Hum. Reprod. Advance Access originally published online on October 30, 2007
Human Reproduction 2008 23(1):211-215; doi:10.1093/humrep/dem341
The vascular endothelial growth factor +405G>C polymorphism in endometriosis
1 Molecular Biology Laboratory, Istituto Auxologico Italiano, Cusano Milanino, Milan, Italy 2 Università degli Studi di Milano, Milan, Italy 3 Fondazione Ospedale Maggiore Policlinico, Mangiagalli e Regina Elena, Milan, Italy 4 Ospedale Macedonio Melloni, Milan, Italy
5 Correspondence address. Tel: +39-02-619112576; Fax: +39-02-619113033; E-mail: a.diblasio{at}auxologico.it
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BACKGROUND: Vascular endothelial growth factor (VEGF) is a potent stimulus of angiogenesis potentially contributing to the pathogenesis of endometriosis. The aim of this study was to investigate the potential association between the single nucleotide polymorphism +405G>C of the VEGF gene with the risk of endometriosis, for the first time in the Caucasian population.
METHODS: The polymorphism +405G>C of the VEGF gene was examined in n = 203 Italian women affected by endometriosis and in n = 140 women without laparoscopic evidence of the disease. All the women were genotyped by PCR-restriction fragment length polymorphism from venous blood samples. We then performed a meta-analysis including results from the present study and from the two previously published studies on this topic.
RESULTS: The distribution of the three different genotypes significantly differed between women with and without the disease (P = 0.03). The odds ratio (95% confidence interval) for endometriosis in women carrying the C allele was 1.8 (1.2–2.8). The Breslow–Day test revealed statistically significant heterogeneity among the studies performed so far thus indicating inconsistency among studies and excluding the possibility of obtaining a common estimation of the effect.
CONCLUSIONS: Results obtained herein are in keeping with those obtained previously and support a role for the +405G>C VEGF polymorphism in endometriosis development, although a further, larger study is required to confirm our findings. However, this effect may depend on the population studied. Ethnicity and the characteristics of endometriosis are likely to influence this association.
Key words: endometriosis/vascular endothelial growth factor/405G>C/polymorphism
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Introduction |
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In the pathogenesis of endometriosis, retrograde menstruation may be regarded as a necessary, although not sufficient, factor to explain the peritoneal implantation and diffusion (Vignali et al., 2002
). Sampson’s hypothesis supporting retrograde menstruation as the critical phenomenon in the development of endometriosis does not explain why the process—a physiological occurrence—does not result in the disease in the majority of the women. Thus, mechanisms underlying the disease are still poorly elucidated although familial aggregation and a higher rate of concordance in monozygotic twins provide support for a contribution of genetic factors in the pathogenesis of this disorder (Simpson et al., 2003; Barlow and Kennedy, 2005). A risk of 4–7% has been recognized for first-degree relatives (Simpson et al., 2003), indicating that, as for most adult-onset conditions, the disease is characterized by a polygenic and multifactorial etiology.
In this context, the identification of gene variants responsible for inherited susceptibility to the disease represents one of the main tasks of the current research. At present, however, an unequivocal consensus regarding the association of any potential candidate gene and this disease is lacking. Meta-analyses and systematic reviews of association studies applied to endometriosis for genes involved in steroid biosynthesis/response and in detoxification processes showed strong conflicting results and substantial heterogeneities among studies (Guo, 2005, 2006a,b).
