1 Department of Obstetrics and Gynecology, College of Medicine, University of Ulsan, Asan Medical Center and 2 Department of Obstetrics and Gynecology and 3 The Institute of Reproductive Medicine and Population, Medical Research Center, Seoul National University College of Medicine, Korea
4 To whom correspondence should be addressed at: Department of Obstetrics and Gynecology, The Institute of Reproductive Medicine and Population, Medical Research Center, Seoul National University College of Medicine, 28 Yungun-dong, Chongno-ku, Seoul 110744, Korea. E-mail: ymchoi{at}snu.ac.kr
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Abstract |
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Key words: angiogenesis/endometriosis/polymorphism/VEGF
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Introduction |
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Vascular endothelial growth factor (VEGF), also known as vascular permeability factor, is a heparin-binding glycoprotein with potent angiogenic and endothelial cell-specific mitogenic activities. Some investigators have demonstrated higher peritoneal concentrations of VEGF in women with advanced stage endometriosis (McLaren et al., 1996; Shifren et al., 1996
), and others have shown increased VEGF mRNA and protein expression in the eutopic endometrium from subjects with endometriosis (Donnez et al., 1998
; Tan et al., 2002
). Recently, it has been demonstrated that use of antihuman VEGF effectively interferes with the maintenance and growth of endometriosis by inhibiting angiogenesis in a nude mouse model (Nap et al., 2004
).
Based upon the genetic predisposition (Campbell and Thomas, 2001; Zondervan et al., 2001
; Bischoff and Simpson, 2004
) and the possible pivotal role of VEGF in the pathogenesis of endometriosis, it is necessary to investigate whether the VEGF gene polymorphisms are associated with susceptibility to endometriosis. Hsieh et al. (2004)
have shown that individuals with T/T homozygosity and the T allele of the VEGF-460 gene have a higher risk of advanced endometriosis in a Taiwanese population. However, there have been no other reports demonstrating or supporting the hypothesis that the VEGF gene polymorphism is associated with susceptibility to endometriosis.
The present study was designed to explore the association between the VEGF gene polymorphism and the risk of advanced endometriosis in a Korean population. We investigated the frequency of 5'-untranslated region 460 and +405 polymorphisms in patients with and without advanced endometriosis.
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Materials and methods |
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None of the subjects had received hormone therapy during the previous 12 months. All of the patients who had the diagnosis of minimal or mild endometriosis were excluded in both the case and control groups. The indications for surgery amongst the endometriosis group were dysmenorrhoea (n = 65), pelvic pain (n = 57), adnexal mass (n = 49), infertility (n = 32), and others (n = 12). Pelviscopic surgery, exploratory laparotomy and transabdominal hysterectomy were performed in 141 (65.6%), 47 (21.8%) and 27 (12.6%) patients respectively in the endometriosis group.
Patients with leiomyoma, adenomyosis, invasive carcinoma of the uterine cervix or ovarian cancer were excluded from the control group. The indications for surgery or diagnostic laparoscopy in the control group were benign ovarian cyst (n = 76), infertility (n = 60), pelvic pain or dysmenorrhoea (n = 56) and carcinoma in situ of the uterine cervix (n = 27). Pelviscopic surgery, exploratory laparotomy, transabdominal hysterectomy and diagnostic laparoscopy were performed in 87 (39.7%), 38 (17.3%), 34 (15.6%) and 60 (27.1%) patients respectively in the control group. The review board for human research of Seoul National University Hospital approved this project, and informed written consent was obtained from each woman. Ages ranged from 18 to 50 years (31.0 ± 7.2, mean ± SD) in the endometriosis group, from 20 to 54 years (43.4 ± 10.1) in the control group, and from 40 to 49 years (43.3 ± 2.6) in the fertile women group.
Genomic DNA analysis
Peripheral blood was drawn from each patient and collected in an EDTA-containing tube. Genomic DNA was extracted from the peripheral blood with the Wizard DNA Purification Kit (Promega, Madison, WI, USA).
