Endometriosis is a multifactorial

Transcription

1 Glutathione transferase polymorphisms and risk of endometriosis associated with polychlorinated biphenyls exposure in Italian women: a gene environment interaction Susanna Vichi, Ph.D., a Emanuela Medda, B.Sc., b Anna Maria Ingelido, Ph.D., c Annamaria Ferro, M.D., d Serena Resta, M.D., d Maria Grazia Porpora, M.D., d Annalisa Abballe, Ph.D., c Lorenza Nistico, M.D., Ph.D., b Elena De Felip, Ph.D., c Simonetta Gemma, Ph.D., a and Emanuela Testai, Ph.D. a a Department of the Environment and Primary Prevention, Unit of Mechanisms of Toxicity, Italian National Institute for Health; b National Centre for Epidemiology, Surveillance and Health Promotion, Unit of Genetic Epidemiology, Italian National Institute for Health; c Department of the Environment and Primary Prevention, Unit of Toxicological Chemistry, Italian National Institute for Health; and d Department of Gynaecology, Obstetrics and Urology, Sapienza University of Rome, Rome, Italy Objective: To investigate the occurrence of a gene environment interaction between glutathione transferase (GST) gene polymorphisms (GSTM1, GSTT1, GSTP1, and GSTA1) and serum polychlorinated biphenyls (PCBs) levels. This is suggested as possible risk factors for endometriosis, a multifactorial gynecological disease. Design: Case-control study conducted from 2002 to Setting: Policlinico Umberto I, Sapienza University of Rome and Italian National Institute for Health, Rome. Patient(s): Italian women (N ¼ 343), with laparoscopic diagnosis and histologic confirmation of the presence (cases, N ¼ 181) or the absence (controls, N ¼ 162) of endometriosis. Intervention(s): Genomic DNA extraction, multiplex polymerase chain reaction (PCR), and restriction fragment length polymorphism analysis. Determination of serum concentrations of selected PCBs by ion-trap mass spectrometry (subgroup, 63 cases and 63 controls). Main Outcome Measure(s): Endometriosis diagnosis by laparoscopy, GST genotypes, serum PCB levels. Result(s): The genotype distributions of GSTM1, GSTA1, and GSTP1 did not show any statistically significant difference between cases and controls. The GSTT1 null genotype was negatively associated with the disease. The GSTP1 wild-type genotype in the presence of medium-high blood levels of PCB153, total PCBs, or of high levels of PCB180 significantly increased the risk of endometriosis, suggesting a multiplicative interaction. Conclusion(s): The GSTs polymorphisms per se do not increase the risk of developing endometriosis. However, a gene environment interaction was observed for GSTP1 Ile/Ile and GSTM1 null genotypes, modulating the effect of PCB153, PCB180, and of total PCBs on disease risk. (Fertil Steril Ò 2012;97: Ó2012 by American Society for Reproductive Medicine.) Key Words: Endometriosis, GST polymorphism, PCBs, biomonitoring, gene environment interaction Endometriosis is a multifactorial gynecologic disease defined as the presence of hormonally dependent endometrial glandular and stromal cells outside uterus. Because of its negative impact on the quality Received August 2, 2011; revised and accepted February 21, 2012; published online March 14, S.V. has nothing to disclose. E.M. has nothing to disclose. A.M.I. has nothing to disclose. A.F. has nothing to disclose. S.R. has nothing to disclose. M.G.P. has nothing to disclose. A.A. has nothing to disclose. L.N. has nothing to disclose. E.D.F. has nothing to disclose. S.G. has nothing to disclose. E.T. has nothing to disclose. Study carried out within the framework of the Research Project Exposure to Organohalogenated Compounds as a Risk Factor for Women's Reproductive Health funded by the Italian National Institute for Health and the Ministry of Health. Reprint requests: Simonetta Gemma, Ph.D., Department of the Environment and Primary Prevention, Unit of Mechanisms of Toxicity, Italian National Institute for Health, Viale Regina Elena, Rome, Italy ( simonetta.gemma@iss.it). Fertility and Sterility Vol. 97, No. 5, May /$36.00 Copyright 2012 American Society for Reproductive Medicine, Published by Elsevier Inc. doi: /j.fertnstert of women's life in terms of pain and infertility and the economic burden for diagnosis, pharmacologic and surgical treatments, as well as for assisted fertilization practice, endometriosis has been recognized as a health priority. In industrialized countries endometriosis occurs in 6% 10% of the general female population; in women with pain, infertility, or both, the frequency is 35% 50% (1). Nevertheless these numbers are likely an underestimation, as among asymptomatic fertile women undergoing tubal sterilization procedures incidence ranges from 4% 43% (2). VOL. 97 NO. 5 / MAY

2 ORIGINAL ARTICLE: ENVIRONMENT AND EPIDEMIOLOGY The etiopathogenesis of this disease remains still unclear. The hypothesis of Sampson that retrograde menstruation triggers endometriosis is not supported by the high prevalence of this event (90%), indicating the involvement of additional risk factors. Endometriosis is characterized by a general inflammatory response in the peritoneal cavity with production of reactive oxygen species (ROS), which might act by increasing growth and adhesion of endometrial cells in the peritoneal cavity, with progression of endometriosis and infertility (3). Contrasting results on the association between inflammationinduced oxidative stress and endometriosis have been reported (4). Hormonal imbalance, genetic predisposition, failure of host immune response, and environmental factors have been suggested to concur to its onset and progression (5), but the association still remains controversial and no single