Recently, two independent groups from South India and Korea have explored the frequencies of the +405G/C polymorphism in the 5' untranslated region of the vascular endothelial growth factor (VEGF) gene in endometriosis based on the strong rationale that neoangiogenesis mediated by this specific factor represents a critical event in ectopic tissue survival, migration and invasion (Bhanoori et al., 2005; Kim et al., 2005). Moreover, the +405G/C variant has a functional significance on VEGF protein production (Watson et al., 2000), i.e. known to be altered in various diseases such as pre-eclampsia, psoriasis and diabetic retinopathy (Ray et al., 2004; Banyasz et al., 2006; Buraczynska et al., 2006; Young et al., 2006). On the basis of the results obtained in endometriosis, both groups agreed in supporting an association between the +405G/C polymorphism and the disease susceptibility (Bhanoori et al., 2005; Kim et al., 2005). However, the analysis of genotype and allele frequencies demonstrated a clear formal discordance between the two reports since the frequency of the +405G allele was increased in affected women in the study by Bhanoori et al. (2005), but decreased in the study by Kim et al. (2005). In view of this controversy surrounding the role of VEGF +405G>C polymorphism in endometriosis development, we aimed to further investigate this previously reported association (i) in the Caucasian population for the first time and (ii) in an attempt to provide a summary estimate for studies performed so far.
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Materials and Methods |
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Subjects
The VEGF +405G>C polymorphism (dbSNP reference number 2010963) was evaluated in women who attended the endoscopic surgical service of the II Department of Obstetrics and Gynecology of the Fondazione Ospedale Maggiore Policlinico, Mangiagalli e Regina Elena to undergo gynecological laparoscopy between January 2001 and July 2004. Individuals studied were of Italian Caucasian origin. During the study period, 343 women were enrolled. Endometriosis was documented in 203 women (59%). Stage of the disease was found to be minimal or mild (stage I–II) in 78 cases (38%) and moderate–severe (Stage III–IV) in 125 cases (62%). Other gynecological benign pathologies were present in 20 of these 203 women with endometriosis (myomas in 10 cases, paraovarian cysts in three cases, sero-mucinous cysts in four cases and dermoid cysts in three cases). The presence of endometriotic ovarian cysts was observed in 117 (58%) of these 203 women. Deep peritoneal endometriosis, defined as lesions infiltrating to a depth of at least 5 mm beneath the peritoneal surface, was observed in 45 (22%) women. One hundred and forty women who underwent laparoscopy and in whom no endometriosis was found served as control group. Specifically, they included 33 cases with a regular pelvis, 38 cases of serous or mucinous cysts, 16 cases of dermoid cysts, 31 cases of uterine myomas, 1 case of ovarian fibroid, 14 cases of uterine malformations, 6 cases of paraovarian cysts and 1 case of appendicitis. The mean age + SD of patients with endometriosis and controls was 32.1 + 5.6 and 34.4 + 7.8 years, respectively.
All the women underwent complete pre-surgery clinical examination before the diagnostic-operative laparoscopy. Indications to laparoscopy included chronic pelvic pain, infertility, ovarian cysts and myomas. None of the women was taking medications except for non-steroidal anti-inflammatory drugs. Diagnosis of ovarian endometriotic cysts and deep peritoneal lesions was always confirmed histologically. Diagnosis of superficial peritoneal lesions was based on direct visualization when endometriotic implants presented as typical lesions. Lesions whose aspect was dubious were always removed in order to confirm their endometriotic nature by histologic examination. Three physicians active in the evaluation and treatment of endometriosis staged all patients according to the revised American Society for Reproductive Medicine classification (ASRM) (American Society for Reproductive Medicine, 1997). Patients with pelvic inflammatory disease were excluded from both cases and controls because of difficulty in the clear visualization of the pelvis. Women were informed that blood would be used for research purposes and gave written consent. Approval for this study was granted by the local Human Institutional Investigation Committee.
Molecular analysis of the VEGF +405G>C polymorphism
Peripheral blood was drawn from each patient before surgery and collected in an EDTA-containing tube. Genomic DNA was extracted from blood with the Nucleon BACC2 kit (Amersham Biosciences, Munich, Germany). The fragment containing the single nucleotide polymorphism +405G>C in the 5'-untranslated region of the VEGF gene was amplified from genomic DNA (50 ng) by PCR using primers (5'-ATTTATTTTTGCTTGCCATT-3'; 5'-GTCTGTCTGTCTGTCCGTCA-3') according to the procedure described by Watson et al. (2000). PCR was performed in a total volume of 25 µl containing 50 ng genomic DNA, 5 pmol of each primers, 1x Taq polymerase buffer (1.5 mM MgCl2) and 0.5 units of Red Taq (Sigma). The PCR amplification was carried out for 35 cycles (denaturation at 94°C for 45 s, annealing for 1 min at 55°C, extension at 72°C for 45 s and final extension for 10 min at 72°C). PCR products of 304 bp were digested with 2 IU of restriction enzyme BsmFI (New England Biolabs, USA) at 65°C overnight and analysed by 2% agarose gel electrophoresis followed by ethidium bromide staining and ultraviolet visualization. The +405G allele was cut into two fragments of 203 and 101 bp, whereas the +405C allele remained uncut. Genotyping error rate was determined by genotyping duplicate individuals for the same marker (Leal, 2005). The resulting genotype error rate was c. 0.3%.
