Increased dioxin-like compounds in the serum of women with peritoneal endometriosis and deep endometriotic (adenomyotic) nodules

Transcription

1 Increased dioxin-like compounds in the serum of women with peritoneal endometriosis and deep endometriotic (adenomyotic) nodules Jean-François Heilier, Pharm.D., a Fabienne Nackers, M.D., b Violaine Verougstraete, M.D., Ph.D., a René Tonglet, M.D., Ph.D., b Dominique Lison, M.D., Ph.D., a and Jacques Donnez, M.D., Ph.D. c,d a Industrial Toxicology and Occupational Medicine Unit, b Epidemiology Unit, and c Department of Gynaecology, Université Catholique de Louvain, Brussels, Belgium; and d Cliniques Universitaires St-Luc, Brussels, Belgium Objective: To investigate the possible association between the body burden of dioxin-like compounds and endometriotic disease. Design: Case-control study. Setting: Gynecology ward in a university hospital. Patient(s): Seventy-one women with peritoneal endometriosis (n 25) or deep endometriotic (adenomyotic) nodules (n 25) and controls (n 21). Intervention(s): Collection of 200 ml of blood (fasted) and face-to-face interview. Main Outcome Measure(s): Assessment of dioxin (PCDD), furan (PCDF), and dioxin-like PCB serum concentrations (picograms toxic equivalent [TEQ]/g lipids). Result(s): Age and body mass index were traced by linear multiple regression as determinants of total TEQ levels. After standardization for these variables (30 years and 22.5 kg/m 2 ), the mean TEQ levels were (controls), (peritoneal endometriosis), and (deep endometriotic [adenomyotic] nodules) pg TEQ/g lipids. Logistic regression analysis indicated a significantly increased risk of deep endometriotic (adenomyotic) nodules (odds ratio [OR], 3.3; 95% confidence interval [CI], ) for an increment of 10 pg in total TEQ levels/g lipids. An increased risk was also found for peritoneal endometriosis (OR, 1.9; 95% CI, ) for total TEQ levels and for dioxins alone (OR, 3.2; 95% CI, ). Conclusion(s): The results provide the first epidemiological evidence of an association between increased PCDD/PCDF and PCB body burden and endometriosis. (Fertil Steril 2005;84: by American Society for Reproductive Medicine.) Key Words: Dioxins, PCDD, PCDF, PCBs, dioxin-like PCBs, endometriosis, deep endometriotic (adenomyotic) nodules Endometriosis is characterized by the extrauterine growth of endometrial tissue, which causes internal bleeding, inflammation, and scarring (1). Its prevalence and severity are increasing in developing countries (2). Two distinct clinical entities with different histopathogeneses have recently been identified, that is, peritoneal endometriosis and deep endometriotic (adenomyotic) nodules of the rectovaginal septum (3 5). While it is clear that hormones, especially estrogens, play an important role in the pathogenesis of both diseases, their exact etiologies remain unclear. Several investigators have proposed that endocrine-disrupting compounds (EDCs), such as dioxins or dioxin-like polychlorinated biphenyls (PCBs), could play a role in the onset or the growth of endometriosis (6, 7). Dioxins and PCBs are lipophilic compounds that accumulate and persist in the environment and the food chain. Received July 9, 2004; revised and accepted April 4, This work was supported by grant no from the Fonds de la Recherche Scientifique Médicale, Belgium. Reprint requests: Jean-François Heilier, TOXI 30.54, Clos Chapelle aux champs, Woluwe B-1200, Belgium (FAX: ; jean-francois.heilier@toxi.ucl.ac.be). Dioxins are made up of 210 chlorinated hydrocarbon compounds divided into 75 polychlorinated-dibenzo-dioxins (PCDDs) and 135 polychlorinated-dibenzo-furans (PCDFs). In the past, their environmental release resulted from the synthesis of organochlorine compounds and numerous anthropogenic combustion processes in the presence of chlorine at relatively low temperatures. The so-called Seveso dioxin (2,3,7,8, tetrachlorodibenzodioxin [TCDD]) is the most toxic dioxin congener. Seventeen