Increased exposure to dioxin-like compounds is associated with endometriosis in a case control study in women

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1 Reproductive BioMedicine Online (2010) 20, ARTICLE Increased exposure to dioxin-like compounds is associated with endometriosis in a case control study in women Peter Simsa a,b, Attila Mihalyi a, Greet Schoeters c, Gudrun Koppen c, Cleophas M Kyama a,d, Elly M Den Hond c, Vilmos Fülöp b, Thomas M D Hooghe a,d, * a Leuven University Fertility Center, Department of Obstetrics and Gynaecology, University Hospital Gasthuisberg, Leuven B-3000, Belgium; b Department of Obstetrics and Gynaecology, National Institute of Health, Budapest 1139, Hungary; c Flemish Institute of Technological Research (VITO), Environmental Toxicology, Mol, Belgium; d Division of Reproduction, Institute of Primate Research, PO Box 24481, Karen, Nairobi, Kenya * Corresponding author. address: thomas.dhooghe@uz.kuleuven.ac.be (TM D Hooghe). Peter Simsa graduated in 1998 and then did research in the area of breast cancer and B cell infiltration at Semmelweis University, Hungary. From 2004 to 2007, he worked as an international research scholar on toxicological aspects of endometriosis under supervision of Professor Dr Thomas D Hooghe (Leuven University, Belgium). He is currently finalizing his PhD thesis in Hungary and studying immunological aspects of recurrent spontaneous abortion. Abstract Although endometriosis is thought to be an environmental disorder initiated by dioxin exposure, this association is controversial. This study was performed to test the hypothesis that endometriosis occurs more often in women exposed to higher concentrations of dioxin-like compounds (DLC) than in those women exposed to lower concentrations. Plasma samples collected prior to laparoscopic surgery from 96 women with endometriosis and 106 control patients with a normal pelvis were measured for DLC concentrations using the dioxin-responsive chemical-activated luciferase expression bioassay. The results showed that concentration (mean ± SD) of DLC was marginally higher in patients with endometriosis (22.3 ± 9.3 pg CALUX-TEQ/g lipid) than in controls (20.5 ± 10.8 pg). After categorization of patients in a group with low plasma concentrations (<25th centile) and a group with high plasma concentrations (>75th centile) of DLC, the age-adjusted odds ratio to have endometriosis was 2.44 (95% CI ; P = 0.04) for women with high concentrations of DLC and it increased to 3.01 (95% CI ; P = 0.03) when only women with moderate severe endometriosis were considered. In conclusion, women exposed to higher plasma concentrations of DLC were at higher risk of having endometriosis than women exposed to lower concentrations of DLC within normal environmental concentrations. RBMOnline ª 2010, Reproductive Health Care Ltd. Published by Elsevier Ltd. All rights reserved. KEYWORDS: case control, dioxin-like compounds, DR-CALUX bioassay, endometriosis, environment /$ - see front matter ª 2010, Reproductive Health Care Ltd. Published by Elsevier Ltd. All rights reserved. doi: /j.rbmo

2 682 P Simsa et al. Introduction Endometriosis is a common gynaecological disease with a prevalence of 7 10% among women of reproductive age (Wheeler, 1989, 1992) and is frequently associated with pelvic pain (Vessey et al., 1993) and subfertility (Carter, 1994). The most accepted hypothesis on the pathogenesis of endometriosis is the retrograde menstruation theory (Sampson, 1927). This theory states that menstrual endometrial tissue regurgitates into the peritoneum where viable endometrial cells attach to the pelvic peritoneum and invade the underlying tissue (D Hooghe et al., 2006). Although retrograde menstruation occurs in 70 90% of women of reproductive age, not all develop endometriosis (Halme et al., 1984). Therefore, other factors must play a role in the pathogenesis of endometriosis, like genetic background, malfunctioning inflammatory/immunological mechanisms and potentially environmental factors (Bischoff and Simpson, 2004; Mihalyi et al., 2005). The main group of environmental pollutants that have been proposed to play a role in the pathogenesis of endometriosis include polyhalogenated aromatic hydrocarbons, a class of widespread environmental contaminants consisting of polychlorinated dibenzo-p-dioxins (PCDD), dibenzofurans and 12 polychlorinated biphenyls (PCB) (Schecter et al., 2006). Dioxins and chemically related compounds appear in the environment as a result of human combustion activities. They are involved in the pathogenesis of cancer, developmental malformations and immunosuppression in animal models (Bock and Kohle, 