Polymorphisms in VEGF and KDR genes in the development of endometriosis : a systematic review

Objectives: to review studies that used case-control design to verify the association of polymorphisms in VEGF and KDR genes in the development of endometriosis. Methods: the systematic review selected articles published until September 1, 2015 from PubMed, MEDLINE, BVS, SciELO databases, considering the following key words: endometriosis and (“polymorphism” or "SNP" or "genetic polymorphism") and ("VEGF" OR "Vascular endothelial growth factor" or "VEGFR-2" or "Vascular endothelial growth factor2" or "KDR" or "Kinase Insert Domain Receptor"). Results: 106 articles were identified, only 11 were eligible. Discrepant results were observed regarding polymorphisms in VEGF gene in the development of endometriosis, which can be explained by methodological differences, sample size, eligible control type, using the unadjusted risk estimates and the heterogeneity of the studied population. Only one study investigated polymorphisms in KDR gene in the development of endometriosis, however it was ineligible for this review. Conclusions: to avoid discrepancy in the results, we suggest that the ideal control group should be formed by fertile women and free of gynecological diseases. Multicentric studies with adequate design, involving different population besides the combined analysis on polymorphisms in VEGF and KDR genes are still necessary to contribute in the understanding of this disease, which are social, clinical and economical problems.


Introduction
Endometriosis is a gynecological disease characterized by the presence of functional endometrial tissue outside the uterine cavity, representing one of the most common gynecological disorders. 1The real profile of patients with endometriosis is uncertain, although there is a consensus that it is present in at least 10% of the women at reproductive age, 2,3 30 to 50% of infertile women 4 and 3 to 5% of postmenopausal women. 57][8] Endometriosis causes physical, mental and social consequences to women, bearing in mind that their psyche, interpersonal and martial relationships are affected by the problems of the symptoms, in particularly, by the difficulty of bearing children. 9here are several theories explaining about the appearance of endometriosis; however its etiology is still not clear.The most widely accepted theory until today is Sampson´s proposal, 10 who described that the endometrial tissue, by backward menstrual flow, returns to the uterine tubes and adheres to the peritoneal cavity.For this to occur, the angiogenesis process is essential and it is characterized by the growth of new blood vessels from pre-existing ones. 11Among the pro-angiogenic factors, the vascular endothelial growth factor (VEGF) is highlighted and plays an important role in the development of endometriosis. 11In 2008, our group observed an increased distribution of VEGF and its receptor VEGFR-2 in samples of endometriosis of the ovary, bladder and the recto-sigmoid, when it was compared to control and the largest distribution of VEGF and VEGFR-2 was observed in the endometriosis of the recto-sigmoid,which is the most severe. 12EGF is encoded by a gene with the same name, located on chromosome 6p21.3,composed by eight exons, 13 while its receptor VEGFR-2 is encoded by KDR gene (kinase insert domain receptor), located on chromosome 4q11-q12, composed by 30 exons. 148 Recently, our group observed a positive association for VEGF rs1570360 SNP in the development of endometriosis (OR: 1.90; CI95%: 1.23-2.97) in Brazilian women. 8][21][22][23][24][25][26][27][28][29][30][31][32][33][34][35][36][37] However, the results of the analysis are still controversial.In this context, the aim of this study was to review all the studies that used case-control design to investigate the magnitude of SNPs association of VEGF and/or KDR genes susceptibility to develop endometriosis, in order to discuss the possible causes of the conflicting results already described.