Genotyping of each polymorphism was carried out by PCRRFLP analysis, as previously described by Watson et al. (2000) for the 460 C/T polymorphism and as described by Awata et al. (2002)
for the +405 C/G polymorphism, with minor modifications. The PCR primers for the 460 C/T and the +405 C/G polymorphisms were 5'-TGTGCGTGTGGGGTTGAGCG-3' (forward) and 5'-TACGTGCGGACAGGGCCTGA-3' (reverse) and 5'-TTGCTTGCCATTCCCCACTTGA-3' (forward) and 5'-CCGAAGCGAGAACAGCCCAGAA-3' (reverse), respectively. Following an initial denaturation step (5 min at 94°C), samples were subjected to 30 rounds of PCR at 94°C for 30 s (460 C/T) or 1 min (+405 C/G), 60°C (460 C/T) or 62°C (+405 C/G) for 30 s (460 C/T) or 1 min (+405 C/G), and 72°C for 60 s with a final extension time of 5 min at 72°C. The PCR products were digested with either 2 IU of restriction enzyme BstU1 (New England Biolabs, USA) at 60°C overnight for the 460 C/T polymorphism or with BsmFI (New England Biolabs) at 65°C overnight for the +405 C/G polymorphism, separated by 3% (460 C/T) or 2% (+405 C/G) agarose gel electrophoresis, and identified using ethidium bromide staining. The 460C allele was cut into two fragments of 155 and 20 bp, while the 460T allele remained uncut (175 bp). The +405G allele was cut into two fragments of 273 and 196 bp, while the +405C allele remained uncut (469 bp).
Statistical analysis
The numbers for the endometriosis and control groups were initially chosen to detect a difference between the two groups assuming that odds ratio is 2.0 with a power of 0.8 and 5% type I error. Genotype distributions were examined for significant departure from HardyWeinberg equilibrium by a goodness-of-fit 2-test.
2-Analysis was used to evaluate differences in the proportions of the genotypes between the endometriosis and control groups. Haplotype frequencies, the disequilibrium coefficient (r2) and the standardized disequilibrium coefficient (D) were evaluated utilizing Haploview program (Version 3.2, available at http://www.broad.mit.edu/mpg/haploview/index.php) and haplotype association was analysed by univariate logistic regression. P < 0.05 was considered significant.
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Results |
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The distribution of genotypes and allele frequencies of the 460 C/T polymorphism in the endometriosis group did not differ from those in the control and the fertile women group (Table I). However, genotype distribution of the +405 C/G polymorphism was significantly different between patients with and without endometriosis (P = 0.01) and between patients with endometriosis and the fertile women (P = 0.02) (Table II). Patients with endometriosis showed a higher incidence of the +405 CC genotype compared with the controls and the fertile women (23.3 versus 13.2 and 10.0%, P = 0.007 and 0.016, odds ratio: 1.98 and 2.73, 95% CI: 1.203.28 and 1.176.33 respectively) (Table II). The allele frequencies of the +405 C/G polymorphism were not different between patients with and without endometriosis and between patients with endometriosis and the fertile women (Table II). The distribution of genotypes and allele frequencies of the +405 C/G polymorphism in patients with stage III endometriosis did not differ from those with stage IV endometriosis.
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Haplotype frequencies of two VEGF biallelic polymorphisms were determined using Haploview program. Disequilibrium coefficient (r2) was 0.25 and standardized disequilibrium coefficient (D) was 0.96. Four haplotypes were estimated to be present, and the haplotype frequencies of two VEGF biallelic polymorphisms were not significantly different between patients with and without endometriosis (Table III). Among the 79 women with +405 CC genotype in the endometriosis and control groups, 77 (97.5%) had 460 TT genotype, whereas among 233 women with 460 TT genotype in both groups, only 77 (33.0%) had +405 CC genotype.
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Discussion |
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Analysing the 460 C/T and +405 C/G polymorphisms of the VEGF gene, we found that genotype distribution of the +405 C/G polymorphism was significantly different between patients with and without endometriosis, and women with the +405 CC genotype had a significantly increased risk of endometriosis compared with those without the genotype. The findings of the present study are not consistent with the previous report by Hsieh et al. (2004), which demonstrated a significant association between the 460 C/T polymorphism and susceptibility to endometriosis. The discrepancy might be due to ethnic composition differences between the two studies. However, the previous report by Hsieh et al. (2004) included no subjects with the 460 CC genotype and the frequency of the 460 CC, 460 CT and 460 TT genotypes of the VEGF gene polymorphism in the control group were not in HardyWeinberg equilibrium (P = 0.00006). The results of the present study may be more reliable, since the distribution of the genotypes of the control subjects used in an association study should not show a significant deviation from HardyWeinberg equilibrium. Considering that all the women except two with +405 CC genotype in the endometriosis and control groups of the present study had 460 TT genotype, it is also possible that the original reported association with the 460TT genotype resulted from skewed distribution of genotype in the endometriosis patients in their report, in which most of the 460 TT genotype might have had +405 CC genotype.
