Vascular endothelial growth factor +405 G/C polymorphism and endometriosis risk: a comprehensive meta-analysis

Endometriosis, a prevalent condition associated with infertility, is characterized by the presence of tissues resembling the endometrium outside the uterus[1]. These sites often include the bladder, colon, and pelvic region, including the ovaries, ligaments, and peritoneal surfaces[2]. This disorder affects approximately 5%–10% of women in their reproductive years and 176 million women worldwide[2]. It is often accompanied by pelvic pain and infertility and is an estrogen-dependent inflammatory disease. Typical symptoms include pelvic pain, dysmenorrhea, dyspareunia, heavy menstrual bleeding, and fertility difficulties[3]. Endometriosis is difficult to diagnose and treat because of the significant knowledge gap regarding its cellular and molecular pathways. Diagnosing endometriosis is challenging and requires surgical visualization and sampling, together with histological examination, for confirmation. Furthermore, the categorization and staging are not always correlated with the clinical presentation, responsiveness to treatment, or prognosis[4,5]. Although the precise cause of endometriosis is still mostly unknown, hypotheses include coelomic metaplasia and lymphatic and vascular metastases[6]. The well-established theory of “retrograde menstruation” involves the overflow of endometrial tissue-containing menstrual debris via the fallopian tubes into the pelvic peritoneal cavity[7]. The pathophysiology of endometriosis involves immunological factors and vascularization hormones during the development and establishment of lesions[8]. Vascular endothelial growth factor (VEGF), also referred to as vascular permeability factor, was first identified by Ferrara and Henzel[9]. VEGF enhances vascular permeability by activating prostacyclin and nitric oxide[10]. Additionally, it is an angiogenic cytokine that is essential for both normal and pathological angiogenesis and has specific mitogenicity for endothelial cells[11]. The 14 kb coding region of VEGF, which has eight exons and seven introns, is located on chromosome 6p12[12]. Vascular endothelial growth factor receptor-1 (VEGFR-1), VEGFR-2, and neuropilin are receptors that interact with VEGF[13]. Variations in VEGF protein synthesis have been linked to single nucleotide polymorphisms (SNPs) in VEGF[13]. SNPs are pivotal in understanding the genetic intricacies of endometriosis. SNP markers allow researchers to identify genetic variants associated with disease onset and progression. Association studies have identified SNPs and mutations in several genes that potentially increase the susceptibility to endometriosis, although their exact function remains unknown. Several VEGF SNPs have been identified. Exon 1 of VEGF contains the +405 G/C polymorphism, which corresponds to the 5’ untranslated region (UTR) of the gene. It has been investigated in connection with the risk of endometriosis and is linked to differential VEGF expression. Different alleles (G or C) at this location may affect VEGF expression and the onset or course of endometriosis[14,15]. Several studies have examined the association between VEGF polymorphisms and endometriosis, and VEGF + 405G/C has been thoroughly examined for possible associations with endometriosis risk[13,16–28]. However, the evidence is still ambiguous, and their conclusions are debatable because of diverse ethnic groups and small sample sizes. Thus, we included both the findings of prior research and additional studies that had not been incorporated into meta-analyses, to delve deeper into the correlation between VEGF polymorphisms and the susceptibility to endometriosis and to specifically investigate the “C” allele as a potential risk factor for developing endometriosis.