theory seems to cover all the aspects of this disease. The hypothesis for a role of persistent environmental contaminants, such as polychlorinated dibenzo-p-dioxins (PCDDs) and polychlorinated biphenyls (PCBs), as risk factors in endometriosis, has been initially based on data on primates (6). Some criticisms (7) were related to bias and methodological deficiencies of the study. However, PCDDs and PCBs can interfere with synthesis, function, storage, or metabolism of hormones, bind to estrogen or androgen receptors. Many studies on the possible association between PCB serum levels and endometriosis, although controversial, indicated a slight increase of risk associated to PCB exposure (8 14). A genetic contribution to endometriosis etiopathogenesis was suggested by a 6% increased risk for close relatives, although no simple Mendelian inheritance was shown. Genetic susceptibility was explored also by studying mutations in genes responsible for detoxication, such as glutathione transferase (GST), as a possible risk factor to endometriosis (15 22). The GSTs, phase II enzymes, are critical in controlling free radical-associated injuries, making the association between the endometriosis and any alterations in their enzymatic activity biologically plausible. Many functional polymorphic variants at the four major loci have been described, including deletions for GSTM1 and GSTT1 and single-nucleotide polymorphisms in exon 5 of GSTP1 gene (an A/G transition causing a Ile/Val substitution in position 105 of the protein) (23) and in the proximal promoter of GSTA1 (C-69T) (24). The GSTM1 and GSTT1 gene deletion results in the absence of protein expression in 42% 60% and 13% 26%, respectively, of the Caucasians (25), lacking the related enzymatic activity. The GSTP1 Ile/Val and GSTP1 Val/Val enzymes (present in 30% 50% and 4% 16% of the Caucasians) show substrate-dependent alteration of its catalytic activity (23). The GSTA1 (C-69T) T allele (present in homozygosis in 14% 18% and in heterozygosis in 44% 49% of the Caucasians) significantly lowers enzyme hepatic expression (24, 26). Because endometriosis has the characteristics of a polygenic and multifactorial disease, it is likely that both environmental and genetic determinants interact. Therefore the concurrent contributions of both factors need to be investigated, as well as the presence of a gene environment interaction accounting for multiplicative joint effects. To this aim a case-control study was carried out on Italian women aged years, to examine whether functionally relevant polymorphisms in GSTA1, GSTT1, GSTM1, and GSTP1 genes are associated with the risk of endometriosis per se and in association with exposure to PCBs. MATERIALS AND METHODS Study Population and Data Collection Three hundred forty-three (181 cases and 162 controls) Italian women, undergoing laparoscopy for suspected endometriosis or other benign gynecologic conditions, were enrolled in a case-control study from 2002 to 2005 at the Department of Gynaecology and Obstetrics, of the Policlinico Umberto I, Sapienza University of Rome. All patients met the inclusion criteria: years of age, residence in Rome in the past 5 years, nulliparity/no breastfeeding history, absence of immunologic, hormonal disorders, or chronic diseases, and no occupational exposure to PCBs or pesticides. All enrolled women signed an informed consent form; a physician unaware of the indications to laparoscopy administered a questionnaire before surgery that documented age, education, job, medical/gynecologic/obstetric history, height and weight, and smoking habits. Among the 386 women characterized by these features, 343 underwent (incidental) laparoscopy and fully met the inclusion criteria. Before laparoscopy, a fasting blood specimen (1 ml) was collected from the cubital vein in vacutainer tubes to be used for DNA extraction. In a subset of women giving their consent (63 cases and 63 controls) the amount of blood collected was 30 ml to analyze genotype and serum PCB levels. For the subset, the body mass index (BMI) was calculated and a more detailed questionnaire was used to obtain information on potential confounders relevant for the exposure part (i.e., metabolic diseases, gravidity, parity, dietary, and weight changes in the past years) (Supplemental Table 1, available online). A detailed medical and gynecologic history was taken, and all patients underwent clinical and ultrasound examinations. More detailed information on questionnaire design, and diagnostic methodologies were previously described (8). A 10-mm laparoscopy was performed under general anesthesia. All women undergoing laparoscopy also had endometrial implants excised for histology to confirm the presence of endometriosis. The disease was staged according to the revised American Society of Reproductive Medicine classification. The control group consisted of women without complaints of infertility or pelvic pain, undergoing laparoscopy for benign gynecologic conditions (benign adnexal masses [61.7%], fallopian tubes abnormalities/diseases [24.7%], uterine myomas [13.6%]), that are not expected to share etiology with endometriosis and had no visual evidence of histologic features of endometriosis in random peritoneal biopsies. DNA Extraction Blood samples from cases and controls were collected in ethylenediaminetetraacetic acid (EDTA) vacutainer tubes and 1144 VOL. 97 NO. 5 / MAY 2012

