linear regression tests. We have suggested that for the recessive model, the publication bias might be owing to the limited number of the selected studies. Therefore, the negative results in our meta-analysis are possibly due to the limited number of publications to determine statistical significance.
To the best of our knowledge, there is no earlier study on the analysis of thrombophilia gene polymorphisms in RPL in the Iranian population. However, in interpreting results of this meta-analysis, some limitations should be addressed. First, there was no sufficient number of relevant studies to explore more comprehensive association between the thrombophilic genes and RPL in the Iranian patients. Second, in this meta-analysis, only published studies were searched. It is possible that some important unpublished studies that meet our inclusion criteria were missed and ignored in the literature search. Therefore, inevitable publication bias might be exist, which could eventually help explain the possible existence of publication bias in the recessive model. Third, the Iranian population is mixed of different ethnicities, including Persian, Azeri, Kurdish, Lurs, Gilaki, Balochi, etc. However, we did not conduct subgroup analyses because insufficient data were available from the primary literature search. Moreover, due to limited individual data, a more precise analysis on other covariates, such as age, number of abortions, and environmental factors should be performed. As a result, more studies with large sample sizes in view of these factors are also desired. Finally, due to the lack of the original data, we did not take potential interactions among gene-gene (especially thrombo-philic genes interactions), gene-environment, or even different polymorphism loci of the same gene, which all may affect RPL risk in the population.
In conclusion, based on the available evidence, the current meta-analysis demonstrates that there is a significant association between the MTHFR C677T, MTHFR A1298C, Prothrombin G20210A, FVL G1691A, and PAI-1 4G/5G polymorphisms and RPL risk in the Iranian population. In addition, the direction of further research with a larger sample size should focus not only on the simple relationship of thrombophilic genes polymorphisms and RPL risk but also on gene-gene and gene-environment interactions.