Study design This study was a comprehensive meta-analysis that investigated the impact of the VEGF +405G/C polymorphism on the vulnerability to endometriosis. Fourteen case-control studies published between 2005 and 2024 were included, with a total sample size of 3417 endometriosis cases and 3531 controls. Statistical analyses were performed using the SPSS software (version 11.0), and odds ratios (ORs) and 95% confidence intervals (CIs) were computed to evaluate associations between VEGF polymorphisms and the endometriosis risk across five genetic models. The protocol for this study aligned with previously published methodologies[13,16–26]. Literature search strategy A literature search of the PubMed database was conducted in June 2024, to identify relevant studies. The search included keywords related to the polymorphisms of interest, as well as the conditions of endometriosis. The keywords used were “VEGF,” “Vascular endothelial growth factor A,” or “Vascular endothelial growth factor” in combination with “Endometriosis,” “polymorphism,” “Single nucleotide polymorphism,” “SNP,” “Mutant,” and “Gene variation.” Inclusion and exclusion criteria To test the meta-analysis research hypothesis, the following requirements had to be met by the included studies: - A case-control study of the correlation between the VEGF +405 G/C polymorphism and endometriosis risk; - All patients were diagnosed with endometriosis confirmed by pathological or histological examination; - Provided the genotypic or allelic frequencies of the investigated VEGF polymorphisms in cases and controls; - Full text in English. Exclusion criteria: - Not relevant to the VEGF +405 G/C polymorphism; - No control population; - Abstracts, reviews, comments, letters, duplicate publications, conference presentations, meta-analyses, and editorials. Data extraction Data from published studies were extracted to obtain the necessary information. The following information was extracted from each article: first author, year of publication, ethnicity, number of cases and controls, and genotype and allelic frequencies. Statistical analysis All statistical analyses were performed using the SPSS software (version 11.0). ORs and 95% CIs were computed to assess the strength of the associations between VEGF+ 405 G/C polymorphisms and the risk of endometriosis across the genetic models. The P value for each study was determined using the χ2, and P values ≤0.05 were considered significant. The pooled OR was calculated by examining five genetic models for each polymorphism: - Allelic model (risk allele vs. non-risk allele), G vs. C; - Recessive model (homozygous risk allele genotype vs. heterozygous genotype + homozygous non-risk genotype), CC vs. CG + GG; - Dominant model (homozygous risk allele + heterozygous genotype vs. homozygous non-risk allele genotype), CC + GC vs. GG; - Homozygous model (GG vs. CC); - Heterozygous model (GG vs. GC).

Characteristics of studies included in the meta-analysis Fourteen studies met the inclusion criteria[13,16–28]. The total numbers of endometriosis patients and healthy controls were 3417 and 3531, respectively. In the meta-analysis, the +405 G/C polymorphism was studied in all 14 articles. The included studies were published from 2005 to 2015. All patients fulfilled the diagnostic criteria for endometriosis, as confirmed by pathological or histological examinations. The study selection flowchart and characteristics of eligible studies included in the meta-analysis are shown in Fig. 1 and Table 1, respectively. | Author | Year | Ethinicity | Case/ctrl | Case | Control | Case | Control | |||||| |---|---|---|---|---|---|---|---|---|---|---|---|---|---| | CC | GC | GG | CC | GC | GG | C | G | C | G | |||| | Kim et al. [26] | 2005 | Korean | 215/219 | 50 | 89 | 76 | 29 | 116 | 74 | 181 | 241 | 174 | 264 | | Bhanoori et al. [27] | 2005 | Indian | 215/210 | 4 | 71 | 140 | 18 | 79 | 113 | 79 | 351 | 57 | 153 | | Ikuhashi et al. [16] | 2007 | Japanese | 146/181 | 22 | 76 | 48 | 31 | 94 | 56 | 120 | 120 | 156 | 206 | | Gentilini et al. [17] | 2008 | Italian Caucasian | 203/140 | 28 | 106 | 69 | 14 | 59 | 67 | 162 | 244 | 87 | 193 | | Zhao et al. [18] | 2008 | Australian | 949/946 | 85 | 422 | 442 | 74 | 413 | 459 | 592 | 1306 | 561 | 1331 | | Cosín et al. [20] | 2009 | Caucasian | 186/180 | 18 | 91 | 77 | 16 | 80 | 84 | 127 | 245 | 112 | 248 | | Toktam et al. [21] | 2010 | Iranian | 150/144 | 22 | 80 | 48 | 13 | 48 | 83 | 75 | 133 | 124 | 176 | | Attar et al. [22] | 2010 | Turkish | 52/60 | 29 | 16 | 7 | 21 | 30 | 9 | 74 | 30 | 72 | 48 | | Altinkaya et al. [19] | 2011 | Turkish | 98/94 | 25 | 57 | 16 | 84 | 10 | 0 | 107 | 89 | 178 | 10 | | Emamifar et al. [23] | 2012 | Iranian | 480/600 | 153 | 230 | 97 | 66 | 276 | 258 | 536 | 408 | 804 | 792 | | Saliminejad et al. [24] | 2013 | Iranian | 135/173 | 23 | 57 | 55 | 27 | 86 | 60 | 103 | 140 | 167 | 206 | | Vanaja et al. [13] | 2013 | Indian | 302/324 | 23 | 101 | 178 | 23 | 