Association of the Vascular Endothelial Growth Factor Gene Polymorphisms (–460C/T, +405G/C and +936T/C) with Endometriosis: A Meta-Analysis

Introduction

Endometriosis is a common gynecological disease defined by the presence of ectopic endometrial glands and stroma, which is associated with both pelvic pain and infertility. Up to 10% of women of reproductive age may be affected. It is a multifactorial and polygenic disease in which angiogenesis may be implicated (Shifren et al., 1996; Donnez et al., 1998; McLaren, 2000; Tan et al., 2002; Gilabert-Estelles et al., 2007). Endometrial angiogenesis is promoted by numerous inducers and growth factors, including vascular endothelial growth factor (VEGF). VEGF increases vascular permeability and induces endothelial cell proliferation, migration, differentiation and capillary formation (Ferrara, 2004). Several studies have investigated and observed that VEGF mRNA and protein levels were significantly higher in women with endometriosis (Donnez et al., 1998; Di Carlo et al., 2009), which supported a key role for VEGF in the pathological angiogenesis in endometriosis (Shifren et al., 1996; Kupker et al., 1998; Fasciani et al., 2000; Tan et al., 2002; Khan et al., 2003; Matalliotakis et al., 2003; Gilabert-Estelles et al., 2007). After the attachment of endometrial cells, high VEGF levels could provoke an increase in the subperitoneal vascular network and facilitate implantation and viability of endometrial cells (Donnez et al., 1998). Moreover, it has been demonstrated that anti-human VEGF antibody could effectively interfere with the maintenance and growth of endometriosis by inhibiting angiogenesis in a nude mouse model (Nap et al., 2004).

The human VEGF gene is located on Chromosome 6p21.3 (Vincenti et al., 1996) and consists of eight exons exhibiting alternate splicing which results in a family of proteins (Tischer et al., 1991). Several transcription factor-binding sites are found in the VEGF 5′-untranslated region (5′-UTR) and variation within the region increases the transcriptional activity (Fukumura et al., 1998; Lambrechts et al., 2003). Three functional VEGF polymorphisms in 5′ or 3′-UTR [–460C/T (rs833061), +405G/C (rs2010963) and +936T/C (rs3025039)] have been found to be associated with variation in VEGF protein production (Renner et al., 2000; Watson et al., 2000) and have been related to several diseases in which angiogenesis is involved (Krippl et al., 2003; Lin et al., 2003; Lee et al., 2005). Up to now, a number of molecular epidemiological studies have been conducted to examine the association between VEGF–460C/T, +405G/C, +936T/C polymorphisms and risk of endometriosis in diverse populations (Hsieh et al., 2004; Bhanoori et al., 2005; Kim et al., 2005; Ikuhashi et al., 2007; Gentilini et al., 2008; Kim et al., 2008; Zhao et al., 2008; Cosin et al., 2009; Liu et al., 2009; Attar et al., 2010; Lamp et al., 2010; Altinkaya et al., 2011), however, there is no conclusive result available. This discrepancy exists partially because of the possible small effect of the polymorphism on endometriosis risk and the relatively small sample size in each of the published studies. Therefore we performed this meta-analysis to derive a more precise estimation of these associations. The goal of this study is to pool together data from multiple investigations, to analyze the heterogeneity among different investigations and to derive a more precise estimation of these associations.

Materials and Methods

Results

Discussion

The present meta-analysis, including 2690 cases and 2803 controls, explored the association between the VEGF–460C/T, +405G/C, +936T/C polymorphisms and endometriosis risk. In the overall analysis, no significant association was observed between the –460C/T and +405G/C polymorphisms and risk of developing endometriosis. By contrast, a significant association was found between the +936T/C polymorphism and the risk of developing endometriosis in CT versus CC, CT/TT versus CC and T versus C genetic models. Stratified analysis showed that significantly strong association between +936T/C polymorphism and endometriosis was present only in stage III–IV (OR = 1.32 for dominant mode; OR = 1.30 for T vs. C allele), but not in stage I–II disease. However, no significantly increased risk of endometriosis was found in any of the genetic models in Asian or in Caucasian subjects. Our results indicate that the VEGF+936T allele is a genetic risk factor for developing endometriosis, especially for stage III–IV disease.