Methods
To identify all the studies published until September 1, 2015 on the associations among SNPs of VEGF and/or KDR and the development of endometriosis, an evaluation on titles and abstracts of the studies was performed by two researchers in an independent form and "blinded" in PubMed, Medline, BVS and SciELO databases using the following keywords: ("endometriosis") AND ("polymorphism" OR "SNP" OR "genetic polymorphism" OR "genetic polymorphisms") AND ("VEGF" OR "Vascular endothelial growth factor" OR "VEGFR-2" OR "Vascular endothelial growth factor-2" OR "Vascular endothelial growth factor-2" OR "KDR" OR "Kinase Insert Domain Receptor").The studies were selected according to the inclusion and exclusion criteria, pre-selected and described as followed.
The inclusion criteria for this review were casecontrol design studies and that evaluated associations among polymorphisms of VEGF and/or KDR genes and the development of endometriosis.As for the period of the publication, articles that were published until September 2015 were included without any restriction of languages.
The exclusion criteria of the study were: (I) the absence of a control population; (II) incomplete data associations; (III) duplicated data; (IV) metaanalysis, review, letters, commentaries or editorial articles; (V) those that did not analyze SNPs in VEGF and/or KDR; (VI) and did not include patients with endometriosis; (VII) those that did not have access to the complete text.
After performing a search strategy in PubMed, Medline, BVS and SciELO databases in articles which studied SNPs in VEGF and/or KDR genes with the development of endometriosis, the following information was extracted: the first author, Cardoso JV et al. publication year, journal; country where the study was conducted; number of cases and controls; mean age of cases and controls with their respective deviation standard and/or interval; selection on criteria/eligibility and exclusion of cases of endometriosis and controls; endometriosis staging; control source; type of SNP; genotyping technique used to identify the studied SNPs; data of allelic and genotypic frequency of the SNPs and statistic data of odds ratio (OR) with its respective 95% confidence intervals (CI95%).

Evaluation on quality in the included studies
Two reviewers independently evaluated the methodological quality of 19 studies included in this review, using STROBE (Strengthening the Reporting of Observational Studies in Epidemiology), a criteria instrument. 38Twenty and two evaluated items received a score from 0 to 1.These items were related to the title and the abstract of the article (item 1), the introduction (items 2 and 3), the methods (items 4 to 12), the results (items 13 to 17), the discussion (items 18 to 21) and the financial information (item 22).After evaluating all the criteria, each article received a score from 0 to 22 of each researcher.The final score was performed an average of the two grades transformed into percentage to better evaluate the quality of the articles.Previously, the reviewers described if the articles reached a percentage higher than 50%, they would be consi-dered satisfactory.

Results
Figure 1 shows a flowchart on the selection of articles for this review.The search strategy identified 100 publications on VEGF gene and 6 publications on KDR, no articles were found evaluating simultaneously the influence of SNPs in both genes with the development of endometriosis.After removing the duplicated articles, 34 were selected for reading the titles and abstracts.However, 14 studies were excluded: 5 because they were meta-analyzes, 2 because they were reviews, one because it was a criticism on the meta-analysis published before, one because it explored SNPs in IL-10 gene instead of polymorphisms in VEGF and/or KDR genes, 4 because they studied patients with adenomyosis and one because it evaluated women with pterygium instead of patients with endometriosis.After the selection of the abstracts, a study was excluded because the text was not completely available. 19fter a thorough reading of 19 articles eligible for this review, the inclusion and exclusion criteria on cases and controls were evaluated and 8 studies were still excluded: 5 used endometriosis cases without histopathological confirmation of the disease; 20,25,28,30,34 2 which included controls did not have surgery procedures (laparoscopy and/or laparotomy) in confirming the diagnosis of endometriosis; 22,29 and 1 which included controls only performed an ultrasound and was diagnosed negative for the disease. 17Thus, 11 case-control studies were included in this review that evaluated SNPs only in VEGF gene susceptibility to develop endometriosis from 2005 to 2015. 8,21,23,24,26,27,31- 33,36,37In relation to evaluate the quality of 11 studies selected for this review, all the articles reached a percentage greater than 50% 38 and the average score ranged between 12 to 21 points (55-96%).The studies that had the lowest and the highest score, according to the STROBE criteria were Kim et al. 24 and Perini et al., 8 respectively (Table 1).
In this review 4205 women´s data were included (2096 with endometriosis and 2109 controls) from Asian countries, 21,24,27,32 North America, 33 North Africa, 8 Europe, 23,26,37 Euro-Asian countries 31 and Africa. 36able 1 describes the inclusion and exclusion criteria of the group with endometriosis, emphasizing that all the studies considered the women eligible who had surgical diagnosis of the disease with histopathological confirmation.Whereas the staging of the disease was found in 4 (36.3%)studies that included patients in stage I, II, III or IV; 24,31,33,36 3 (27.3%)grouped into stages I-II or III-IV; 8,23,26 2 (18.2%) included only women in stages I and II; 32,37 one (9.1%)included patients in stage III and IV 21 and another (9.1%) showed no information on surgical staging of the disease. 27In relation to the exclusion criteria of the group with cases of endometriosis, 2 (18.2%) studies did not inform any criteria, 27,33 while others, a total of 9 (81.8%)considered patients who had a history of angiogenesis-dependent diseases or that it could be associated to polymorphisms of VEGF, and women who presented adenomyosis, a pelvic inflammatory disease, bilateral tubal occlusion and myoma.
For the control group (Table 2), all the studies included women with surgical diagnosis of endometriosis, although 3 (27.3%)studies included women who underwent surgery for tubal ligation, 8,26,32 3 (27.3%)included women who underwent surgery to remove cyst in the ovary; 8,21,23 2 (18.2%) included women who underwent surgery to remove myoma; 8,23 2 (18.2%) included women who underwent hysterectomy 21,27 and 6 (54.5%) included Excluded studies for using cases without any histological confirmation of endometriosis (n=5) Excluded studies for using controls that were not submitted to surgery for negative diagnose confirmation on endometriosis (n=3) Excluded study lack of a thorough text (n=1) Selected studies for full text analysis VEGF (n=19) and KDR (n=1) genes   women who were submitted to surgery because of various problems such as appendicitis, carcinoma in situ of the cervix, dermoid cysts, parovarium cysts, serous cysts and mucinous tumors, dysmenorrhoea, pelvic pain, fibroma ovarian, dysfunctional bleeding uterine, hydrosalpinges, infertility and malformation of the uterine and normal pelvis. 8,21,23,27,31,37The exclusion criteria for the control group was one (9.1%)study that did not inform any of the exclusion criteria. 33The remaining 10 (90.9%) considered almost the same exclusion criteria of the group with cases of endometriosis. 8,21,23,24,26,27,31,32,36,37Rheumatoid arthritis, GCA, diabetic retinopathy, psoriasis and Behçet's disease.Endocrine therapy before surgery, endometriosis, inflammatory disease and myoma.