dioxin congeners, including TCDD, which preferentially bioaccumulate in the food chain, were found to bind to a specific receptor (aryl hydrocarbon receptor [AhR]), which mediates most of their toxic effects through the activation of several genes, including cytochromes P450 (8, 9). Several PCBs have been synthesized and used for a number of industrial applications. Twelve PCBs share with dioxins an activity on the AhR (dioxin-like PCBs) (10). All compounds able to bind to the AhR (7 PCDD, 10 PCDF, and 12 dioxin-like PCBs) have been classified relative to the TCDD and received a toxic equivalency factor (TEF), which reflects their respective biological potency. Concentrations of these compounds are frequently expressed as picograms /05/$30.00 Fertility and Sterility Vol. 84, No. 2, August 2005 doi: /j.fertnstert Copyright 2005 American Society for Reproductive Medicine, Published by Elsevier Inc. 305

2 of toxic equivalent [TEQ], which is the sum of the products of the concentration of each compound multiplied by its TEF (11). In industrialized countries, humans are exposed to dioxins and PCBs mainly via food. They accumulate in fat tissue with age, but parity and breast-feeding contribute to reducing the body burden (12). The possible relationship between peritoneal endometriosis and dioxins has been studied experimentally in nonhuman primates. An association between endometriosis and chronic exposure of rhesus monkeys to TCDD and also dioxin-like PCBs (which were paired with TCDD for that study) has been reported by Rier et al. (13, 14). Yang and coworkers also found that TCDD facilitated the survival and growth of endometrial implants in cynomolgus monkeys (15). Several epidemiological studies have been conducted to investigate the potential relationship between endometriosis and organochlorine body burden, but their results have been inconclusive. In a cohort study, Eskenazi et al. (16) followed 601 Italian women who were 30 years of age when they were acutely exposed in Seveso. The investigators found a nonsignificant increased risk of endometriosis in women who had been highly exposed to the dioxin ( 100 parts per trillion [ppt] TCDD soon after the accident). Three casecontrol studies have been published. In Israel, Mayani and coworkers determined TCDD levels in the serum of infertile women who underwent diagnostic laparoscopy. They found a significantly higher proportion of women with detectable serum levels of TCDD ( 0.4 ppt) among those with endometriosis (18%), compared with women without the disease (3%) (17). In Belgium, Pauwels et al. (18) measured dioxin-like compounds in the serum of infertile women with the CALUX assay. After separating two groups (cutoff value 100 pg TEQ/g lipids), they found a statistically nonsignificant association between blood dioxin TEQ content and endometriosis, which was confirmed by laparoscopy (crude odds ratio [OR], 4.33; 95% confidence interval [CI], ). In Canada, Lebel et al. (19) conducted a similar study focusing on chlorinated pesticides (DDT, Mirex [Allied Chemical Corp.], Aldrin [Shell International Chemical Co. Ltd.]) and non-dioxin-like PCBs. They did not detect any association between organochlorine body burden and endometriosis, but dioxins and the majority of dioxin-like PCBs were not determined. In these case-control studies, endometriosis was not classified according to its specific localization (peritoneal or deep nodules) and infertile women served as controls. In a previous report, we showed that women with deep endometriotic (adenomyotic) nodules had increased serum levels of PCBs (20). Therefore, the purpose of the present study was to investigate the possible association between PCDDs/PCDFs or dioxin-like PCBs and endometriotic diseases, considering peritoneal endometriosis and deep endometriotic (adenomyotic) nodules separately. Furthermore, the control group was recruited from a population of women who did not present for infertility. MATERIALS AND METHODS