2006; Schecter et al., 2006). Dioxin (2,3,7,8 tetrachlorodibenzo-p-dioxin; TCDD) and the chemically related PCB have been ranked as the 72nd and fifth most hazardous chemicals, respectively, on the Priority List of Hazardous Substances of the Agency for Toxic Substances and Disease Registry (2008). Their high toxicity is linked to their potency to activate the aryl hydrocarbon receptor. Dioxins have also been postulated to stimulate the development of endometriosis via their immunosuppressive effects and their interference with the oestrogen signalling pathway. The immunosuppressive effect of high doses of dioxins is well documented (Oh et al., 2005). Firstly, dioxin exposure may lead to inhibition of leukocyte phagocytic function, which is possibly important in the prevention of endometriosis by the elimination of menstrual debris (Levin et al., 2005). Additionally, dioxins can decrease immunological memory (Lawrence and Vorderstrasse, 2004), induce apoptosis in both T cells (Ahmed et al., 2005) and B cells (Puebla-Osorio et al., 2004), inhibit T-lymphocyte function and decrease natural killer cell activity in plasma and peritoneal fluid (Neubert et al., 1991). Furthermore, dioxin may stimulate the activity of peritoneal fluid macrophages and their local production of pro-angiogenic factors, cytokines (e.g. interleukin-1) and growth factors (Clark et al., 1991; Koninckx, 1999). The combined effect of immune dysfunction and peritoneal inflammation could favour the development of endometriosis. Furthermore, cellular changes or genetic predisposition may predestine an individual to the immunological modulation caused by dioxin exposure (Pauwels et al., 2001; Rier and Foster, 2002). Local oestrogen production can be increased following dioxin exposure and facilitate development of endometriotic lesions by elevating mrna expression of aromatase, the key catalytic enzyme in oestrogen synthesis (Attar and Bulun, 2006). Dioxins and PCB are known to interfere with oestrogen concentrations. Both agonistic and antagonistic effects have been ascribed to dioxins and PCB by direct interference with the oestrogen receptor or by the interaction between the activated aryl hydrocarbon receptor (AHR)/aryl hydrocarbon receptor nuclear translocator heterodimer (Mimura and Fujii-Kuriyama, 2003) and the oestrogen receptor a and b, leading to oestrogen-dependent gene activation (Ohtake et al., 2003). A connection between endometriosis and TCDD was firstly observed in rhesus monkeys, but this initial study has been debated ever since (Guo, 2004; Rier et al., 1993). It is still not clear if TCDD play a role in the pathogenesis of endometriosis in women (Foster, 2008; Heilier et al., 2008). The results available in the present literature are remarkably conflicting with regard to their power, design, methodology and interpretation (Birnbaum and Cummings, 2002; Boyd et al., 1995; Eskenazi et al., 2002; Fierens et al., 2003; Gerhard and Runnebaum, 1992; Guo, 2004; Lebel et al., 1998; Mayani et al., 1997; Pauwels et al., 2001; Rier and Foster, 2002). Indeed, dioxin concentrations were similar in women with and without endometriosis in most studies (Boyd et al., 1995; Fierens et al., 2003; Lebel et al., 1998; Pauwels et al., 2001) except in one study with a low number of patients (79 patients including 44 cases and 35 controls) (Mayani et al., 1997). In another study, the concentration of organochlorine pollutants was found to be higher in women with endometriosis than in controls (Gerhard and Runnebaum, 1992). In two studies, the odds ratio (OR) to have endometriosis was non-significantly elevated to 4.60 (95% CI ) (Pauwels et al., 2001) or to 2.10 (95% CI ) (Eskenazi et al., 2002) in women with a plasma concentration higher than 100 pg CALUX- TEQ/g lipid when compared with women with a lower concentration of dioxin-like compounds (DLC) in plasma lipids. The current study was designed to test the hypothesis that endometriosis is found more often in women with exposure to higher concentrations of DLC than in women exposed to lower concentrations of DLC. Materials and methods Patient recruitment and power calculation In this case control study, the concentration of DLC was analysed in plasma lipid samples from patients who had undergone laparoscopic surgery because of infertility with/without pain between 2001 and The plasma samples were stored in the tissue bank of the Leuven University Fertility Centre, Belgium. All patients had signed an informed consent agreeing that biological samples would be collected for scientific purposes during laparoscopy. This consent form had been approved by the Institutional Review Board (IRB) of University Hospital Gasthuisberg. Power calculation in a previous study had demonstrated that 85 cases and 85 controls were required to detect with a power of 90% and a significance level of 0.05 a significant four-fold risk (OR = 4.00) for endometriosis in women with higher exposure to DLC (>100 pg TEQ (toxic equivalents)/g plasma