3 Fertility and Sterility stored at 20 C until subjected to analysis. Genomic DNA extraction from whole blood was obtained using the QIAamp DNA blood mini kit (Qiagen) according to the manufacturer's instructions. Genotyping of the four loci was performed on the entire study population sample (181 cases and 162 controls) by personnel blinded to case-control status. To validate genotyping procedures 5% of samples were typed twice. GSTM1 and GSTT1 Genotyping The detection of GSTM1 and GSTT1 homozygote null genotype (-/-) was performed simultaneously in a single assay by a multiplex polymerase chain reaction (PCR) reaction as previously described (27). The absence of GSTM1-orGSTT1- specific PCR products indicated that GSTM1 or GSTT1 genes were deleted on both alleles (null). In contrast, PCR products indicated the presence of at least one copy of GST gene (positive: -/þ or þ/þ). GSTP1 Genotyping The GSTP1 polymorphism (rs1695) was analyzed by restriction fragment length polymorphism following the method of Harries et al. (28) with minor modifications. The homozygous wild-type (Ile/Ile), homozygous mutant (Val/Val), and the heterozygous (Ile/Val) genotypes were characterized by the presence of one band of 176 bp, two bands of 81 and 95 bp, and three bands of 176, 81 and 95 bp, respectively. GSTA1 Genotyping The GSTA1 C-69T polymorphism (rs ) was determined by restriction fragments length polymorphism following the method by Coles et al. (24) with minor modifications. The point mutation C-69T introduces a restriction site for EarI. Individuals homozygous for the wild-type allele (base C) showed one 480-bp band, homozygous for the mutant allele (base T) showed two bands of 100 and 380 bp, heterozygous were characterized by the presence of three bands of 480, 380, and 100 bp. Organochlorinated Persistent Pollutants Analysis In the subset of women (63 controls and 63 cases) enrolled in the study, serum levels of 11 organochlorinated persistent pollutants were determined by ion-trap mass spectrometry. The PCB congeners were selected for the analysis on the basis of their abundance in human tissues, and of toxicological relevance. They were the six non-dioxin-like congeners (NDL- PCBs) currently referred to as indicator (29), namely PCBs 28, 52, 101, 138, 153, and 180, known to account for 50% 80% total PCBs in human tissues (30), plus NDL-PCB170, and the dioxin-like congeners most abundant in human tissues, that is PCBs 105, 118, 156, and 167. Total PCB serum concentration was calculated as the sum of all of the 11 congeners analyzed. Serum samples (10 ml) were added with a 13 C mixture of labeled PCBs (28, 52, 101, 118, 138, 153, 156, 180) and allowed to rest overnight. Samples were added with 15 ml of formic acid:2-propanol (4:1, vol/vol), sonicated, and extracted by manual shaking with N-hexane. After centrifugation, the organic phase was removed and collected. The extraction process was repeated four times, and the N-hexane aliquots were pooled in a centrifuge tube and concentrated. After acidic treatment with concentrated H 2 SO 4, the organic phase was reduced in volume and transferred into a 1-mL vial to undergo instrumental analysis. Instrumental analysis was carried out by ion-trap mass spectrometry (Thermofinnigan Polaris Q) coupled to high resolution gas chromatography and used in the MS MS mode. The isotope dilution technique was applied throughout. The PCBs are highly lipophilic compounds, known to be strongly associated to serum lipids, therefore concentrations of total cholesterol, phospholipids, and triglycerides were determined by enzymatic methods, as previously described (8). The PCB concentrations were expressed in per gram of serum fat. This choice is in line with the approach established by World Health Organization for the studies of Persistent Organic Pollutants in biological samples, and specifically targeted to monitor possible differences of exposure between groups of individuals. Statistical Analysis The c 2 test was applied to verify that observed and expected genotype frequencies of GSTP1 and GSTA1 loci were in Hardy-Weinberg equilibrium. For GSTM1 and GSTT1, frequency of two genotypes (-/þ and þ/þ) could not be observed, thus deviations from Hardy-Weinberg proportions were not tested. Genotypic distributions among cases and controls were compared by c 2 test. A P value %.05 was considered as statistically significant. Crude odds ratio (OR) with 95% confidence intervals (CI) were also estimated for all GST polymorphisms. Based on published data on enzymatic activity or protein expression, GSTM1, GSTT1, and GSTA1 were grouped in high risk and low risk genotypes (GSTM1 -/þ and þ/þ; GSTT1 -/þ and þ/þ; GSTA1 C/T and C/C). Conflicting data on GSTP1 Ile105Val did not allow us to choose a reference genotype a priori. In the sample of case and control women in which exposure was estimated (n ¼ 126), the distribution of each PCB found to be associated with the disease (PCB118, PCB153, PCB138, PCB170, PCB180, and total PCBs) was divided into tertiles. After evaluation of the risk conferred by each stratum, women were grouped at low or medium-high exposure for PCB118, PCB153, PCB138, PCB170, and total PCBs. For exposure to PCB180 women were grouped in low-medium and high serum level. Crude ORs and 95% CIs were estimated for the second and third tertile of each analyte (8). The homogeneity of the association between PCB levels and the disease across GST genotype strata was verified using the heterogeneity test and by introducing a first-order multiplicative term in logistic regression model. Unconditional logistic regression models were used to estimate the magnitude of the association (OR) between the risk factors and the outcome controlling for the effects of all other variables in the model. Multiplicative term VOL. 97 NO. 5 / MAY