Participants The present study was conducted prospectively between January 2001 and June Patients with deep endometriotic (adenomyotic) nodules had pelvic pain and severe dysmenorrhea and dyspareunia, and in all cases clinical examination revealed a nodular lesion (2 3 cm in size) either in the rectovaginal septum or in the retrocervical space where the vagina is attached. Magnetic resonance imaging and rectal sonography confirmed the lesion according to the classification recently described (21). Patients were admitted to the university hospital for surgical treatment and histological examination of the excised nodular lesions, which confirmed the diagnosis of deep endometriotic (adenomyotic) nodules, as described by Donnez et al. (22). In parallel, we recruited patients with peritoneal endometriosis who were hospitalized for surgical treatment during the same period. This group consisted of patients with minimal or moderate endometriosis (23), and the presence of deep endometriotic lesions was formally excluded to compare distinct clinical entities. The control group was made up of healthy women who were recruited concurrently from the consultations of the gynecologists who referred the deep endometriotic (adenomyotic) nodule cases. This group included women of similar age with no clinical suspicion of either peritoneal endometriosis or deep endometriotic (adenomyotic) nodules and no complaints of infertility, pelvic pain, or dysmenorrhea and with a normal pelvic examination, unremarkable vaginal echography, and a CA-125 level under 35 U/mL. The first 71 women (21 controls, 25 cases of peritoneal endometriosis, and 25 cases of deep endometriotic [adenomyotic] nodules) who volunteered to provide a 200-mL blood sample for PCDD/PCDF and PCB analysis were included in the study group (fewer than 10% refused to participate). The protocol of this study was approved by the Ethics Committee of the Université Catholique de Louvain, and all patients signed an informed consent form. After overnight fasting, a 200-mL blood sample was collected in a Teruflex BB*D456M bag without anticoagulant and centrifuged within 24 hours. Serum was transferred into organic solvent-cleaned glassware and kept frozen ( 20 C) until analysis. Chemical Analyses Sample cleanup was performed by automated extraction (Power-Prep System, FMS, Waltham, MA) on C18 and silica (acid, basic, and neutral) columns. The 17 PCDDs/ 306 Heilier et al. PCDD/PCDF, endometriosis, and adenomyosis Vol. 84, No. 2, August 2005

3 PCDFs (2,3,7,8-TCDD; 1,2,3,7,8-PeCDD; 1,2,3,4,7,8- HxCDD; 1,2,3,6,7,8-HxCDD; 1,2,3,7,8,9-HxCDD; 1,2,3, 4,6,7,8-HpCDD; OCDD; 2,3,7,8-TCDF; 1,2,3,7,8-PeCDF; 2,3, 4,7,8-PeCDF; 1,2,3,4,7,8-HxCDF; 1,2,3,6,7,8-HxCDF; 1,2,3,7, 8,9-HxCDF; 2,3,4,6,7,8-HxCDF; 1,2,3,4,6,7,8-HpCDF; 1,2,3, 4,7,8,9-HpCDF; OCDF) as well as 12 dioxin-like PCBs (nos. 77, 81, 126, 169, 105, 114, 118, 123, 156, 157, 167, and 189 as classified by the International Union of Pure and Applied Chemistry) with AhR activity were determined by gas chromatography high-resolution mass spectrometry (GC- HRMS), using a Hewlett-Packard 6890 Series gas chromatograph (Palo Alto, CA), hyphenated to a MAT95XL highresolution (10 4 ) mass spectrometer (Finnigan, Bremen, Germany). Isotopic dilution was used for quantification (24). Concentrations of free cholesterol, bound cholesterol, phospholipids, and triglycerides were determined by enzymatic methods. Total lipid concentration was calculated according to Akins (25). PCDD/PCDF and PCB levels were expressed as toxic equivalent (TEQ) per gram of serum lipids (11, 26, 27). Statistical Analyses SAS/STAT version 8 software (SAS Institute, Cary, NC) was used for frequency, variance, and multiple and logistic regression analyses. One-way analysis of variance or Kruskall-Wallis analysis was performed to detect any possible differences between parameter means. Statistical significance of differences between group means was assessed by the Student-Newman-Keuls