We tried to recruit pre-menopausal women aged 4050 years as control subjects, and the mean age of the control subjects was higher than that of the endometriosis patients. As described by Hadfield et al. (2001), recruiting women from this age group has the merit of maximizing the probability that they were unaffected by endometriosis, i.e. to avoid including younger women who might develop the disease in later life. As of April, 2005, we have no other data about the allele frequencies for +405 C/G or the frequency of +405 CC genotype in the Korean population except ours and Han et al.s (2004)
. The frequency of +405 CC genotype in healthy individuals of the previous report by Han et al. (2004)
was similar to the frequency reported for the endometriosis patients and higher than that of the control group in the present study. However, the frequency of +405 CC genotype of the control group (13.2%) in the present study is slightly higher than that in the control group of the previous study by Awata et al. (2002)
(10.3%) in a Japanese population. Despite few data among the general population in Korea, the control subjects in the present study do not seem to have a different frequency of the +405 CC genotype from the general population, as the frequency of +405 CC genotype in the 70 women with proven fertility without any gynaecological disease was 10.0%.
Although it is unclear how the polymorphisms in the untranslated region of the VEGF gene influence its protein production, several reports have demonstrated that single-nucleotide polymorphisms of the VEGF gene are associated with VEGF synthesis. Watson et al. (2000) have demonstrated that the genotype for the +405 polymorphism in the VEGF gene is significantly correlated with VEGF production from stimulated peripheral blood mononuclear cells. Awata et al. (2002)
have shown the association of the +405 CC genotype with a higher serum VEGF concentration in a normal Japanese population. A recent study suggested that genetic polymorphisms including +405 region are correlated with VEGF protein expression in cancer cells and tumour angiogenic activity (Koukourakis et al., 2004
). Transfection analysis of the human VEGF promoter revealed that estrogen has a direct transcriptional effect on VEGF gene expression and that estrogen-regulated transcription requires a variant estrogen response element (Mueller et al., 2000
). Considering that the +405 site is located adjacent to the +410 estrogen response element and that carriage of the 460/+405 polymorphism significantly alters VEGF promoter activity and responsiveness (Stevens et al., 2003
), it may be suggested that the +405 polymorphism itself has an influence on the transcriptional activity by possible alteration of response to estrogen.
Individuals with a specific single nucleotide polymorphism of the VEGF gene may have a higher risk of developing endometriosis through increased expression of VEGF in various cells. An elevated concentration of VEGF in the peritoneal fluid due to increased production of VEGF by activated peritoneal macrophages can be a critical process in the pathogenesis of endometriosis. Alternatively, increased expression of VEGF in eutopic endometrial cells may lead to implantation and proliferation of endometrial cells at ectopic sites through retrograde menstruation. Based on the findings of the previous report by Awata et al. (2002), it is also possible that individuals with the +405 CC genotype have an increased risk of endometriosis due to increased serum levels of VEGF, although contradictory results have been reported as to whether circulating VEGF is modulated in endometriosis (Gagné et al., 2003
; Matalliotakis et al., 2003
).
There is accumulating evidence to support the hypothesis that angiogenesis is of pivotal importance in the development of endometriosis. The present study has shown that the subjects with a specific genotype in the VEGF gene polymorphism had a significantly increased risk of endometriosis than those without the genotype. These findings strongly suggest that endometriotic lesions could become established in certain groups of women in whom angiogenic activity is enhanced due to the presence of specific genotypes. Further studies on the functional relevance of the VEGF polymorphism in endometriosis are necessary to confirm these observations.
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Acknowledgements |
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References |
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Submitted on February 15, 2005; resubmitted on May 13, 2005; accepted on May 19, 2005.