Vascular endothelial growth factor (VEGF), which is an important factor in regulating local angiogenesis by stimulating the proliferation of vascular endothelial cells, has emerged as likely being involved in the pathogenesis of endometriosis. Peritoneal fluid and endometrium VEGF concentrations have been reported to increase in women with endometriosis 1.
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 with 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 heterogeneity among studies 2.
On the basis of the genetic predisposition and the possible important function of VEGF in the pathogenesis of endometriosis, it might be essential to investigate whether VEGF gene polymorphisms are associated with susceptibility to the disease. The present study is designed to examine the connection between the nucleotide polymorphisms [single-nucleotide polymorphisms (SNPs)] and the risk of endometriosis. The frequency of the VEGF 5′-untranslated region +405C/G polymorphisms will be investigated in patients with and without endometriosis.
Materials and methods
The present study was conducted on 100 women who were divided into two groups (cases and control) on the basis of the following criterion: laparoscopic diagnosis of endometriosis (indications for laparoscopy included chronic pelvic pain, infertility, ovarian cysts, and myomas). Women who fulfilled the above-mentioned criterion served as cases and those without evidence of endometriosis served as controls.
We excluded women having rheumatoid arthritis, giant cell arthritis, diabetic retinopathy, psoriasis, or Behcet’s disease because of the possibility of association between these diseases and genetic polymorphisms. Patients with pelvic inflammatory disease were excluded from both cases and controls. Written informed consent was obtained from all participants.
All women were subjected to full history taking and clinical examinations. Endometriosis was diagnosed by direct visualization of implants by laparoscopy and classified according to the Revised American Fertility Society (R-AFS) classification (1996) as follows: stage I (minimal), stage II (mild), stage III (moderate), and stage IV (severe). This classification depends on the size, site, and depth of pelvic and extrapelvic lesions; scores are given depending upon severity.
The study cases were divided into two groups: women having minimal (stage I) or mild (stage II) endometriosis at early stage, and women having moderate (stage III) or severe (stage IV) endometriosis at advanced stage. Both cases and controls were selected from the Department of Obstetrics & Gynecology in Kasr El-Aini Hospital, Cairo University, and from National Research Center clinics.
Molecular analysis of the VEGF +450C/G polymorphism
Venous blood samples (EDTA) of 8 ml were taken from the peripheral blood of each individual. Genotyping for the +405G>C polymorphism was performed using real-time Taqman-based assays (USA SNP ID rs2010963; Applied Biosystems, Foster City, California, USA). DNA was extracted using the QIAamp DNA Blood Mini Kit (Qiagen Straβe 1, Hilden, Germany) according to the manufacturer’s ‘Blood and Body Fluid Spin Protocol’.
PCR amplification was performed by 7500 fast real-time PCR (Applied Biosystems) using the following primers and probe: SNP ID rs2010963.
Data were analyzed using IBM SPSS Advanced Statistics version 20.0 (SPSS Inc., Chicago, Illinois, USA). Numerical data were expressed as mean and SD. Qualitative data were expressed as frequency and percentage. The χ2-test (Fisher’s exact test) was used to examine the relation between qualitative variables. For quantitative data, comparison between two groups was made using the independent sample t-test. Binary logistic regression was used to estimate the risk of gene expression in the study and control groups A P-value less than 0.05 was considered significant.
The present study was conducted on 100 women who were divided into two groups (50 women served as cases and another 50 women served as controls). Demographic characteristics were similar in the two groups. There was no significant difference between cases and controls with regard to mean age (28.7 and 28.6, respectively; P=0.897). The women in the case group were classified according to early stage (I and II) [30 women (60%)] and advanced stage (III and IV) [20 women (40%)] of endometriosis.
Among both cases and controls, the genotypic distribution of the individual SNPs as well as of the VEGF alleles was not in Hardy–Weinberg equilibrium (P<0.05) (Table 1).
The +405 genotype frequencies among the cases were as follows: GG=70%, GC=10%, and CC=20%; the G and C allele frequencies were 76 and 24%, respectively (Table 2). The +405 genotype frequencies among the controls were as follows: GG=40%, GC=30%, and CC=30%; the G and C allele frequencies were 54 and 46%, respectively. The genotype [P<0.05, odds ratio (OR)=5.2 (1.66–16.6)] (Table 2) and allele frequencies [P<0.05, OR=2.45 (1.35–4.47)] (Table 2) of the +405C/G polymorphism showed a significant difference, which resulted from an increased proportion of GC heterozygote carriers (but not CC homozygote carriers) among endometriosis patients as compared with controls. No significant difference was observed between early-stage and advanced-stage endometriosis patients versus controls with respect to +405G/C genotype distributions.
In the present study, the +405 genotype frequencies among the cases were as follows: GG=70%, GC=10%, and CC=20%; the G and C alleles frequencies were 76 and 24%, respectively. The +405 genotype frequencies among the controls were as follows: GG=40%, GC=30%, and CC=30%; the G and C allele frequencies were 54 and 46%, respectively. The genotype [P<0.05, OR=5.2 (1.66–16.6)] and allele frequencies [P<0.05, OR=2.45 (1.35–4.47)] of the +405G/C polymorphism showed a significant difference, which resulted from an increased proportion of GC heterozygote carriers (but not CC homozygote carriers) among endometriosis patients as compared with controls.