test and reported as a letter code. Categorical data were examined after Pearson s 2 procedure. Determinants of PCDDs/PCDFs or dioxin-like PCBs (log-normalized) were traced by multiple regression analysis using, as independent variables, age, body mass index (BMI), tobacco consumption, number of children, and cumulative duration of breast-feeding. Stepwise multiple regression analysis used a P.25 for entry and.05 to stay in the model. P.05 was considered statistically significant (28). Estimated risk of disease expressed as an OR was calculated after identification of significant variables by backward selection (P.05 to stay in the model) in a logistic regression model. The model included PCDDs/PCDFs and dioxin-like PCBs as exposure variables, as well as age, BMI, age at menarche (codification 1 if 12 years vs. 0), cumulative duration of oral contraceptive use, familial history (cod. 1 if the mother had endometriosis vs. 0), and menstrual cycle deregulation (cod. 1 if cycle 27 days vs. 0). Variables were assumed to be normally distributed. The Hosmer and Lemeshow goodness-of-fit test was used to assess the model (29). Biologically relevant interactions were also checked (30). TABLE 1 Characteristics of the study population. Controls Peritoneal endometriosis Deep endometriotic nodules P n Age (y) a Current smokers (%) 9 (43) 7 (28) 6 (24).37 b Pack-years for smokers, median (range) 14.4 ( ) 6.7 (1.2 17) 4.3 ( ).11 c BMI (kg/m 2 ) a Age at menarche a Oral contraceptive use (years) a Menstrual cycle 27 days (%) 7 (33) 11 (44) 5 (20).19 b Family history d (%) 1 (5) 3 (12) 3 (12).65 b Children (%) 0 10 (48) 18 (72) 18 (72) 1 4 (19) 2 (8) 2 (8).40 b 2 7 (33) 5 (20) 5 (20) Breast-feeding e (range) 0.5 (0 16.0) 2.0 (0 10) 1.3 (0 9.5).67 c Note: Values are mean SD unless otherwise specified. a Analysis of variance. b 2 analysis. c Kruskall-Wallis. d Patient s mother had endometriosis. e Cumulative duration of breast-feeding in women with 1 child (months). Fertility and Sterility 307

4 TABLE 2 Nonstandardized dioxin-like PCBs and PCDDs/PCDFs in serum. Controls Peritoneal endometriosis Deep endometriotic nodules P n Lipids (mg/dl) 663 ( ) a 588 ( ) 600 ( ).043 Triglycerides (mg/dl) 115 (89 149) 125 ( ) 136 ( ).484 Dioxin-like PCBs (pg TEQ/g lipids) 8.5 ( ) 11.0 ( ) 12.4 ( ).026 PCDD PDCF (pg TEQ/g lipids) 15.5 ( ) 20.9 ( ) 26.0 ( ).001 a Geometric mean (Geometric SD). RESULTS The main characteristics of the studied population are described in Table 1. Age, age at menarche, cumulative duration of oral contraceptive use, smoking habits, breast-feeding, miscarriage rates, and BMI did not significantly differ among the groups. Both dioxin-like PCB and PCDD/PCDF serum concentrations were higher in women with deep endometriotic nodules than in controls (Table 2). Levels of both organochlorine compounds were also elevated in patients with peritoneal endometriosis. While total blood lipids were slightly higher in controls, no difference in triglyceride levels was found among the three groups, excluding variations in fasting status among the groups (Table 2). As a first step, we attempted to identify the determinants of serum dioxin-like PCB and PCDD/PCDF levels. Age, parity, breast-feeding, and BMI were all considered in a multiple regression analysis to take into account the lipophilic and cumulative properties of PCDDs/PCDFs and dioxin-like PCBs. For both groups of compounds, age was a significant determinant, contributing to increased serum concentrations, while an increased BMI had a slightly negative influence. Parity and breast-feeding were not significantly associated with PCDD/PCDF or dioxin-like PCB levels. After standardization for age (30 years) and BMI (22.5 kg/m 2 ), the three groups were still found to be significantly different for both dioxin-like PCB and PCDD/PCDF levels in serum (Fig. 1). Dioxin-like PCB levels in the serum of patients with deep endometriotic (adenomyotic) nodules were significantly increased compared with