A Meta-Analysis for Association of XRCC3 rs861539, MTHFR rs1801133, IL-6 rs1800795, IL-12B rs3212227, TNF-α rs1800629, and TLR9 rs352140 Polymorphisms with Susceptibility to Cervical Carcinoma

Background: In spite of substantial declines in both incidence and mortality rates in the past 50 years, cervical cancer remains one of the leading causes of cancer associated mortality among women globally. We performed this meta-analysis to explore the role of XRCC3 rs861539, MTHFR rs1801133, IL-6 rs1800795, IL-12B rs3212227, TNF-α rs1800629 and TLR9 rs352140 polymorphism with susceptibility to cervical carcinoma. Methods: The search databases include PubMed, SciELO, MedRxiv, Web of Science, Scopus, Cochrane Library, China National Knowledge Infrastructure, and China Biology Medicine disc up to 30 June 2021. The language is limited to English and Chinese. The comparison between the polymorphisms and cervical cancer was assessed using pooled odds ratio (OR) and 95% confidence interval (CI). The data are statistically analyzed by Comprehensive Meta-Analysis (CMA) 2.0 software. Results: A total of 59 studies including seven studies with 1,112 cases and 1,233 controls on XRCC3 rs861539, 14 studies with 2,694 cases and 3349 controls MTHFR rs1801133, four studies with 1,121 cases and 1,109 controls on IL-12B rs3212227, seven studies with 1,452 cases and 2,186 controls on IL-6 rs1800795, 20 studies with 4,781 cases and 4909 controls on TNF-α rs1800629, and seven studies with 1743 cases and 2292 controls on TLR9 rs352140 were included. There was a significant association between XRCC3 RS861539, TNF-α rs1800629, and IL-6 rs1800795 polymorphisms and an increased risk of cervical carcinoma in overall population. However, the MTHFR rs1801133, IL-12B rs3212227 and TLR9 rs352140 polymorphisms were not associated. Conclusion: The pooled analysis showed that XRCC3 RS861539, TNF-α rs1800629, and IL-6 rs1800795 were associated with cervical carcinoma susceptibility, but not MTHFR rs1801133, IL-12B rs3212227 and TLR9 rs352140 polymorphisms.

To date, the occurrence and development of cervical cancer is suggested to be associated with persistent HPV infection Zarchi et al., 2010;Pal and Kundu, 2020). The DNA of HPV integrates into the host cell genome and disrupts the open reading frame and causes overexpression of E6 and E7 genes (Binesh et al., 2012;Soheili et al., 2016). However, the specific molecular mechanisms and potential single gene require further studies. Familial based studies and evaluation of inherited genetic variations revealed that host genetic factors have a role in cervical cancer pathogenesis (Yang et al., 2020;Ahmadi et al., 2021). Recently, a comprehensive review study showed that CDK1, CCNB1, ITGB1, FN1, MMP9 and STAT1 played different roles in the progression of cervical cancer through different signaling pathways (Sayad, Ahmadi, Nekouian, et al., 2020;Yi et al., 2020). Moreover, studies have shown that cervical cancer has a heritable genetic component. However, as other complex disease, the identified loci only explain a minority of the risk of cervical cancer. This gap of the heritability explained by genetic markers genome-wide association studies and the heritability identified in familial studies has been termed as 'missing heritability' (Chen et al., 2016;Leo et al., 2017;Sayad, Ahmadi, Moradi, et al., 2020). Thus, our understanding of the genetic basis of cervical cancer is still limited. In this meta-analysis, we explore the role of XRCC3 rs861539, MTHFR rs1801133, IL-6 rs1800795, IL-12B rs3212227, TNF-α rs1800629, and TLR9 rs352140 polymorphisms in susceptibility to cervical cancer.

Eligibility criteria
To obtain the papers, the following criteria should be met to include the papers in our study: 1) studies with case-control or cohort design; 2) studies reported original data; 3) studies appraised association of XRCC3 rs861539, MTHFR rs1801133, IL-6 rs1800795, IL-12B rs3212227, TNF-α rs1800629 and TLR9 rs352140 polymorphisms with cervical cancer; and 4) studies with available and sufficient data for calculating an odds ratio (OR) with 95% confidence interval (CI). The following exclusion criteria were also used: 1) studies on other polymorphism at XRCC3, MTHFR, IL-12B, IL-6, and TNF-α genes; 2) animal studies or in vitro studies; 3) studies with in sufficient data on genotype frequencies or which the number of genotypes and alleles could not be ascertained; 4) linkage studies; 5) family based studies including sibling, twins and trios-parents studies; 6) abstracts, case reports, commentaries, editorials, conference articles, reviews, proceedings and meta-analyses; and 7) duplicates studies or overlapping data.