3 Endometriosis and increased exposure to dioxin-like compounds 683 lipids) when compared with women with lower exposure to DLC. (Pauwels et al., 2001) For economic reasons (cost of CALUX assay), this study limited the sample size to 200 patients. For statistical reasons, an equal number (n = 24) of cases were selected for each of the four stages of endometriosis, according to the revised American Fertility Society classification (rafs; AFS, 1985). Patients with a laparoscopically normal pelvis without evidence of endometriosis (n = 106) served as controls, whereas patients with laparoscopically and histologically proven endometriosis (n = 96) were selected as cases. The latter included women with minimal (rafs sti, n = 24), mild (rafs stii, n = 24), moderate (rafs stiii, n = 24) and severe endometriosis (rafs stiv, n = 24), according to the rafs in ASRM (1997). Among the cases, 24 patients had only deep infiltrating endometriosis (two sti, five stii, nine stiii and eight stiv) and 27 had clear peritoneal endometriosis (five sti, 15 stii, five stiii and three stiv). Sample collection Blood samples were collected before laparoscopic operation into vacutainers containing potassium-edta and were processed within a maximum of 1 hour. Blood samples were centrifuged for 10 min at 1000 g at 4 C, aliquoted and labelled. The samples were kept at 80 C until analysis. Dioxin-responsive chemical-activated luciferase expression bioassay For the dioxin-responsive chemical-activated luciferase expression assay (DR-CALUX), exposure to DLC was assessed via the in-vitro activation of AHR of cultured H4IIE rat hepatoma cells by the DLC present in 5 ml of plasma (CALUX assay; BioDetection Systems BV, Amsterdam, The Netherlands). This method involves n-hexane extraction, followed by lipid destruction and removal of acid labile matrix components and non-stable polyhalogenated aromatic hydrocarbons by passage through a silica column containing concentrated H 2 SO 4 (33%, w/v). The extract is then quantitatively transferred to a conical vial for evaporation until almost dry, followed by adding 7.5 ll dimethylsulphoxide (Acros Organics, Geel, Belgium) and dilution to a total volume of 750 ll with minimal essential medium (Gibco, Merelbeke, Belgium). In the CALUX assay, rat hepatoma H4IIE cells, transfected with an AHR-controlled luciferase reporter gene construct, are grown in 96-well plates in minimal essential medium with 10% fetal calf serum (Gibco) at 37 C and 5% CO 2. When the cell layer reaches 70 80% confluency, the cells are dosed with 100 ll sample extract in triplicate together with TCDD standards and then incubated at 37 C for 24 h. After removal of the medium, cells are washed with phosphate-buffered saline without Ca 2+ /Mg 2+ (Gibco) and 30 ll of a cell-lysis reagent (Promega, Leiden, The Netherlands) is added. The culture plates are shaken for 45 min and stored at 80 C for 1 h at least. The luciferase activity is determined after the cells in the culture plates are thawed on ice and 100 ll luciferin assay mix (Promega) is added at room temperature. The light production is measured by a Victor 2 Luminometer (EG and G Wallac, Oosterhout, The Netherlands). On each 96-well plate, standards of TCDD from 0.1 to 32.2 ng/l are added to construct a TCDD-based calibration curve which is then used to quantify the corresponding CALUX-TEQ content of the individual plasma samples measured. Results are expressed in pg CALUX-TEQ/g plasma lipid. The limit of detection was calculated as the light signal measured from the dimethylsulphoxide control plus three times its standard deviation on each culture plate, viz ngteq/l plasma lipid. Statistical analysis Statistical analysis was performed using Statistical Package for Social Sciences version 14 (SPSS Inc, Hungary) and Statistica version 7 (StatSoft Inc, USA). Since the measured DLC concentrations were not normally distributed (Kolmogorov Smirnov test), the Mann Whitney U-test was used to compare cases with controls. A Kruskal Wallis test was applied to reveal differences in exposure level between controls and women with different stages of endometriosis. The contribution of age to the concentration of DLC in plasma was calculated by linear regression analysis. After logarithmic transformation, the data were analysed using analysis of variance (ANOVA) and