controls but did not significantly differ from those of patients with endometriosis (Fig. 1A). PCDD/PCDF levels in the serum of patients with both diseases were significantly increased compared with controls and differed between the two groups of patients (Fig. 1B). Similar differences were found when the analysis was conducted separately in women with or without children (not shown). The distribution patterns of both PCDDs/PCDFs and dioxin-like PCBs were similar among the three groups (Fig. 2). FIGURE 1 Dioxin-like polychlorobiphenyls (PCBs) (A) and polychlorodibenzodioxins (PCDDs)/polychlorodibenzofurans (PCDFs) (B) in serum, standardized for age (30 years) and body mass index (BMI) (22.5 kg/m 2 ). Bars represent means and standard deviations (SDs). Bars with the same letter are not significantly different (Student-Newman-Keuls test). 308 Heilier et al. PCDD/PCDF, endometriosis, and adenomyosis Vol. 84, No. 2, August 2005

5 FIGURE 2 Congener profiles for dioxin-like polychlorobiphenyls and dioxins (geometric means nonstandardized). As a second step, we assessed the association between PCDD/PCDF and/or dioxin-like PCB body burden and disease. Age, BMI, cumulative oral contraceptive use (31), age at menarche (32), menstrual characteristics (32), and family history (33) were considered as putative confounding factors in the studied relationships on the basis of literature data. Logistic regression analysis identified total TEQ (dioxin-like PCBs and PCDDs/PCDFs) as a risk factor, but none of the above variables was found to be a statistically significant confounder and none of the interaction terms tested (product of PCDD/PCDF or PCB concentration with, respectively, cumulative oral contraceptive use, age at menarche, menstrual characteristics, or family history) was retained in the model. An increase of 10 pg in total TEQ/g lipids in serum (PCDDs/PCDFs dioxin-like PCBs) was associated with a significantly increased risk (OR, 2.6; 95% CI,.3 5.3) of Fertility and Sterility 309

6 TABLE 3 Logistic regression models and odds ratio (OR) for the total of PCDDs/PCDFs and dioxin-like PCBs. P Wald 2 OR (95% CI) a P c Peritoneal endometriosis vs. controls b ( ).53 Deep endometriotic nodules vs. controls ( ).12 Both diseases vs. controls ( ).51 Note: Significant variable is of TEQ compounds. OR for 10 pg/g lipids increase. Level of statistical significance set at P.08. c P Hosmer-Lemeshow. either endometriosis or deep endometriotic nodules. When considering only deep endometriotic nodules, the risk was also significant (OR, 3.3; 95% CI, ). A marginally significant association was observed between total TEQ levels and the risk of endometriosis (OR, 1.9; 95% CI, ) (Table 3). When considering PCDDs/PCDFs and dioxin-like PCBs separately (Table 4), significant associations were observed between PCDDs/PCDFs and disease (alone or together), whereas for dioxin-like PCBs, only the relationship involving patients with deep endometriotic nodules was significant. DISCUSSION The role of endocrine disruptors and, more specifically, organochlorines in the onset or growth of endometriosis has been suspected on the basis of experimental data since the early 1990s (34). Several epidemiological studies have been conducted to examine the role of dioxin exposure in infertile women (17 19) or after accidental exposure like in Seveso (16), but the results have been inconclusive (35). The results presented here provide the first epidemiological evidence of a clear association between increased PCDD/PCDF and PCB body burden and the risk of endometriosis and/or deep endometriotic (adenomyotic) nodules. The main strength of the present study is that patients with endometriosis and deep endometriotic (adenomyotic) nodules were differentiated and controls were not recruited from an infertility clinic. Indeed, since we show here that PCDDs/ PCDFs and dioxin-like PCBs are significantly increased in patients with deep endometriotic (adenomyotic) nodules, failure