Quantitative Data Synthesis XRCC3 RS861539
The summary for the association of XRCC3 RS861539 polymorphism with cervical cancer risk are shown in Table 3. Pooled data revealed that XRCC3 RS861539 polymorphism was significantly associated with susceptibility to cervical cancer under the heterozygote genetic model (TC vs. CC: OR = 1.00, 95% CI 1.066-1.585, p = 0.009). Moreover, stratified analysis by ethnicity revealed that the polymorphisms was significantly associated with cervical cancer in Asian women under three genetic models, i.e., allele (T vs. C: OR= 1.302, studies. We did not define any minimum sample size to include in this meta-analysis. The ''mixed'' group means mixed or unknown populations. If more than one study was published by the same author(s) using repeated or overlapped data, the studies with the largest sample size or the most recently published study was included to the meta-analysis. If selected articles did not reported necessary data the corresponding authors was contacted by email to request the missing data.

Statistical Analysis
The comparison between the XRCC3 rs861539, MTHFR rs1801133, IL-6 rs1800795, IL-12B rs3212227, TNF-α rs1800629 and TLR9 rs352140 polymorphism and cervical cancer was assessed using pooled odds ratio (OR) and 95% confidence interval (CI). The pooled data were calculated under five genetic models, i.e., allele (B vs. A), homozygote (BB vs. AA), heterozygote (BA vs. BB), dominant (BB+BA vs. AA) and recessive (BB vs. BA+AA), in which a ''A'' denotes a major allele; ''B'' denotes a minor allele. The χ 2 test and I 2 statistics were used to assess whether there was between-study heterogeneity, in which P >.10 and I 2 < 50% could be considered that there was no statistical heterogeneity between the research results. The fixed effect model was selected for data consolidation; P ≤.10 and I 2 ≥50% could be considered that there was statistical heterogeneity between the research results, and a random effect model was used for data consolidation. If there was significant heterogeneity among studies, the random effects model (DerSimonian and Laird) was used; otherwise, the fixedeffects model (Mantel and Haenszel) was acceptable. The Hardy-Weinberg equilibrium of the control group was evaluated using the χ 2 test, and the expected and actual genotype frequencies of the control group were compared. In this meta-analysis, P-values of ≤.05 were considered statistically significant (Azadi-Yazdi et al., 2017;Mirjalili et al., 2019;Dastgheib et al., 2020). In addition, the sensitivity analysis was performed by excluding HWE-violating studies. Potential publication bias was evaluated using the Egger's linear regression test and Begg's quantitative test. The asymmetric plot of Egger's test and the P-value of Begg's test less than 0.05 were considered a significant publication bias. If there was evidence of publication bias (P<0.05), trim and fill method was applied to adjust for the effect of publication bias (Jafari et al., 2020). All statistical analyses were performed using Comprehensive Meta-Analysis (CMA) Software version 2.0 (Biostat, Englewood, USA). All tests were two-sided, and the P< 0.05 was considered statistically significant.

Characteristics of the Enrolled Studies
The flowchart of selection of studies and reasons for exclusion is presented in Figure 1. There were 1,013 articles relevant to our search words and 395 duplicated studies were excluded. Then, 207 studies removed after reading titles and abstracts. Following the inclusion exclusion criteria 352 studies were excluded. Finally, a

MTHFR rs1801133
The summary for the association of MTHFR rs1801133 polymorphism with cervical cancer risk are shown in Table 3. Pooled results showed that the MTHFR rs1801133 polymorphism was not associated with cervical cancer in overall population and by ethnicity.

IL-12B rs3212227
The summary for the association of IL-12B rs3212227 polymorphism with cervical cancer risk are shown in Table 4. Pooled data showed that the IL-12B rs3212227 polymorphism was not associated with cervical cancer globally. Stratified analysis by ethnicity revealed that this polymorphism was associated with cervical cancer in Asian women under two genetic models i.e., heterozygote (CA vs. AA: OR = 1.349, 95% CI 1.032-1.762, p=0.028) and dominant (CC+CA vs. AA: OR = 1.340, 95% CI 1.041-1.725, p =0.023).

TLR9 rs352140
The summary for the association of TLR9 rs352140 polymorphism with cervical cancer risk are shown in Table 4. Pooled results showed that the TLR9 rs352140 polymorphism was not associated with cervical cancer in overall population and by ethnicity.

Test of heterogeneity
In the current meta-analysis the χ 2 test and I 2 statistics were used for assessing the heterogeneity of the included studies. Results indicate that there was statistical heterogeneity for XRCC3 rs861539, MTHFR rs1801133, IL-6 rs1800795, IL-12B rs3212227, TNF-α rs1800629, and TLR9 rs352140 polymorphisms under most genetic models. Thus, the random effect model was used for evaluating the pooled OR and 95% CI for those models. Subgroup analyses showed that ethnicity of participants  Table 4. Summary Risk Estimates for Association of IL-6 rs1800795, TNF-α rs1800629 and TLR9 rs352140 Polymorphisms with Cervical Cancer Risk might contribute to part of heterogeneity.