Statistical analysis
The primary end-point of the study was to evaluate whether the VEGF +405G>C polymorphism does influence the occurrence of endometriosis in general. The secondary aims were to evaluate the best fitting model of inheritance and whether this polymorphism influenced the occurrence of specific forms of the disease. The sample size used in this study was calculated based on the primary outcome. The expected prevalence of the C allele in the control group was 30%. Assumptions to determine this sample size included types I and II error of 0.05 and 0.20, respectively. We established as clinically relevant a 50% increase/decrease in the risk to develop endometriosis. On the basis of these assumptions, we have calculated that the number of patients to be recruited in each study group was 
150. Statistical analysis was done using the Statistical Package for the Social Sciences (SPSS 14.0, Chicago, IL, USA). Alleles and genotypes frequencies were compared between groups using the Fisher’s exact test. The odds ratio (OR) was used to measure the strength of the association between allele frequencies and endometriosis. Goodness-of-fit 
2 test was used to test for Hardy–Weinberg equilibrium in both cases and controls.
Since available evidence in the literature regarding the influence of VEGF +405G>C polymorphism in the pathogenesis of endometriosis is controversial (Bhanoori et al., 2005; Kim et al., 2005), we also performed a meta-analysis including results from the present study and from previously published studies on this topic. Search strategies used were online query of the MEDLINE. We included only English language medical papers. Keywords used were endometriosis combined with VEGF or vascular endothelial growth factor. The presence of heterogeneity of polymorphism effect among studies was tested using the Breslow–Day 2 test. The Mantel–Haenszel method was used to obtain the common OR and its 95% confidence interval (CI).
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Results |
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Table I shows the genotype and allele frequencies of the VEGF +405G>C gene polymorphism in women with and without endometriosis. The frequency of the C allele in our control population (31%) was perfectly in line with the frequency reported for a group of healthy English individuals (32%) (Freathy et al., 2006
). The obtained genotype distribution was in good agreement with those expected by Hardy–Weinberg equilibrium in both cases and controls. The genotype frequency pattern showed dominance of the C/G heterozygotes in women affected, whereas the G/G homozygotes predominate in controls (Table I). The distribution of the three different genotypes significantly differed between women with and without the disease (P = 0.034). This effect was particularly evident for the dominant model. According to this model, the OR (95%CI) for endometriosis in women carrying the C allele was 1.8 (1.2–2.8). Conversely, no significant results were obtained assuming a recessive model of inheritance (Table I). The allele distribution was also statistically different (P = 0.019). The OR (95%CI) for endometriosis in women carrying the C allele was 1.5 (1.1–2.0). We then analysed the endometriosis group according to the severity of the disease (ASRM classification, presence of endometriotic cysts, presence of deep peritoneal implants and adhesions score). Overall, we failed to document an association with specific forms and/or with the severity of endometriosis (Table II).
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We identified two previous studies on the relationship between endometriosis and the +405G/C VEGF polymorphism (Kim et al., 2005
; Banhoori et al., 2005). Both studies focused on women with endometriosis stage III–IV. As a consequence, in order to prevent heterogeneity, we decided to perform a meta-analysis focusing exclusively on women with advanced stage of the disease and, consequently, data from stage I–II cases were excluded. Results from a meta-analysis combining results from the two previous studies and from our data are shown in Table III. Breslow–Day test revealed statistically significant heterogeneity, thus supporting inconsistency among studies (Table III). Common OR was not reported since the value of this parameter is questionable in this situation.