167 | 134 | 147 | 457 | 213 | 435 | | Perini et al. [25] | 2014 | Brazilian | 181/110 | 23 | 75 | 83 | 16 | 56 | 38 | 121 | 241 | 88 | 132 | | Henidi et al. [28] | 2015 | Tunisian | 105/150 | 24 | 44 | 37 | 21 | 68 | 61 | 92 | 118 | 100 | 190 | Meta-analysis The association between the VEGF +405G/C polymorphism and the risk of endometriosis was analyzed using several genetic models: recessive (CC vs. CG + GG), dominant (CC + CG vs. GG), homozygous (GG vs. CC), heterozygous (GC vs. GG), and allelic (C vs. G). The pooled ORs with 95% CIs and corresponding P values are summarized in Table 2. Forest plots were generated to visually summarize the pooled ORs and corresponding 95% CIs for the included studies (Figs. 2 and 3). | Case | Control | OR | CI | P value | | |---|---|---|---|---|---| | Recessive model | ||||| | CC | 529 | 453 | 0.8035 | 0.7018–0.92 | 0.00151 | | CG + GG | 2888 | 3078 | ||| | Dominant model | ||||| | GG | 1373 | 1496 | 1.0944 | 0.9947–1.2041 | 0.064 | | CC+ GC | 2044 | 2035 | ||| | Allelic model | ||||| | C | 2516 | 2893 | 1.0552 | 0.98–1.13 | 0.124 | | G | 4123 | 4384 | ||| | Homozygote model | ||||| | GG | 1373 | 1496 | 0.7859 | 0.6796–0.9088 | 0.00114 | | CC | 529 | 453 | ||| | Heterozygote model | ||||| | GG | 1373 | 1496 | 0.9946 | 0.8979–1.1017 | 0.412 | | GC | 1515 | 1582 | Recessive model (CC vs. CG + GG) A significant association was found between the VEGF +405G/C polymorphism and an increased risk of endometriosis in the recessive model (OR: 0.8035, 95% CI: 0.7018–0.92; P = 0.0015). This suggests that individuals with the CC genotype have a higher risk of developing endometriosis than those with the CG or GG genotypes. The forest plot for the recessive model (Fig. 2) clearly demonstrates this association, and most studies had ORs of <1, indicating that the CG or GG genotypes may confer a protective effect against endometriosis. Homozygous model (GG vs. CC) Similarly, a significant association was observed in the homozygous model in which the GG genotype was compared with the CC genotype. The pooled OR was 0.7859 (95% CI: 0.6796–0.9088, P = 0.00114), indicating that the GG genotype was associated with a lower risk of endometriosis than the CC genotype. The corresponding forest plot (Fig. 3) supports this finding, with consistent results across the included studies. Dominant, heterozygous, and allelic models No significant associations were found in the dominant (OR: 1.094, 95% CI: 0.9947–1.2041; P = 0.064), heterozygous (OR: 0.9946, 95% CI: 0.8979–1.1017; P = 0.412), or allelic (OR: 1.0552, 95% CI: 0.98–1.13; P = 0.124) models. These results suggested that the VEGF +405G/C polymorphism did not significantly influence the risk of endometriosis in these genetic models.

Endometriosis, a prevalent inflammatory condition affecting reproductive-aged women[2], manifests as elevated levels of VEGF in the serum and peritoneal fluid. This heightened presence of VEGF implicates its involvement in critical pathological processes, such as tissue invasion and angiogenesis. VEGF, a potent angiogenic agent, facilitates neovascularization, which is crucial for the implantation of endometrial cells at ectopic sites[29]. Moreover, the development and spread of endometriotic lesions rely heavily on angiogenesis, which is regulated by various factors including VEGF[30,31]. Specifically, VEGFR-2 signaling is a central transduction pathway in angiogenesis[32]. Women with endometriosis exhibit elevated expression of VEGF in the glandular epithelium and VEGFR2 in the endometrial blood vessels, compared to controls[31]. Furthermore, VEGF, which is responsive to estrogen, plays a pivotal role in both normal and aberrant angiogenesis in the human endometrium; its levels fluctuate throughout the menstrual cycle and remain elevated in endometriosis[32]. The complex interaction between these factors highlights the diverse roles of VEGF in the development of endometriosis and offers possibilities for targeted treatment approaches. VEGF is located on chromosome 6p21.3 and consists of eight exons exhibiting alternate splicing that form a family of proteins[32]. At least 30 SNPs exist in VEGF[33]. Several transcription factor binding sites are found in the VEGF 5’-UTR, and transcriptional regulation of the gene is very complex[34]. Polymorphisms within the 5’-UTR lead to differences in VEGF expression between individuals and may influence the etiology of a variety of VEGF-associated pathological conditions[26]. Notably, numerous investigations have examined the association between VEGF and the risk of endometriosis, particularly the +405G/C polymorphism. However, the study findings have been contradictory. Ikuhashi et al.[16], Zhao et al.[18], Cosín et al.[20], Toktam et al.[21], Saliminejad et al.[24], Perini et al.[25], and Henidi et al.[28] found no significant association between the VEGF polymorphism and the risk of endometriosis. Conversely, Kim et al.