to detect a clear association between endometriosis and PCDD/PCDF exposure in previous studies could have been due to the inclusion of patients with deep endometriotic (adenomyotic) nodules in the control group (i.e., infertile patients with no apparent endometriosis at laparoscopy). The controls included in the present study did not have deep endometriotic (adenomyotic) nodules but, because of the absence of laparoscopic examination, it cannot be formally ascertained that all these women were completely free of endometriosis. It is, however, reasonable to assume that in TABLE 4 Logistic regression models and odds ratio (OR) for PCDDs/PCDFs and dioxin-like PCBs. Significant variable(s) P Wald 2 OR (95% CI) a P b Dioxins ENDO vs. CTRL PCDDs/PCDFs ( ).55 DEN vs. CTRL PCDDs/PCDFs ( ).07 BD vs. CTRL PCDDs/PCDFs ( ).83 Dioxin-like PCBs ENDO vs. CTRL Dioxin-like PCBs DEN vs. CTRL Dioxin-like PCBs ( ).21 BD vs. CTRL Dioxin-like PCBs ( ).76 Note: BD both diseases; CTRL controls; DEN deep endometriotic nodules; ENDO peritoneal endometriosis. a OR for 10 pg/g lipids increase. b P Hosmer-Lemeshow. 310 Heilier et al. PCDD/PCDF, endometriosis, and adenomyosis Vol. 84, No. 2, August 2005

7 this group of asymptomatic women, certainly if vaginal echography and CA-125 levels were normal, the prevalence of active endometriosis must have been low (2). In any case, misclassification would only have led to underestimating the risk. Another strength of the present study is that we used gas chromatography hyphenated to high-resolution mass spectrometry for the determination of all relevant PCDD/PCDF and dioxin-like congeners. The levels measured in the present study are higher than those reported by Pauwels et al. with the CALUX assay in a Belgian population (endometriosis, 29 pg TEQ/g lipids, and controls, 27 pg TEQ/g lipids; vs. endometriosis, 33.4 pg TEQ/g lipids, and controls, 29.1 pg TEQ/g lipids in our study [median values]) (18). This may be explained by the fact that the CALUX assay has been reported to be prone to underestimation because of the presence of high levels of non-dioxin-like PCBs in blood samples (36). In addition, the GC-HRMS method allowed us to study the distribution of the different congeners. Since these patterns did not differ from group to group, the origin of the quantitative difference does not seem to stem from a specific cause or source. Since parity and breast-feeding have been reported to reduce dioxin body burdens in humans (12), it might be suspected that the association observed in the present study reflects a consequence of these gynecological diseases. Indeed, patients with endometriosis or deep endometriotic (adenomyotic) nodules included in the present study had lower parity and a shorter duration of breast-feeding than controls. This hypothesis can, however, be excluded for two reasons. First, neither parameter was found to significantly influence dioxin and PCB levels in the multiple regression analysis, probably because the study population was relatively young and did not include many multiparous women. Second, the differences in dioxin levels among the three groups were still observed when the analysis was conducted separately in women with or without children. The biological plausibility of a causal relationship between dioxins and related compounds and the risk of endometriosis is supported by experimental data (14, 15). Several putative mechanisms of action (activation of procarcinogens, alteration of synthesis or metabolism of estrogens, immunosuppression, etc.) that may explain the role of dioxins and related compounds in endometriosis and/or deep endometriotic (adenomyotic) nodules have been proposed (for a review, see Rier et al. [35]). Two hypotheses among those most frequently cited are of particular interest. First, it is well known that peritoneal endometriosis and deep endometriotic (adenomyotic) nodules are estrogen-dependent diseases (7) and are associated with higher local estrogen