4 ORIGINAL ARTICLE: ENVIRONMENT AND EPIDEMIOLOGY represents a combination of exposure and potential effect modifier and was added to the logistic model to allow the assessment of interaction. Confidence limits around this estimate were obtained using the coefficient and its related standard error. Age (years), BMI (kilograms per square meter), evidence of relevant weight modification (>10 kg) in the past 5 years (yes vs. no), and smoking habits (ex-smokers vs. nonsmokers; current smokers vs. nonsmokers) were tested as covariates. Age and BMI were evaluated as continuous variables. In addition, a 2 4 table approach was used (31) according to which, beside the effect of each individual factor, any excess risk due to the gene environment combination was also calculated. Case-only analysis, a powerful strategy to detect gene environment interactions, in which controls group is used to validate the independence assumption between gene and environmental exposure, was performed. Case-only ORs for the relevant interactions were estimated by unconditional logistic regression adjusted for age. Our sample size (181 cases and 162 controls) had 80% probability to detect a risk equal to 1.7, 1.9, and 1.9, for GSTM1, GSTT1, and GSTA1 high risk homozygous genotypes, respectively (alpha ¼ 0.05, 1-sided; recessive mode of inheritance). The same power was obtained at the GSTP1 locus for a risk of 1.85 conferred by the homozygous genotype (2-sided, unchanged other parameters). The subsample of 63 cases and 63 controls had 80% power to detect a gene environment interaction of approximately 7.5, 10, 11.5, and 7 between each of the GSTM1, GSTT1, GSTA1, and GSTP1 high risk genotypes and medium-high levels of each PCB. Statistical analysis was performed with STATA software (version 9) and QUANTO (version 1.2.4; was used for power calculation. According to the rules of the Italian National Institute for Health this study has been authorized and approved by the Head of the Department of the Units involved in the study and by the President of the Institute (authorization ID number 36006). RESULTS All participants (N ¼ 343) were of Italian ethnicity. There was no significant difference in age distribution between cases (mean, years) and controls (mean, years). More than 50% of cases and controls were in the age range of years; the rest of the individuals were equally distributed (20% each) in the lower and higher age intervals. Sociodemographic, clinical characteristics, and all the information used to obtain data on potential confounders relevant for the exposure part related to the subset analyzed for serum PCB levels and GST loci (N ¼ 126) are described in Supplemental Tables 1 and 2, available online. Genetic Analysis The GSTM1, GSTT1, GSTP1, and GSTA1 genotype distributions were studied in 181 women with endometriosis and in 162 controls (Supplemental Table 3, available online). Genotypes of GSTP1 and GSTA1 loci were in Hardy-Weinberg equilibrium in case and control subjects. Women with GSTM1 null genotype have a nonsignificant increased risk of developing endometriosis compared with those carrying at least one allele. The GSTT1 homozygous null genotype confers a significant reduced risk of disease (Supplemental Table 3). The GSTP1 Ile/Ile frequency was higher in cases (48.6%) than in controls (42.0%). Women homozygous for the Ile variant appear to have a higher risk of endometriosis (OR ¼ 1.31; 95% CI: ) than subjects who carry at least one Val allele (Supplemental Table 3, right side). Genotype frequencies of GSTA1 did not differ between cases and controls, therefore no significant association was found with endometriosis (Supplemental Table 3). Genotype distribution of GSTM1, GSTT1, GSTP1, and GSTA1 were similar between the subgroups of women that were also analyzed for PCBs (N ¼ 126) and those who were not (N ¼ 217). In addition, we did not detect significant differences in the GSTs genotype distribution among the four stages of the disease. PCB Analysis The PCB exposure has been studied on a subset of the enrolled women (63 patients and 63 unaffected women). The PCB congeners 118, 138, 153, 170, and 180 makes up 70% or more of total amount. Therefore they represent the majority of the PCB exposure. Among the 11 analyzed congeners, only medium-high levels of PCB congeners 118, 138, 153, and 170 and high level of PCB180 are associated with a significant increased risk of endometriosis compared with the low ones (OR range, ) (Table 1). The risk of endometriosis appears to be also significant for total PCBs, obtained as the sum of all the 11 analyzed congeners (OR ¼ 4.6; 95% CI: ). The cutoff points of the tertiles are indicated in Table 1 (see Table footnotes). Interaction Between GST Genotype and PCB Exposure The association between levels of serum organochlorine and risk of endometriosis was evaluated after stratification by GST genotypes for the assessment of gene environment interaction (Table 2 and 3). Women with medium-high levels of PCB118, PCB153, PCB138, PCB170, total PCBs, and GSTM1 null genotype showed a higher (3.5<OR<8.3) risk of developing the disease compared with women with at least one GSTM1 functional allele (1.4<OR<2.6) (Table 2). The risk pattern is consistent for all the congeners tested. Although the strata heterogeneity tests were not significant, the absence of a functional GSTM1 allele is likely to be an effect modifier of the exposure to these PCBs. Results were confirmed by logistic regression analysis, suggesting the presence of a multiplicative gene environment interaction after age adjustment (.10<P<.52). The BMI, weight modification, and smoking habit were excluded as covariates because they had no confounding effect. Different from the previous PCBs, GSTM1 risk genotype seems not to be an effect modifier of the exposure to high levels of PCB180 (Table 2) VOL. 97 NO. 5 / MAY 2012