[26] proposed that the higher incidence of advanced-stage endometriosis in the Korean population could be associated with the VEGF +405 G/C polymorphism. According to Bhanoori et al.[27], women with endometriosis have a lower frequency of the VEGF +405C allele than control subjects. Gentilini et al.[17] validated the hypothesis that the VEGF +405 G/C polymorphism contributes to the development of endometriosis. Altinkaya et al.[19] identified a potential connection between the Turkish population’s risk of both early- and advanced-stage endometriosis and the GC genotype and G allele. Attar et al.[22] noted that the G allele is protective and suggested a link between the endometriosis risk and the CC genotype. According to Emamifar et al.[23], the VEGF +405 G/C polymorphism may be associated with an increased risk of endometriosis in women in northern Iran. Vanaja et al.[13] showed that women carrying the +405 GG genotype are significantly more likely to develop endometriosis than those without this genotype. Given the controversial results of published studies, we conducted a meta-analysis to explore the association between the VEGF +405 G/C (rs2010963) polymorphism and the risk of endometriosis based on 14 independent publications comprising 3417 endometriosis cases and 3531 healthy controls. In all the genetic models analyzed, both the recessive and homozygous models exhibited statistical significance, indicating a notable association. This suggests that women with the VEGF +405 CC genotype have a significantly higher risk of developing endometriosis than those without this genotype. Attar et al.[22] established a correlation between the +405 CC genotype and elevated serum VEGF levels in a Turkish population. Furthermore, our study is unique in detecting an association with endometriosis in both the recessive (CC vs. CG + GG) and homozygous (GG vs. CC) models. Our findings align with those of earlier studies indicating a potential association between the VEGF +405 G/CSNP and endometriosis risk. A 2012 meta-analysis of nine studies revealed similar outcomes and found a statistically significant difference in the frequency of the CC genotype and C allele at the VEGF + 405 G/C site between the two groups (OR: 1.45, 95% CI: 1.10–1.91; P = 0.009). These results suggest that women with the +405 C allele have a significantly higher risk of developing endometriosis[35]. Our findings diverge from those of previous research that failed to identify an association between the VEGF +405 G/C polymorphism and endometriosis. A 2015 meta-analysis of 11 studies reported no significant association between endometriosis and polymorphisms in any model[36]. This outcome aligns with the conclusions drawn in two studies from 2012[37,38] and a subsequent meta-analysis from the same year that included 14 studies, which found no association[39]. However, this meta-analysis had certain limitations. First, the number of studies and participants in specific subgroups in this meta-analysis was limited. Consequently, further studies with larger sample sizes and more comprehensive data are warranted. Second, stratified analyses that consider various factors, such as age, geographic distribution, and stage of endometriosis, would be beneficial, as these may introduce specific confounding biases. Unfortunately, insufficient data were available to conduct these stratified analyses.

In conclusion, this meta-analysis indicated a correlation between the VEGF + 405 G/C polymorphism and susceptibility to endometriosis in both heterozygous and recessive models. However, further research is required to validate these findings and explore the underlying mechanisms involved in the association between VEGF polymorphisms and the susceptibility to endometriosis. Acknowledgments None. Author contributions K.R. contributed to the execution of experiments, analysis and interpretation of data, statistical analysis, and drafting of the manuscript. K.V.V. contributed to the statistical analysis, analysis and interpretation of data, and drafting of the manuscript. M.B. contributed to the conception and design of the study, analysis and interpretation of data, statistical analysis, and drafting of the manuscript. All authors have seen and agreed to the Author contributions statement. Funding(s) None. Conflicts of interest All authors declare no conflicts of interest. Ethics approval The study is based on the analysis of published patient data. These datasets are freely available. Given their pre-existing nature, there is no direct interaction with human subjects, thereby circumventing the necessity for ethical committee approval. Data availability statement Only published data was used. The data used in our study were obtained from previously published research articles available on PubMed. Since this was a meta-analysis, we only used publicly accessible data from peer-reviewed studies, which can be accessed through the original publications.

Endometriosis; VEGF; Polymorphism; Meta-analysis