impregnation (37). Recently, Ohtake et al. reported that genes under the control of activated estrogen receptors could also be regulated by the dioxin-activated AhR because of some sharing of their signaling pathways (38). Second, it has been proven that both endometriosis and deep endometriotic (adenomyotic) nodule tissues express significant P450 aromatase transcript and protein levels (39). This enzyme catalyzes the transformation of endogenous androgens into estrogens and might contribute to the onset or growth of the diseases. Since the expression of some P450 cytochromes is regulated by the AhR activated by dioxins, this might be a relevant avenue for future research, although there is at present no conclusive evidence of a role of dioxins in the expression of P450 aromatase specifically (7). The fact that, in the present study, both peritoneal endometriosis and deep endometriotic (adenomyotic) nodules appear to be influenced by dioxins and PCBs might suggest that, despite their different histological origin, both diseases share a common etiological pathway. In conclusion, the present study is the first to demonstrate that PCDD/PCDF and dioxin-like PCB impregnation is associated with peritoneal endometriosis and deep endometriotic (adenomyotic) nodules and represents a risk factor for these diseases. This study was conducted in Belgium, a country where the prevalence of endometriosis, and especially deep endometriotic (adenomyotic) nodules, is high (2, 40), but it remains to be seen whether a similar association exists in other countries. Acknowledgments: The authors thank K. Rozenkranz, M.D., O. Donnez, M.D., F. Grandjean, M.D., L. De Buyl, M.D., A.-T. Ha, M.Sc., and R. Rousseau, M.Sc., for assistance with the data collection. REFERENCES 1. Sampson JA. Peritoneal endometriosis due to menstrual dissemination of endometrial tissue into the peritoneal cavity. Am J Obstet Gynecol 1927;14: Donnez J, Van Langendonckt A, Casanas-Roux F, Van Gossum JP, Pirard C, Jadoul P, et al. Current thinking on the pathogenesis of endometriosis. Gynecol Obstet Invest 2002;54: Donnez J, Nisolle M, Smoes P, Gillet N, Beguin S, Casanas-Roux F. Peritoneal endometriosis and endometriotic nodules of the rectovaginal septum are two different entities. Fertil Steril 1996;66: Nisolle M, Donnez J. Peritoneal endometriosis, ovarian endometriosis, and adenomyotic nodules of the rectovaginal septum are three different entities. Fertil Steril 1997;68: Donnez J, Donnez O, Squifflet J, Nisolle M. The concept of adenomyotic disease of the retroperitoneal space is born. Gynaecol Endosc 2001;10: Birnbaum LS, Cummings AM. Dioxins and endometriosis: a plausible hypothesis. Environ Health Perspect 2002;110: Kitawaki J, Kado N, Ishihara H, Koshiba H, Kitaoka Y, Honjo H. Endometriosis: the pathophysiology as an estrogen-dependent disease. J Ster Biochem Mol Biol 2002;83: Hahn ME. Evolutionary and physiological perspectives on Ah receptor function and dioxin toxicity. In: Schecter A, Gasiewicz TA, eds. Dioxins and health. 2nd ed. Hoboken, NJ: Wiley-Interscience, 2003: Mimura J, Fujii-Kuriyama Y. Functional role of AhR in the expression of toxic effects by TCDD. Biochim Biophys Acta 2003;1619: Agency for Toxic Substances and Disease Registry. Polychlorinated biphenyls. Update November van den Berg M, Birnbaum L, Bosveld AT, Brunstrom B, Cook P, Feeley M, et al. Toxic equivalency factors (TEFs) for PCBs, PCDDs, PCDFs for humans and wildlife. Environ Health Perspect 1998;106: Fertility and Sterility 311