5 Fertility and Sterility TABLE 1 Raw ORs and PCB levels in cases and controls. Controls (N [ 63) Cases (N [ 63) N % N % OR 95% CI P value i PCB118 c Low Medium High Medium-high a PCB153 d Low Medium <.01 High <.01 Medium-high a <.01 PCB138 e Low Medium High Medium-high a <.01 PCB170 f Low Medium High Medium-high a <.01 PCB180 g Low Medium High High b Total PCBs h Low Medium <.01 High <.01 Medium-high a <.01 Note: CI ¼ confidence interval; OR ¼ odds ratio; PCB ¼ polychlorinated biphenyl. a Versus low level (first tertile). b Versus low/medium level (first/second tertile). c Low %13.2 ng/g fat, medium ng/g fat, high >24.2 ng/g fat. d Low %62 ng/g fat, medium ng/g fat, high >104 ng/g fat. e Low %33.6 ng/g fat, medium ng/g fat, high >56 ng/g fat. f Low %5.37 ng/g fat, medium ng/g fat, high >12.4 ng/g fat. g Low %33.2 ng/g fat, medium ng/g fat, high >60.4 ng/g fat. h Low %208 ng/g fat, medium ng/g fat, high >305 ng/g fat. i Chi-square test. To estimate and compare risks conferred by either gene and/or environmental exposures, a 2 4 table was used. Reference category consists of women with the low-risk genotypes and low PCB (or low-medium for PCB180) blood concentrations. As shown in Table 4 (left side), the presence of GSTM1 null genotype did not confer risk per se; medium-high PCB exposure (or high for PCB180) increased the risk of endometriosis in the absence of GSTM1 null genotype (1.4<OR<3.1) and even higher ORs were associated with the occurrence of both risk conditions (3.1<OR<5.1). Although confidence intervals are quite wide, the lower limits are above 1 (except for GSTM1 genotype and PCB118). Table 3 shows that, in the presence of the GSTP1 Ile/Ile genotype, women with medium-high PCB blood levels (or high for PCB180) had higher ORs (2.6<OR<30.6) compared with those with at least one Val allele (0.6<OR<2.3). The differences between strata were detectable for all PCBs tested and reached the statistical significance for PCB153, PCB180, and total PCBs. For these PCB categories, the additional risks due to the presence of both medium-high PCB levels and of GSTP1 Ile/Ile genotype, as estimated by multivariate logistic regression, were statistically significant, indicating a multiplicative interaction (P¼.04, P¼.0023, and P¼.0022). Evidence of a gene environment interaction for risk of endometriosis was also given by the combined ORs for PCB exposures and GSTP1 Ile/Ile genotype (Table 4, right side). Odds ratios due to genotype alone indicate that Ile/Ile genotype is not a risk factor for endometriosis per se, but increases the risk of endometriosis due to medium-high PCBs (or high for PCB180) exposure, resulting in significantly higher ORs (4.8<OR<9.3). The case-only results were very similar to case-control analysis and revealed, after age adjustment, an interaction between GSTP1 and PCB153 (OR ¼ 3.6; 95% CI: ) or PCB180 (OR ¼ 5.9; 95% CI: ) and between GSTP1 and total PCB (OR ¼ 8.5; 95% CI: ). In addition, a significant interaction (OR ¼ 4.6; 95% CI: ) between GSTM1 and PCB153 was found. Stratified analysis for GSTT1 and GSTA1 genotypes showed inconclusive results with no evidence for gene environment VOL. 97 NO. 5 / MAY

6 ORIGINAL ARTICLE: ENVIRONMENT AND EPIDEMIOLOGY TABLE 2 Serum PCB concentrations and risk of endometriosis stratified by glutathione transferase M1 genotypes. GSTM1 genotypes PCB concentration Controls (N [ 63) Cases (N [ 63) N % N % OR 95% CI Heterogeneity test PCB118 Positive a Low Medium-high Null b Low P¼.15 Medium-high PCB153 Positive a Low Medium-high Null b Low P¼.11 Medium-high PCB138 Positive a Low Medium-high Null b Low P¼.48 Medium-high PCB170 Positive a Low Medium-high Null b Low P¼.42 Medium-high PCB180 Positive a Low-medium High Null b Low-medium P¼.57 High Total PCBs Positive a Low Medium-high Null b Low P¼.20 Medium-high Note: CI ¼ confidence interval; OR ¼ odds ratio; PCB ¼ polychlorinated biphenyl. a ( /þ and þ/þ). b ( / ). interaction, possibly because few women were included in the risk genotype strata (N ¼ 10). DISCUSSION Although a number of studies in the literature attempted to find a possible association between endometriosis and exposure to persistent organochlorinated compounds or to single GST polymorphisms, to our knowledge this is the first study exploring the possibility of an interaction between GST genes, as well as between GST genes and PCB congeners. To evaluate whether the presence of low activity alleles may predispose women to endometriosis, through promotion of an inflammation process, we considered four functional polymorphisms of the main GST isoform involved in ROS detoxication. Genotype frequencies of the four loci tested in our study are in agreement with existing data on Caucasians (25, 32), as represented by our enrolled population. For GSTM1 and GSTT1 null genotypes, the absence of a positive association with endometriosis, as observed in our study, is consistent with results from a group of United Kingdom women (16, 33), and with those reported by other investigators in Japanese, Indian, and Korean women (15, 22, 34). Other studies on different ethnic groups (Slavic, French, Turkish, Indian, Asiatic, and Russian women) reported a positive, although in some cases not significant, association between endometriosis and the GSTM1 null genotype (15, 19, 35 39), or the GSTT1 null genotype (37, 38, 40). When the available data were pooled in a meta-analysis, no association between endometriosis risk and GSTM1 polymorphism was found. A slight (29%) increase of risk was reported