Sensitivity Analysis
The process of performing a meta-analysis involves a sequence of decisions and it is important to perform a sensitivity analysis in order to assess the impact of different decisions on pooled data. Thus, a sensitivity analysis was carried out by excluding a single study in turn on pooled ORs. The results showed that no individual study had an influence on the pooled OR all involved polymorphisms at XRCC3 rs861539, MTHFR rs1801133, IL-6 rs1800795, IL-12B rs3212227, TNF-α rs1800629, and TLR9 rs352140 polymorphisms, suggesting the stability of our findings. Moreover, excluding HWE deviated studies suggested that there were no independent studies that significantly influenced our pooled data.

Publication Bias
Begg's funnel plot and Egger's test were used for evaluating publication bias. As shown in Tables 3 and  4, the Egger's test results showed that there was no publication bias for the XRCC3 rs861539, MTHFR rs1801133, IL-6 rs1800795, IL-12B rs3212227, TNF-α rs1800629, and TLR9 rs352140 polymorphisms under all five genetic models. Moreover, Begg's funnel did not statistically revealed a significant publication bias in any of the models for all involved polymorphisms. Thus, the publication bias tests revealed that our pooled ORs were reliable.

Discussion
Genetic factors have been shown to influence the susceptibility of patients to various diseases and have attracted increasing attention (Motamedi et al., 2012;Mazaheri et al., 2014;Kabiri Rad et al., 2018). Several efforts have been made to identify the genetic susceptibility factors underlying development of cervical cancer. However, only a few polymorphisms have shown consistency among studies. In this meta-analysis, the association of XRCC3 rs861539, MTHFR rs1801133, IL-6 rs1800795, IL-12B rs3212227, TNF-α rs1800629 and TLR9 rs352140 polymorphisms with susceptibility to cervical carcinoma was assessed by including all relevant studies. Yuan et al., (2021) in a meta-analysis based on 15 studies with 5,740 cases and 9,931 controls revealed that there was no significant association between the XRCC3 Thr241Met and the risk of gynecological malignancies. However, their subgroup analysis by ethnicity showed that XRCC3 Thr241Met was associated with an increased risk of gynecological malignancies in Asians. Moreover, their stratified analysis by cancer type indicated that XRCC3 Thr241Met associated with cervical cancer in Asians (CT vs. CC: OR=1.50, 95%CI=1.04-2.14; TT vs. CC: OR=3.14, 95%CI=1.38-7.14; CT+TT vs. CC: OR=1.64, 95% CI=1.17-2.31). Abbas et al., (2010) in a case-control study with 260 cervical cancer cases and 265 controls evaluated the association of XRCC1+399A/G, XRCC2+31467G/A and XRCC3+18067C/T polymorphisms with cervical cancer in Indian women. Their results showed that XRCC2+31479G/A and XRCC3+18067C/T polymorphisms were not associated with cervical cancer. However, they showed that he XRCC1+399A/G is linked with cervical cancer in the Indian population. Al-Harbi et al., (2017) in a study among 232 cervical cancer cases and 313 control subjects evaluated the association of CDKN1A C31A, ATM G1853A, HDM2 T309G, TGFB1 T10C, XRCC1 G399A, and XRCC3 C241T with cervical cancer among Saudi Arabian women. They showed that the TGFB1 T10C and XRCC1 G399A polymorphisms were associated with cervical cancer risk.
Our pooled results showed that the MTHFR rs1801133 polymorphism was not associated with cervical cancer in overall population and by ethnicity. Silva et al., (2019)reported that there were no differences in the genotypic and allelic distribution of MTHFR C677T polymorphism between remission (with the presence of pre-neoplastic lesions) and Persistence (with the presence of pre-neoplastic lesions). The same authors, in another study showed that MTHFR C677T polymorphism was not associated with cervical cancer and HPV infection (Silva et al., 2019). However, Sohrabi et al., in a case-control study evaluated the association of MTHFR A1298C and C677T variants among in 50 cervical intraepithelial neoplasia cases, 98 HPV-positive subjects and 47 non-cancerous/non-HPV patients as healthy controls. Their results showed that MTHFR 1298 CC is more likely to be a potential risk factor for HPV-cervical cancer progression (Sohrabi et al., 2020). Zhou et al., (2020) indicated that the MTHFR rs4846048 enhanced the risk of cervical cancer through association with miR-522. Gong et al., (2018) reported that MTHFR C677T polymorphism was not associated with the risk of cervical cancer or cervical intra-epithelial neoplasia, while, the MTHFR A1298C polymorphism could increase the risk of both cervical cancer and cervical intra-epithelial neoplasia.