8 12. Abraham K, Papke O, Gross A, Kordonouri O, Wiegand S, Wahn U, et al. Time course of PCDD/PCDF/PCB concentrations in breastfeeding mothers and their infants. Chemosphere 1998;37: Rier SE, Martin DC, Bowman RE, Dmowski WP, Becker JL. Endometriosis in Rhesus monkeys (Macaca mulatta) following chronic exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin. Fundam Appl Toxicol 1993;21: 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: Yang JZ, Agarwal SK, Foster WG. Subchronic exposure to 2,3,7,8- tetrachlorodibenzo-p-dioxin modulates the pathophysiology of endometriosis in the cynomolgus monkey. Toxicol Sci 2000;56: Eskenazi B, Mocarelli P, Warner M, Samuels S, Vercellini P, Olive D, et al. Serum dioxin concentrations and endometriosis: a cohort study in Seveso, Italy. Environ Health Perspect 2002;110: Mayani A, Barel S, Soback S, Almagor M. Dioxin concentrations in women with endometriosis. Hum Reprod 1997;12: Pauwels A, Schepens PJ, 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: Lebel G, Dodin S, Ayotte P, Marcoux S, Ferron LA, Dewailly E. Organochlorine exposure and the risk of endometriosis. Fertil Steril 1998;69: Heilier JF, Ha AT, Lison D, Donnez J, Tonglet R, Nackers F. Increased serum polychlorobiphenyl levels in Belgian women with adenomyotic nodules of the rectovaginal septum. Fertil Steril 2004;81: Squifflet J, Feger C, Donnez J. Diagnosis and imaging of adenomyotic disease of the retroperitoneal space. Gynecol Obstet Invest 2002;54: Donnez J, Nisolle M, Gillerot S, Smets M, Bassil S, Casanas-Roux F. Rectovaginal septum adenomyotic nodules: a series of 500 cases. Br J Obstet Gynaecol 1997;104: American Society for Reproductive Medicine. Revised American Society for Reproductive Medicine classification of endometriosis: Fertil Steril 1997;67: Focant JF, De Pauw E. Fast automated extraction and clean-up of biological fluids for polychlorinated dibenzo-p-dioxins, dibenzofurans and coplanar polychlorinated biphenyls analysis. J Chromatogr B 2002; 776: Akins JR, Waldrep K, Bernert JT Jr. The estimation of total serum lipids by a completely enzymic summation method. Clin Chim Acta 1989;184: Brown JF Jr, Lawton RW. Polychlorinated biphenyl (PCB) partitioning between adipose tissue and serum. Bull Environ Contam Toxicol 1984; 33: Patterson DG Jr, Turner WE, Isaacs SG, Alexander LR. A method performance evaluation and lessons learned after analyzing more than 5,000 human adipose tissue, serum, and breast milk samples for polychlorinated dibenzo-p-dioxins (PCDDs) and dibenzofurans (PCDFs). Chemosphere 1990;20: Kleinbaum DG, Kupper LL, Muller KE, Nizam A, eds. Applied regression analysis and other multivariate methods. 3rd ed. Pacific Grove, CA: Duxburry Press, Hosmer DW, Lemeshow S, eds. Applied logistic regression. New York: Wiley, Kleinbaum DG, Klein M, eds. Logistic regression: a self-learning text. 2nd ed. New York: Springer, Mahmood TA, Templeton A. Prevalence and genesis of endometriosis. Hum Reprod 1991;6: Cramer DW, Wilson E, Stillman RJ, Berger MJ, Belisle S, Schiff I, et al. The relation of endometriosis to menstrual characteristics, smoking, and exercise. JAMA 1986;255: Moen MH, Magnus P. The familial risk of endometriosis. Acta Obstet Gynecol Scand 1993;72: Gerhard I, Runnebaum B. [The limits of hormone substitution in pollutant exposure and fertility disorders]. Zentralbl Gynakol 1992;114: Rier S, Foster WG. Environmental dioxins and endometriosis. Toxicol Sci 2002;70: Brown DJ, Chu M, Van Overmeire I, Chu A, Clark GC. Determination of REP values for the CALUX bioassay and comparison to the WHO TEF values. Organohalogen Compounds 2001;53: Takahashi K, Nagata H, Kitao M. Clinical usefulness of determination of estradiol level in the menstrual blood for patients with endometriosis. Nippon Sanka Fujinka Gakkai Zasshi 1989;41: Ohtake F, Takeyama K, Matsumoto T, Kitagawa H, Yamamoto Y, Nohara K, et al. Modulation of oestrogen receptor signalling by association with the activated dioxin receptor. Nature 2003;423: Kitawaki J, Noguchi T, Amatsu T, Maeda K, Tsukamoto K, Yamamoto T, et al. Expression of aromatase cytochrome P450 protein and messenger ribonucleic acid in human endometriotic and adenomyotic tissues but not in normal endometrium. Biol Reprod 1997;57: Koninckx PR, Braet P, Kennedy SH, Barlow DH. Dioxin pollution and endometriosis in Belgium. Hum Reprod 1994;9: Heilier et al. PCDD/PCDF, endometriosis, and adenomyosis Vol. 84, No. 2, August 2005