for GSTT1 null women, which could be easily questioned, as the analyzed studies appeared to be influenced by publication bias (41). In addition, crucial factors such as size of the groups, case and control definition, exclusion of the disease in control women, and age at diagnosis/enrolment of studied subjects were heterogeneous among studies or not declared. In our study, all enrolled women underwent laparoscopic evaluation and only those with histologic evidence/absence of endometriotic lesions were assigned to a case/control group. In addition, potential bias or methodologic genotyping errors were minimized and age at enrolment was taken into account in the statistical analysis of gene environment interaction. According to Schisterman et al. (42), to avoid biased estimates when assessing health risk of PCBs, statistical analysis and the relations among serum PCBs, serum lipids, and outcome should be carefully considered. However, in our study, whatever the causal scenario was hypothesized, the 1148 VOL. 97 NO. 5 / MAY 2012

7 Fertility and Sterility TABLE 3 Serum PCB concentrations and risk of endometriosis stratified by glutathione transferase P1 genotypes. GSTP1 genotypes PCBs concentration Controls (N [ 63) Cases (N [ 63) N % N % OR 95% CI Heterogeneity test PCB118 Ile/Val, Val/Val Low Medium-high Ile/Ile Low P¼.90 Medium-high PCB153 Ile/Val, Val/Val Low Medium-high Ile/Ile Low P¼.04 Medium-high PCB138 Ile/Val, Val/Val Low Medium-high Ile/Ile Low P¼.23 Medium-high PCB170 Ile/Val, Val/Val Low Medium-high Ile/Ile Low P¼.19 Medium-high PCB180 Ile/Val, Val/Val Low-medium High Ile/Ile Low-medium P¼.0023 High Total PCBs Ile/Val, Val/Val Low Medium-high Ile/Ile Low P¼.0022 Medium-high Note: CI ¼ confidence interval; OR ¼ odds ratio; PCB ¼ polychlorinated biphenyl. standardized model invariably results in an underestimation of the PCBs effect on endometriosis. To evaluate the possibility that, apart from GSTM1 and GSTT1, other GST enzymes could have a role in the development of endometriosis, we analyzed two other polymorphisms at GSTA1 and GSTP1 genes, which, lowering tissue defenses against ROS, may also influence disease susceptibility. To our knowledge no other study analyzed a possible association between GSTA1 C-69T polymorphism and endometriosis; nevertheless it could be functionally relevant, being expressed both in the ovaries and in the uterus (43). Previous data about the association of GSTP1 and endometriosis were inconclusive. No correlation was described in Korean women (20), whereas a study on Turkish women identified the GSTP1 Ile105Val polymorphism as a risk factor for endometriosis (17). Despite the existence of a biological plausibility for studying both enzymes we did not detect a significant association with endometriosis. Power estimates of our sample size cannot rule out that these genes may have small effects on endometriosis risk. Although conflicting results have been published in the past years, our group has recently demonstrated the existence of an association between PCBs and p,p'-dichlorodiphenyldichloroethylene (DDE) serum concentrations and the risk of endometriosis. Because no correlation with dietary habits was found (8), the higher PCB levels in patients with endometriosis could be associated with a different kinetic behavior of PCB congeners, as a result of genetic make-up and/or induction/inhibition phenomena in the tested population. In addition to their involvement in quenching the excess of ROS related to the inflammatory process, having a synergistic effect with the activity of some PCBs themselves, GST may contribute to the detoxication of some hydroxylated PCB congeners, formed by the cytochrome P450 and their further excretion. A significant interaction has been reported for CYP1B1 (cytochrome P450 1B1) and CYP1A1 (cytochrome P450 1A1) gene and PCBs in the development of endometriosis (44). Thus, we evaluated whether there was a detectable synergistic effect between PCB exposure and GST polymorphisms, which was detectable. The novelty of the study is the detection of a significant multiplicative interaction between the GSTP1 Ile homozygous genotype with medium-high levels of PCB153 and of total PCBs, and with high levels of PCB180. Differences between strata, although not significant, were also obtained for the GSTM1 null genotype with medium-high levels of PCB153 and with total PCBs. It may be that GSTM1 modulates the effect of PCB exposure to a lesser extent than GSTP1, which could not be observed with our sample size. The effects detected between the two strata for both GSTM1 and GSTP1 genotypes, are consistent for all the PCBs considered (except GSTM1 and PCB180), suggesting that interactions are unlikely to have occurred by chance. In addition, the case-only VOL. 97 NO. 5 / MAY

8 ORIGINAL ARTICLE: ENVIRONMENT AND EPIDEMIOLOGY TABLE 4 Independent and joint effects of glutathione transferase M1 or glutathione transferase P1 genotype and of PCB exposure on disease risk. PCB concentration GSTM1 genotypes OR 95% CI GSTP1 genotypes OR 95% CI PCB118 Low Positive a Ref Ile/Val, Val/Val Ref Medium-high Positive a Ile/Val, Val/Val Low Null b Ile/Ile Medium-high Null b Ile/Ile PCB153 Low Positive a Ref Ile/Val, Val/Val Ref Medium-high Positive a Ile/Val, Val/Val Low Null b Ile/Ile Medium-high Null b Ile/Ile PCB138 Low Positive a Ref Ile/Val, Val/Val Ref Medium-high Positive a Ile/Val, Val/Val Low Null b Ile/Ile Medium-high Null b Ile/Ile PCB170 Low Positive a Ref Ile/Val, Val/Val Ref Medium-high Positive a Ile/Val, Val/Val Low Null b Ile/Ile Medium-high Null b Ile/Ile PCB180 Low-medium Positive a Ref Ile/Val, Val/Val Ref High Positive a