In the current study pooled data indicated that the TNF-α rs1800629 polymorphism was associated with cervical cancer risk. Wang et al., (2012) in meta-analysis based 27 studies showed that both TNF-α -238 and -308 G/A polymorphisms could be used to identity individual with elevated susceptibility to cervical cancer in by ethnicity (Bi, 2014). Behboodi et al., (2021) in a study among 153 Iranian cervical cancer cases and 292 free cancer subjects demonstrated that TNF-α rs1800629 was associated with increased level and risk of developing cervical cancer. However, Duvlis et al., (2020) reported that TNF-a-238G/A and TNF-a-308 G/T polymorphisms were not associated with the risk of HPV associated cervical intraepithelial lesions or cervical cancer cases in Macedonian women compared to controls. Moreover, Traore et al., (2020) revealed that TNF-308 G/A or IL18-607C/A polymorphisms were not associated with HPV infection among Burkina Faso women.
Pooled data from all eligible studies showed that the IL-6 rs1800795 polymorphism was significantly associated with cervical cancer risk in overall population and among Asian women. Similarly, Duan et al., (2018) in a meta-analysis based on 7 studies showed that the IL-6 rs1800795 polymorphism is associated with risk of cervical cancer in overall. Pu et al., (2016) in a study with 360 cervical cancer cases and 728 healthy subjects showed that this polymorphism is risk factor for cervical cancer development in Chinese women. de Souza et al., (2006) in a case-control study with 56 cases and 253 controls evaluated the association of IL-6 rs1800795 polymorphism with cervical cancer in a Brazilian population. In 2017, Zidi et al., (2017) in a case-control study evaluated the effects of six different genetic variants atIL-6 with risk of cervical cancer. The study revealed that the IL-6 rs1800795 polymorphism has a has a protective role in cervical cancer development in Tunisian women. de Lima Júnior et al., (2016) revealed that the IL-6 rs1800795 polymorphism was not associated with HPV infection and healthy controls in in Brazilian women. The current meta-analysis data showed that the IL-12B rs3212227 genetic variant was not associated with susceptibility to cervical cancer, while; stratified analysis showed that this polymorphism was associated with cervical cancer risk among Asian women. Zheng et al., (2017) in a metaanalysis based on 33 articles with 10,587 cancer cases and 12,040 healthy subjects assessed the genetic association of IL-12B rs3212227 with cancer risk. Their pooled data indicated that IL-12B rs3212227 polymorphism was associated with cancer risk in overall. However, subgroup analysis by cancer type showed that IL-12B rs3212227 was not associated with cervical cancer. In 2016, Chang et al., (2015) in a meta-analysis based on 5 studies with 2552 cervical cases and 2232 healthy subjects showed that this polymorphism did not associate with risk of cervical cancer. In 2012, de Carvalho et al., found that the IL-12B rs3212227 variant has a protective role in development of cervical cancer in Brazilian women (Do Carmo Vasconcelos De Carvalho et al., 2012). Similarly, Han et al., (2008) found that IL-12B rs3212227 did not associate with cervical cancer risk in Korean population. However,  showed that this polymorphism was associated with increased risk of cervical cancer among polish women.
The current meta-analysis data showed that TLR9 rs352140 was not associated with susceptibility to cervical cancer. Nath et al., (2020) in a study showed that TLR4/9 polymorphisms are associated with increased HPV16/18 infection susceptibility and cervical squamous cell carcinoma risk among the women of Jharkhand state. In another study in India showed the TLR4 and TLR9 polymorphisms and haplotypes with hrHPV infection and cervical cancer risk (Pandey et al., 2019). Martínez-Campos et al., (2017) in a case-control study revealed that TLR9 rs187084 is a risk factor for HPV infection, squamous intraepithelial cervical lesion, and uterine cervical neoplasm in Mexican female population. Jin et al., (2017) also evaluated the association of some variants at Toll-like receptors gene with cervical cancer among 420 Chinese cervical cancer patients and 842 controls. Their results showed that mutant alleles of TLR2 rs3775290, TLR4 rs7873784, and TLR9 rs352140 were associated with increased cervical cancer risk. Similarly, Yang et al., (2020) in meta-analysis based on eleven studies indicated that the TLR9 rs187084 and rs352140 polymorphisms may contribute to development of cervical cancer, but not TLR2-196 to -174 del/ins polymorphism.
In summary, our pooled data indicated that the XRCC3 RS861539, TNF-α rs1800629, and IL-6 rs1800795 genetic variants were associated with susceptibility to cervical cancer globally. However, the MTHFR rs1801133, IL-12B rs3212227 and TLR9 rs352140 variants were not associated. Larger and more rigorous studies among different ethnicities are needed to further evaluate these associations with cervical cancer.