Ikuhashi et al.3 investigated whether polymorphisms in the VEGF gene are associated with endometriosis in a Japanese population. Genotyping of VEGF −460C/T, +405G/C, and +936C/T polymorphisms was performed in 147 endometriosis cases. No significant differences in the frequency and genotype distribution of VEGF −460C/T, +405G/C, and +936C/T polymorphisms were found between the endometriosis patients (all disease stages) and controls, which is in contrast to the results of our study. However, a positive association was found between stage III–IV disease and the VEGF +936T allele (P=0.018).
Toktam et al.4 evaluated 105 women with and 150 women without laparoscopic evidence of endometriosis as cases and controls, respectively, comparing frequencies of the genotype and allele of the +405G>C polymorphism between them using PCR-RFLP. They found that the +405 VEGF genotype frequencies in the case group were as follows: 41.3% G/G, 46.2% C/G, and 12.5% C/C, representing 43, 48, and 13 patients, respectively. The genotype frequencies in the control group were 32% GG, 53.3% GC, and 14.7% CC in 48, 80, and 22 patients, respectively. There were no statistically significant differences between the cases and controls in terms of 405G/C genotype distributions and allele frequencies (P=0.267).
The study assessed the genotyping results of the VEGF gene polymorphisms at −460C/T, −1154G/A, −2578C/A, and +936C/T in 344 north Chinese women with endometriosis who served as the case group and 360 healthy women without endometriosis who served as the control group, using PCR and RFLP analysis. They found no significant difference in allele and genotype distributions of the −460C/T and +936C/T polymorphisms between patients and controls, in contrast to the results of our study. However, the frequencies of the −1154G/A and −2578C/A genotype and allele were significantly different between the two groups, which was similar to our results (all P<0.013). The −2578A/A and −1154A/A genotypes were found less frequently in patients with endometriosis compared with controls.
Bhanoori et al. 5 evaluated 215 women with endometriosis and 210 with no evidence of the disease and reported that the genotype and allele frequencies of the −460C>T polymorphism did not differ significantly between cases and controls. In agreement with the results of our study, the genotype (P=0.002) and allele (P=0.001) frequencies of the +405G>C polymorphism showed a significant difference between cases and controls. The +405GG genotype was found more often in patients with an endometrioma greater than 3 cm compared with controls. The frequency of the −460T/+405C haplotype (P=0.016) was significantly lower in affected women compared with controls.
Altinkaya et al.6 tested the SNPs −460C/T and +405G/C in the 5′-untranslated region of the VEGF gene in 98 affected women and 94 women with no laparoscopic evidence of disease. Endometriosis was also confirmed histologically. Following genomic extraction of genomic DNA, genotyping of the −460C/T and +405G/C polymorphisms of the VEGF gene was performed. The genotype and allele frequencies of the −460C/T polymorphism did not differ significantly between cases and controls. In agreement with the results of our study, the genotype (P<0.001) and allele frequencies (P<0.001) of the +405G/C polymorphism showed a significant difference between cases and controls. Regardless of early or advanced stage, women with endometriosis showed a higher incidence of the +405GC genotype and +405G allele when compared with controls.
Selected 215 women in advanced stage of endometriosis as the case group and 219 women without endometriosis and 70 fertile women as the control group. Genotyping of the −460C/T and +405G/C polymorphisms of the VEGF gene was performed, which showed that the distribution of genotypes and allele frequencies of the −460C/T polymorphism in the endometriosis group did not differ from those in the control group and the fertile women group. However, in accordance with the results of our study, genotype distribution of the +405G/C polymorphism was significantly different between patients with and those without endometriosis (P=0.01) and between patients with endometriosis and fertile women (P=0.02). Patients with endometriosis showed a higher incidence of the +405CC genotype compared with controls and fertile women (P=0.007 and 0.016, respectively).
Gentilini et al. 7 examined 203 Italian women affected by endometriosis and 140 women without laparoscopic evidence of the disease and found that the distribution of the three different genotypes significantly differed between women with and those without the disease (P<0.03), in agreement with our results. The OR (95% confidence interval) for endometriosis in women carrying the C allele was 1.8 (1.2–2.8), confirming the possibility of endometriosis to be almost two times higher in patients carrying the C allele. Moreover, they showed that the existence of this allele could be a risk factor for the implantation of endometrial fragments that are refluxed into the peritoneal cavity.
Our findings were not in line with those of previously stated studies perhaps because of difference in geographical location or the genetic basis of the studied population, as the possibility of a disease occurrence depends both on its prevalence in the population and on environmental factors 8. Race difference and genetic backgrounds are important factors in genetic association studies. Therefore, selection of different control groups might lead to dissimilar results 8. For example, in the present study, the control group comprised women without the disease as confirmed by laparoscopy, whereas in a study on the Japanese population female neonates were selected as the control group 3. In contrast, in a study carried out on the Korean population, the control group was made up of women who had either benign ovarian cysts, infertility, pelvic pain, or dysmenorrhea.
This study showed that the genotype and allele distribution of the +405G>C VEGF gene polymorphism showed significant difference between cases and the controls among Egyptian women. Further studies on a larger scale are needed to confirm or refute that the presence of the +405G/C polymorphism in the VEGF gene has considerable effect on disease onset, severity, and modification.
Conflicts of interest
There are no conflicts of interest.
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