Ile/Val, Val/Val Low-medium Null b Ile/Ile High Null b Ile/Ile Total PCBs Low Positive a Ref Ile/Val, Val/Val Ref Medium-high Positive a Ile/Val, Val/Val Low Null b Ile/Ile Medium-high Null b Ile/Ile Note: CI ¼ confidence interval; OR ¼ odds ratio; PCB ¼ polychlorinated biphenyl; Ref ¼ reference. a ( /þ and þ/þ). b ( / ). design, which is a more robust way of estimating gene environment interaction, yielded the same results. The sample size did not prevent us to detect a significant interaction. Odds ratios and CIs reported in the present study cannot be considered conclusive. Estimates of the actual interaction magnitude could be better defined if further information is accumulated. When the effect of GSTM1 or GSTP1 variant genes was singled out, they did not result per se in an appreciable association with endometriosis, thus providing a possible explanation for inconsistent results observed in previous studies. These polymorphisms can act as modulators of the risk related to environmental exposure. Our results suggest that, with adequately powered sample size, genetic and environmental exposures should be considered simultaneously, therefore disentangling the effect of each factor separately and obtaining accurate estimates. Understanding the roles played by GST enzymes and PCBs may quantify women at high risk and envisage preventive measures. REFERENCES 1. Giudice LC, Kao LC. Endometriosis. Lancet 2004;364: Buck Louis GM, Hediger ML, Peterson CM, Croughan M, Sundaram R, Stanford J, et al. Incidence of endometriosis by study population and diagnostic method: the ENDO study. Fertil Steril 2011;96: Alpay Z, Saed GM, Diamond MP. Female infertility and free radicals: potential role in adhesions and endometriosis. J Soc Gynecol Investig 2006;13: Szczepanska M, Kozlik J, Skrzypczak J, Mikolajczyk M. Oxidative stress may be a piece in the endometriosis puzzle. Fertil Steril 2003;79: Kajihara H, Yamada Y, Kanayama S, Furukawa N, Noguchi T, Haruta S, et al. New insights into the pathophysiology of endometriosis: from chronic inflammation to danger signal. Gynecol Endocrinol 2011;27: Rier SE, Turner WE, Martin DC, Morris R, Lucier GW, Clark GC. Serum levels of TCDD and dioxin-like chemicals in Rhesus monkeys chronically exposed to dioxin: correlation of increased serum PCB levels with endometriosis. Toxicol Sci 2001;59: Guo SW. The link between exposure to dioxin and endometriosis: a critical reappraisal of primate data. Gynecol Obstet Invest 2004;57: Porpora MG, Medda E, Abballe A, Bolli S, De Angelis I, di Domenico A, et al. Endometriosis and organochlorinated environmental pollutants: a casecontrol study on Italian women of reproductive age. Environ Health Perspect 2009;117: Quaranta MG, Porpora MG, Mattioli B, Giordani L, Libri I, Ingelido AM, et al. Impaired NK-cell-mediated cytotoxic activity and cytokine production in patients with endometriosis: a possible role for PCBs and DDE. Life Sci 2006; 79: Reddy BS, Rozati R, Reddy S, Kodampur S, Reddy P, Reddy R. High plasma concentrations of polychlorinated biphenyls and phthalate esters in women with endometriosis: a prospective case control study. Fertil Steril 2006;85: De Felip E, Porpora MG, di Domenico A, Ingelido AM, Cardelli M, Cosmi EV, et al. Dioxin-like compounds and endometriosis: a study on Italian and Belgian women of reproductive age. Toxicol Lett 2004;150: VOL. 97 NO. 5 / MAY 2012

9 Fertility and Sterility 12. Lebel G, Dodin S, Ayotte P, Marcoux S, Ferron LA, Dewailly E. Organochlorine exposure and the risk of endometriosis. Fertil Steril 1998;69: Trabert B, De Roos AJ, Schwartz SM, Peters U, Scholes D, Barr DB, et al. Nondioxin-like polychlorinated biphenyls and risk of endometriosis. Environ Health Perspect 2010;118: Pauwels A, Schepens PJC, D'Hooghe T, Delbeke L, Dhont M, Brouwer A, et al. The risk of endometriosis and exposure to dioxins and polychlorinated biphenyls: a case-control study of infertile women. Hum Reprod 2001;16: Babu KA, Reddy NG, Deendayal M, Kennedy S, Shivaji S. GSTM1, GSTT1 and CYP1A1 detoxification gene polymorphisms and their relationship with advanced stages of endometriosis in South Indian women. Pharmacogenet Genomics 2005;15: Baxter SW, Thomas EJ, Campbell IG. GSTM1 null polymorphism and susceptibility to endometriosis and ovarian cancer. Carcinogenesis 2001;22: Ertunc D, Aban M, Tok EC, Tamer L, Arslan M, Dilek S. Glutathione-S-transferase P1 gene polymorphism and susceptibility to endometriosis. Hum Reprod 2005;20: Guo SW. The association of endometriosis risk and genetic polymorphisms involving dioxin detoxification enzymes: a systematic review. Eur J Obstet Gynecol Reprod Biol 2006;124: Hsieh YY, Chang CC, Tsai FJ, Lin CC, Chen JM, Tsai CH. Glutathione S-transferase M1*null genotype but not myeloperoxidase promoter G-463A polymorphism is associated with higher susceptibility to endometriosis. Mol Hum Reprod 2004;10: Hur SE, Lee JY, Moon HS, Chung HW. Polymorphisms of the genes encoding the GSTM1, GSTT1 and GSTP1 in Korean women: no association with endometriosis. Mol Hum Reprod 2005;11: Jeon MJ, Choi YM, Hong MA, Lee GH, Ku SY, Kim SH, et al. No association between the GSTP1 exon 5 polymorphism and susceptibility to advanced stage endometriosis in the Korean population. Am J Reprod Immunol 2010;63: Kim SH, Choi YM, Lee GH, Hong MA, Lee KS, Lee BS, et al. Association between susceptibility to advanced stage endometriosis and the genetic polymorphisms of aryl hydrocarbon receptor repressor and glutathione- S-transferase T1 genes. Hum Reprod 2007;22: Hayes JD, Strange RC. Glutathione S-transferase polymorphisms and their biological consequences. Pharmacology 2000;61: Coles BF, Morel F, Rauch