Discussion
This review provides a detailed description of the studies that used a case-control design study to investigate the magnitude of association of the SNPs in VEGF and/or KDR genes susceptibility in the deve-lopment of endometriosis.The angiogenesis, through VEGF-KDR signals, is an important event in the development of the disease, 11,12 since endometriosis lesions are characterized by a dense vascularization and requires blood supply suitable for survival. 11Furthermore, an increased expression of VEGF and VEGFR-2 in samples of women with endometriosis was observed when compared to its control. 127][18] SNPs that occur in promoter regions, 5'-UTR and 3'-UTR, affect potential regulatory elements, are sensitive to hypoxia and contribute to high variability of production in VEGF tissues. 18,39,40rs699947, rs833061 and rs1570360 SNPs present in the promoter region of VEGF gene, present higher activity of transcription and are associated to high levels of protein. 41rs2010963 SNP located in region 5'-UTR of VEGF gene affects the efficiency of understanding and is related to the production of VEGF from the mononucleated cells of the peripheral blood. 42So, rs3025039 SNP in region 3'-UTR of VEGF gene influences the concentration of VEGF plasmatic stock. 15rs2305948 and rs1870377 SNPs located in the exons of KDR gene decrease the efficiency of VEGF binding to KDR. 18 The possible effect of rs7667298 SNP located in region 5'-UTR of KDR gene is unclear.However, this SNP is associated to rs2071559 SNP (KDR-604T>C) located in the promoter region.The presence of -604C allele reduces the transcriptional activity of KDR. 18rs1531289 and rs7692791 SNPs located in introns of KDR gene were not yet studied in relation to the functional effects.0][21][22][23][24][25][26][27][28][29][30][31][32][33][34][35][36][37] In 2013, Li et al., 43 conducted a meta-analysis study involving 14 studies which investigated the magnitude of association of 5 SNPs in the VEGF gene (rs699947, rs833061, rs1570360, rs2010963 and rs3025039) in the susceptibility on endometriosis.The increased risk of endometriosis developing was observed in the presence of VEGF + 936C>T SNP, while VEGF -2578C>A and -1154G>A SNPs were protective factors for the development of the disease.Thus, VEGF -460T>C and +405G>C SNPs were not associated to the development of endometriosis. 43For KDR, only one case-control design study evaluated the magnitude of association among five SNPs (rs1531289, rs2305948, rs1870377, rs7692791 and rs7667298) and the development of endometriosis in Chinese women, noticing a negative association in the presence of 1192T allele (rs2305948). 17owever, this study was excluded from this review because included controls performed only an ultrasound to diagnose negative for endometriosis and this method of imaging does not exclude the presence of pelvic endometriosis. 44,45Moreover, until the present moment there are no data in literatures that assess the combined form of SNPs in VEGF and KDR genes and the development of endometriosis.
The controversial results observed in the casecontrol studies can be explained by the sample size, by methodological differences in each study design, mainly, by the eligible control type, by the difference in SNPs frequency in different populations and by the use of the measured of association, the unadjusted odds ratio (OR) used to evaluate the magnitude of association of SNPs in the VEGF and KDR genes in the development of endometriosis.The stratification techniques and multiple regression are two statistical methods which can be used to exclude potential confounding variables and consequently produce the adjusted OR. 46,47 Among the 11 studies included in this review, only 3 (27.3%)used the adjusted OR (Table 4) to evaluate the magnitude of association of SNPs in VEGF gene and the development of endometriosis. 8,26,27The manner in which the variables are measured may insert a bias and distort the estimated measurement of the effect. 48he confounding variables occur when part of the association found is the result of the presence of a third variable that is related to the disease and the exposure of interest result in a change of the estimated risk. 49he analytical observational epidemiological studies of the case-control type are applied to evaluate exposures among similar groups, which present (cases) or not (controls) to the disease. 50They are widely used in studies on rare diseases or in long period of induction and regular expositions, 51 such as in the case of endometriosis.In addition, casecontrol studies tend to contribute in locating important findings and efficient in terms of relatively short time with little financial resource. 48However, casecontrol studies tend to be more susceptible to biases than any other analytical epidemiological designs and how individuals are recruited for the study can distort the estimated measurement of the effect. 51he definition of the case group is a critical point in a case-control study, criteria for diagnosis and eligibility and two fundamental aspects for the selection of the subject.The aim is to ensure that all the true cases have equal probability of joining the group, and that no false case is selected, thus, it may distort the estimated measurement of the association in direction to a null hypothesis. 5223][24][25][26][27][28][29][30][31][32][33][34]36,37 In spite of the available images of the method presented a good accuracy in the endometriosis diagnosis, according to a consensus of the European Society of Human Reproduction and Embryology (ESHRE) and the American Society for Reproductive Medicine (ASRM), the gold standard to diagnose endometriosis is the videolaparoscopy with direct inspection of the pelvic cavity and visu-alization of the implants, as well as the histopathological confirmation of the disease. 