VEGF is an endothelial cell-specific angiogenic protein that appears to play an important role in a variety of oestrogen target tissues in regulating endometrial angiogenesis at a local level (Girling & Rogers, 2005). VEGF gene is a promising candidate gene that appears to play a key role in the pathogenesis of endometriosis, as increased VEGF mRNA expression and elevated protein levels in peritoneal fluid and serum of endometriosis patients have been reported (Donnez et al., 1998; Fasciani et al., 2000; Mahnke et al., 2000; McLaren, 2000; Gilabert-Estelles et al., 2007; Di Carlo et al., 2009). Polymorphisms in VEGF have been reported to have an effect on the regulation of gene expression that results in altered levels of VEGF, and may therefore contribute to the pathogenesis of endometriosis (Watson et al., 2000; Cosin et al., 2009).

The +936C/T polymorphism locates in the 3′-UTR of the VEGF gene (Jin et al., 2005) and the +936T allele has been shown to correlate with lower VEGF plasma levels (Renner et al., 2000; Krippl et al., 2003). Several potential mechanisms have been suggested: (i) the +936C>T transition in the 3′-UTR may lead to the loss of a potential binding site for activator protein 4 (AP-4) (Renner et al., 2000), which is a helix-loop-helix transcription factor, enhancing expression of several viral and cellular genes by binding to specific enhancer sites (Mermod et al., 1988; Hu et al., 1990); (ii) this polymorphism may be in linkage disequilibrium with another unknown polymorphism elsewhere; and (iii) the C>T transition may lead to a change of the binding miRNAs. In order to explore the probable mechanisms, the variant +936C>T in the 3′-UTR was analyzed for putative binding miRNAs through MicroSNiPer software (http://cbdb.nimh.nih.gov/microsniper/) (Barenboim et al., 2010). As shown in Figure S5, +936C>T variation creates a binding site for miR-4475, and deletes a binding site for miR-1973. So, we infer that the genetic variant could regulate VEGF gene expression by the creation/deletion of miRNAs and in turn affect the susceptibility of endometriosis. Whether this indeed is the case requires further investigation.

A recent meta-analysis on this polymorphism included six studies and concluded no overall association between these three polymorphisms and endometriosis (Zhao et al., 2008). Several observations in the study, such as no association of VEGF–460C/T, +405G/C with endometriosis, are similar to ours. However, the association between VEGF+936T/C polymorphism and endometriosis was different from ours. Cumulative analysis clearly states less likelihood of finding no overall association as reported by Zhao et al. Inclusion of few recent studies in our meta-analysis could be responsible for the differences in the overall inference.

Some limitations of this meta-analysis should be acknowledged. First, in the ethnic subgroup analyses, the number of each subgroup was relatively small, not having enough statistical power to explore the real association. Second, our results were based on unadjusted estimates, while a more precise analysis should be conducted if all individual data were available, which would allow for the adjustment by other co-variants including age, body mass index, smoking status, drinking status and other lifestyle factors. Third, publication bias, which can occur when studies with null or unexpected results are not published, is of concern. Therefore, we can't exclude the effect of potential publication bias on this metaanalysis.

In conclusion, this meta-analysis suggests that the VEGF+936T polymorphism is capable of causing endometriosis susceptibility. It is necessary to conduct large sample studies using standardized unbiased genotyping methods, homogeneous patients with endometriosis and well-matched controls. Additionally, concerning endometriosis with multifactorial etiology, more studies or complete case-control studies, especially stratified by different ethnic background, environmental exposure or other risk factors, should be performed to clarify possible roles of VEGF polymorphism in the pathogenesis of endometriosis in the future.

Conflict of Interest

The authors declare no conflict of interest.