C, Huber WW, Yang M, Teitel CH, et al. Effect of polymorphism in the human glutathione S-transferase A1 promoter on hepatic GSTA1 and GSTA2 expression. Pharmacogenetics 2001;11: Garte S, Gaspari L, Alexandrie AK, Ambrosone C, Autrup H, Autrup JL, et al. Metabolic gene polymorphism frequencies in control populations. Cancer Epidemiol Biomarkers Prev 2001;10: Hayes JD, Flanagan JU, Jowsey IR. Glutathione transferases. Annu Rev Pharmacol 2005;45: Palli D, Saieva C, Gemma S, Masala G, Gomez-Miguel MJ, Luzzi I, et al. GSTT1 and GSTM1 gene polymorphisms and gastric cancer in a high-risk italian population. Int J Cancer 2005;115: Harries LW, Stubbins MJ, Forman D, Howard GC, Wolf CR. Identification of genetic polymorphisms at the glutathione S-transferase Pi locus and association with susceptibility to bladder, testicular and prostate cancer. Carcinogenesis 1997;18: Appel KE. Risk assessment of non-dioxin-like PCBs report on a WHO consultation. Fresenius Environ Bull 2003;12: Glynn AW, Wolk A, Aune M, Atuma S, Zettermark S, Maehle-Schmid M, et al. Serum concentrations of organochlorines in men: a search for markers of exposure. Sci Total Environ 2000;263: Botto LD. Flavors in gene-environment interactions. Epidemiology 2007;18: Ahn J, Gammon MD, Santella RM, Gaudet MM, Britton JA, Teitelbaum SL, et al. Effects of glutathione S-transferase A1 (GSTA1) genotype and potential modifiers on breast cancer risk. Carcinogenesis 2006;27: Hadfield RM, Manek S, Weeks DE, Mardon HJ, Barlow DH, Kennedy SH. Linkage and association studies of the relationship between endometriosis and genes encoding the detoxification enzymes GSTM1, GSTT1 and CYP1A1. Mol Hum Reprod 2001;7: Morizane M, Yoshida S, Nakago S, Hamana S, Maruo T, Kennedy S. No association of endometriosis with glutathione S-transferase M1 and T1 null mutations in a Japanese population. J Soc Gynecol Invest 2004;11: Aban M, Ertunc D, Tok EC, Tamer L, Arslan M, Dilek S. Modulating interaction of glutathione-s-transferase polymorphisms with smoking in endometriosis. J Reprod Med 2007;52: Baranov VS, Ivaschenko T, Bakay B, Aseev M, Belotserkovskaya R, Baranova H, et al. Proportion of the GSTM1 0/0 genotype in some Slavic populations and its correlation with cystic fibrosis and some multifactorial diseases. Hum Genet 1996;97: Baranova H, Canis M, Ivaschenko T, Albuisson E, Bothorishvilli R, Baranov A, et al. Possible involvement of arylamine N-acetyltransferase 2, glutathione S- transferase M1 and T1 genes in the development of endometriosis. Mol Hum Reprod 1999;5: Lin J, Zhang X, Qian Y, Ye Y, Shi Y, Xu K, et al. Glutathione S-transferase M1 and T1 genotypes and endometriosis risk: a case-controlled study. Chin Med J 2003;116: Roya R, Baludu GS, Reddy BS. Possible aggravating impact of gene polymorphism in women with endometriosis. Indian J Med Res 2009;129: Ivashchenko TE, Shved NI, Kramareva NA, Ailamazian EK, Baranov VS. Analysis of the polymorphic alleles of genes encoding phase 1 and phase 2 detoxication enzymes in patients with endometriosis. Genetika 2003;39: Guo SW. Glutathione S-transferases M1/T1 gene polymorphisms and endometriosis: a meta-analysis of genetic association studies. Mol Hum Reprod 2005;11: Schisterman EF, Whitcomb BW, Louis GM, Louis TA. Lipid adjustment in the analysis of environmental contaminants and human health risks. Environ Health Perspect 2005;113: Abel EL, Lyon RP, Bammler TK, Verlinde CL, Lau SS, Monks TJ, et al. Estradiol metabolites as isoform-specific inhibitors of human glutathione S-transferases. Chem Biol Interact 2004;151: Tsuchiya M, Tsukino H, Iwasaki M, Sasaki H, Tanaka T, Katoh T, et al. Interaction between cytochrome P450 gene polymorphisms and serum organochlorine TEQ levels in the risk of endometriosis. Mol Hum Reprod 2007;13: VOL. 97 NO. 5 / MAY

10 ORIGINAL ARTICLE: ENVIRONMENT AND EPIDEMIOLOGY SUPPLEMENTAL TABLE 1 Sociodemographic and clinical characteristics of cases and controls. Sociodemographic characteristics Controls (N [ 63) % Cases (N [ 63) % P value a Age (y) (mean SD) Age (y) % R Number of children BMI (mean SD) Age at menarche (y) (mean SD) Breastfed when children Yes No Unknown Relevant weight modifications in the past 5 years (>10 kg) Yes No Smoking status Nonsmokers Ex-smokers Smokers: 1 9 cigarettes/day cigarettes/day R20 cigarettes/day Food consumption [times/month (mean SD)] Milk and dairy products Meat Fish Laparoscopic findings Stage of endometriosis (revised ASRM) I II III IV Ovarian endometrioma Yes No Peritoneal lesions Yes No Type of peritoneal lesion Typical Subtle Both Deep endometriosis Yes No Note: ASRM ¼ American Society for Reproductive Medicine; BMI ¼ body mass index. t-test or Chi-square test e1 VOL. 97 NO. 5 / MAY 2012

11 Fertility and Sterility SUPPLEMENTAL TABLE 2 Serum concentrations (geometric mean [95% CI]; ng/g fat) of PCBs in cases and controls. Controls (N [ 63) Cases (N [ 63) P value a PCB ( ) 24.1 ( ).002 PCB ( ) 96.3 ( ).001 PCB ( ) 48.8 ( ).002 PCB ( ) 46.8 ( ).03 PCB ( ) 9.3 ( ).07 Total PCBs ( ) ( ).0002 Note: CI ¼ confidence interval; PCB ¼ polychlorinated biphenyl. a t-test on log-transformed values. VOL. 97 NO. 5 / MAY e2

12 ORIGINAL ARTICLE: ENVIRONMENT AND EPIDEMIOLOGY SUPPLEMENTAL TABLE 3 Genotypic frequency distributions of glutathione transferase polymorphisms (n [ 343). Gene Genotype Controls % (N [ 162) Cases % Grouped (N [ 181) OR 95% CI P value a genotypes OR 95% CI GSTM1 Positive b Null c GSTT1 Positive b Null c GSTP1 (rs1695) Ile/Ile Ile/Val Ile/Ile 1.31 Val/Val Ile/Val, Val/Val GSTA1 (rs ) C/C C/T C/C, C/T 1 T/T T/T Note: CI ¼ confidence interval; OR ¼ odds ratio. a c 2 test. b ( /þ and þ/þ). c ( / ) e3 VOL. 97 NO. 5 / MAY 2012