53,54Thus, 11 articles were included in this review using the "gold standard" to select the cases (surgical and histopathological confirmation), considered to be a good diagnostic criteria and eligibility, ensuring that all 2096 women included were true cases of endometriosis. 8,21,23,24,26,27,31,32,33,36,37 e selection of the control group requires special care and perhaps is the main challenge to guarantee the validity of the study, considering the complexity of the investigated disease (endometriosis).The control group of studies included in this review was formed by women who could have other diseases, including gynecological disorders associated to the development of endometriosis, along with the process of angiogenesis and/or polymorphisms in VEGF gene. 17,39,55,56pproximately 82% (n=9) of the studies included in this review used the control group that presented other gynecological diseases or had no information about the reason for a surgical procedure performed by a gynecologist (laparoscopy and/or laparotomy), being able to provide low risk estimates, considering these diseases and/or the reason why a woman underwent surgery may be associated to the exposure under investigated (SNPs of VEGF gene).It is fundamental that the control group is directly determined by the definition and selection of the case group sampled by the same source. 5736,37 It has been described in the literature that about 11% of the women with endometriosis have no symptoms of the disease. 6,7n addition, the average time between the onset of the symptoms and the diagnosis of endometriosis is extensively long, ranging between 7 to 12 years. 58e emphasize that 4 articles were excluded from this review, 22,25,29,30 because they did not use adequate control to evaluate the magnitude of SNPs association to VEGF gene and the development of endometriosis.In a study conducted by Zhao et al. 25 the controls were women who reported not having endometriosis and only 27% had a negative diagnosis of endometriosis by laparoscopy or hysterectomy.Toktam et al., 30 used control of hospital basis, however it was not informed whether the women had undergone surgical procedure, they only reported that they had gynecological problems.Lamp et al. 29 used healthy fertile women (with at least 2 children), without clinical suspicion of endometriosis and without performing a surgical procedure to exclude the presence of the disease.Ikuhashi et al. 22 used umbilical cord as control material obtained from newborn females.
The main limitations of the case-control studies are: the lack of validity due to the selected bias; the recruitment of controls are not representative to the population at risk; errors on the measurements associated to the function of different biases between cases and controls and the confounding variables that affect a causal association. 50The differences in the allelic frequency pattern can be observed in the studies with ethnic groups, as well as environmental factors contributing to different phenotypic responses. 59Furthermore, it should be taken into account the sample size to avoid making type I errors, stating that the risk factor is associated to the disease, but, in fact, it is not; or type II errors, stating that the risk factor is not associated with the disease, but, in fact, it is. 50Among the 11 articles included in this review, only 3 (27%) of them informed the calculated sample used in the study. 21,23,26oreover, we emphasize the limited number of cases and controls included in Rotman et al. 33 study (24 cases and 18 controls) and Attar et al. 28 (52 cases and 60 controls) and the latter was excluded from this review because they used cases without histological confirmation of endometriosis.
According to Zondervan et al., 57 the choice of the ideal control group for the study on genetic basis of endometriosis is free of diseases, whereas is submitted to surgery for tubal ligation.Among the 11 studies included in this review, approximately 27% (n=3) used women who underwent tubal ligation in the control group, 8,26,32 and only 2 (18%) studies were formed exclusively by a control group of women who underwent the procedure of tubal ligation. 26,32Corroborating with these findings, we also suggest that the ideal control group perform case-control studies, which evaluates the genetic profile of women with endometriosis and should be informed by women submitted to tubal ligation, because it is possible to make a direct inspection of the pelvic cavity and exclude the presence of endometriosis lesions.
This review had the intention to describe and discuss the inclusion/exclusion criteria used to define the cases group with endometriosis and especially the controls group, in order to verify methodological differences that could contribute to the discrepancies in the results found in the literature.The analytical observational studies on case-control, when they are carefully designed can provide relevant information to identify risk factors, especially in cases of endometriosis, which is a complex disease and multifactorial.The epidemiological analyzes which explore the molecular profile of women with endometriosis may contribute to the etiologic investigation and build instruments for the actions and interventions in the field of the public health.
In conclusion, we suggest broader studies with correct methodological design involving different populations as well as a combined analysis of SNPs in VEGF and/or KDR genes are still necessary in virtue of the fundamental role of these factors in the endometriosis development.We suggest a selection of a control group from a hospital basis, without history of infertility and gynecological disorders, such as the case of women undergoing tubal ligation procedures corresponds to the appropriate control type to evaluate the magnitude of SNPs association of VEGF and/or KDR genes in the endometriosis development.