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Discussion |
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In this study, we confirmed a statistically significant association between endometriosis and the VEGF +405 G>C polymorphism. In keeping with previous findings reported by Kim et al. (2005)
, the risk of endometriosis is almost 2-fold higher in women carrying the C allele. These data are in contrast with results obtained by Bhanoori et al. (2005) in a South Indian population indicating a protective role of the C allele on endometriosis development.
Surprisingly, we failed to observe a biological gradient. The presence of the C allele was not higher in women with advanced stages and/or in those with more invasive forms of the disease. This result is difficult to explain. Some hypotheses may be drawn. First, the study may not be sufficiently powered to provide reliable subgroup analyses. The sample size was calculated based on the primary aim of the study which consisted in comparing the frequency of the polymorphism in women with and without endometriosis in general. Our sample size might thus be insufficient to draw firm conclusions regarding specific subgroups. Nevertheless, we have decided to perform this kind of analysis since the presence of a biological grading represents an important criterion when trying to determine whether an association can be interpreted in terms of causality (Hill, 1965). In general, it is assumed that the absence of a biological gradient is against a causal relationship. We cannot however rule out an alternative explanation. The presence of the C allele could represent a risk factor for the implantation of endometrial fragment refluxed in the peritoneal cavity while having no effect on the development of an invasive form of the disease.
In order to interpret adequately data from epidemiological studies, it is important to establish whether there is a biological plausibility for the reported observations. VEGF is a potent angiogenic factor able to promote neovascularization, which is considered an important phenomenon involved in the implantation of endometrial cells in ectopic sites (Vigano et al., 2004). Of interest, endometriotic implants are able to produce VEGF (Shifren et al., 1996). Moreover, the disease is associated with elevated peritoneal levels of the molecule (McLaren et al., 1996). Several cellular populations contribute to these higher levels; peritoneal macrophages may represent the principal source but other cell types such as endometriotic cells and lymphocytes may have a role. In this context, it is noteworthy that the +405G>C polymorphism is known to affect VEGF production in vivo and in vitro, and this effect seems to be dependent on the cellular population involved (Watson et al., 2000; Awata et al., 2002). However, it is still unclear whether the C variant has a dominant effect on VEGF gene expression as different studies reports opposite results (Watson et al., 2000; Awata et al., 2002; Koukourakis et al., 2004). Since endometriosis is a complex disease involving several cell types, the ultimate effect of the presence of the C allele in this specific context needs to be further investigated.
In medicine, meta-analysis methodology is frequently used to disentangle controversial issues. The ultimate aim of this analysis is to combine results from all available studies in order to obtain a common estimation of the effect. In this study, we have applied this approach to the role of +405G>C VEGF polymorphism in endometriosis development. Unfortunately, this analysis did not allow meaningful conclusions to be drawn since inconsistency among studies emerged. Indeed, the Breslow–Day test was statistically significant for all models of inheritance tested. Whereas results from our study and from the study of Kim et al. (2005) were in line and supported a predisposing effect of the C allele, Bhanoori et al. (2005) reported completely opposite findings. This inconsistency might be first explained by the racial difference. The three studies were conducted in three extremely different regions: Korea, South India and Italy (Bhanoori et al., 2005; Kim et al., 2005). The ‘racial’ issue has a strong impact on genetic association studies. Second, selection criteria may vary. Even if we included only endometriosis stage III–IV in our meta-analysis, we cannot exclude that other confounders, such as selection criteria for surgery, may considerably differ among studies.
In conclusion, the present study sheds light on an open controversy regarding the role of the +405G>C VEGF polymorphism in endometriosis development. In particular, the presence of the C allele is associated with an increased susceptibility to the disease. However, considering the heterogeneity between the present and the two previous studies, these findings need to be further replicated in additional cohorts.
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Submitted on March 5, 2007; resubmitted on August 9, 2007; accepted on August 30, 2007.
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