Figure 1 Flowchart
Figure 1Flowchart of the selected articles included in the review.
and KDR genes in the development of endometriosis

Table 1
Description of the inclusion and exclusion criteria of the case group, established by eligible studies for this review.
GCA = giant cell arthritis; * N is the number of cases of endometriosis included in each article; ** Type of surgery for endometriosis diagnosis; ** Yes = not specified the type of surgery performed; *** Classification of endometriosis staging described by the American Society for ReproductiveMedicine (1996, 1997), the American Society ofFertility (1985, 1997).

Table 2
Description of the inclusion and exclusion criteria of the control group, established by eligible studies for this review.
Tubal ligation(N) Others***(N) GCA = giant cell arthritis; * Type of surgery performed; ** Yes = not specified the type of surgery performed; *** Other reasons for conducting surgical procedure include appendicitis, carcinoma in situ of the cervix, dermoid cysts, parovarium cysts, serous cysts and mucinous tumors, dysmenorrhoea, pelvic pain, ovarian fibroma, dysfunctional uterine bleeding, hydrosalpinx, infertility and malformation of the uterine and normal pelvis.

Table 2
and Description of the inclusion and exclusion criteria of the control group, established by eligible studies for this review.
GCA = giant cell arthritis; * Type of surgery performed; ** Yes = not specified the type of surgery performed; *** Other reasons for conducting surgical procedure include appendicitis, carcinoma in situ of the cervix, dermoid cysts, parovarium cysts, serous cysts and mucinous tumors, dysmenorrhoea, pelvic pain, ovarian fibroma, dysfunctional uterine bleeding, hydrosalpinx, infertility and malformation of the uterine and normal

Table 3
Polymorphisms characteristics of VEGF gene analyzed in eligible studies for this review.

Table 3
Polymorphisms characteristics of VEGF gene analyzed in eligible studies for this review.