analysis of covariance (ANCOVA) tests when age was included as independent variable. Subsequently, the age adjusted OR and 95% CI to have endometriosis was calculated in women with higher dioxin exposure when compared with women with lower dioxin exposure. Therefore, using logistic regression, women with concentrations above the 75th centile of the study population were compared with those below the 25th centile. Results All relevant results are summarized in Tables 1 and 2 and the distribution of the concentrations of DLC in plasma lipids is shown in Figure 1. One woman in the control group showed a zero value and was excluded from analysis as a possible technical failure. The mean age was 31.5 (range 20 46) years and similar for control patients (n = 105) and for women with endometriosis (n = 96) (Table 1). In the total patient population, concentrations of DLC varied between 5.4 and 61.2 pg CALUX-TEQ/g lipids. No association was found between age and concentration of DLC (R 2 < ). The median concentration of DLC was borderline non-significantly higher in women with endometriosis (21.2 pg CALUX-TEQ/g lipid) than in controls (19.4 pg CALUX-TEQ/g lipid). Assuming a normal distribution of the CALUX-TEQ concentrations, this study was sufficiently powered (beta = 80%, alpha = 0.05) to detect a difference in concentration of 4 pg CALUX-TEQ/g lipid between cases and controls. Using a Kruskal Wallis test, the median concentration of DLC was also comparable between controls and women with endometriosis stage I, II, III or IV, or between controls, women with combined endometriosis stage I and II and women with combined endometriosis stage III and IV. Using a Mann Whitney U-test, the median concentration of DLC was higher in women with endometriosis stage III and IV (P = 0.048) or in women with endometriosis stage IV only (P = 0.04) than in controls. Using a Mann Whitney U-test, the median concentration of DLC was similar in women with

4 684 P Simsa et al. Table 1 Serum concentration of dioxin-like compounds in controls and in women with endometriosis, classified according to the staging system of the American Society of Reproductive Medicine (AFS et al., 1985). Total Controls Endometriosis All stages (I IV) Stage I+II Stage III + IV Stage I Stage II Stage III Stage IV Number Age (years) 31.5 (20 46) 32.1 (20 46) 30.9 (24 42) 30.9 (24 41) 30.9 (26 42) 30.7 (25 38) 31.2 (24 41) 31.6 (27 42) 30.2 (26 39) Dioxin (pg CALUX-TEQ/g 20.3 ( ) 19.4 ( ) 21.2 ( ) 19.3 ( ) 22.1 ( ) 18.5 ( ) 22 ( ) 21.4 ( ) 22.1 ( ) lipid) ANOVA test NS b NS c NS d (P-value) ANCOVA test (P-value) a NS b NS c NS d Values are median (range). NS = not statistically significant. a Age adjusted. b Comparison between controls and all women with endometriosis. c Comparison between controls, women with endometriosis stage I + II and women with endometriosis stages III + IV. d Comparison between controls, women with endometriosis stage I, women with endometriosis stage II, women with endometriosis stage III and women with endometriosis stage IV. Table 2 Age-adjusted OR to have endometriosis in women with higher exposure (>75 th centile) versus women with lower exposure (<25 th centile). Endometriosis stage Number of women with DLC measurement (pg CALUX-TEQ/g lipid) <25th centile >75th centile OR (95% CI) Control All stages (I IV) ( ) 0.04 Stage I + II ( ) NS Stage III + IV ( ) 0.03 Stage I ( ) NS Stage II ( ) NS Stage III ( ) NS Stage IV ( ) 0.04 a DLC = dioxin-like compounds; kns = not statistically significant. a Numbers are too low in the <25th centile for meaningful statistical comparison. P-value peritoneal endometriosis only (N = 27; median 23.0, range pg CALUX-TEQ/g lipid) and in women with deep infiltrating endometriosis only (N = 24; median 21.4, range pg CALUX-TEQ/g lipid) (Table 2). Statistical analysis of the log-transformed TEQ data with ANOVA and ANCOVA including age as covariable revealed no difference between the groups (Table 1), although the P- value for ANCOVA approached statistical significance. Since the maximal concentration of DLC in patients was lower than 100 pg TEQ/g lipid, it was impossible to determine the OR for endometriosis in women with exposure above this concentration compared with women with lower exposure, as planned in the study design. Alternatively, the patient population was dichotomized defining lower and higher concentrations based on the 25th and 75th centiles of the whole group. The 25th and 75th centiles were, respectively, 14 and 25 pg CALUX-TEQ/g lipid. Using this categorisation, the OR for endometriosis was 2.44 (95% CI ; P= 0.04) for women with higher compared with lower concentrations of DL-compounds versus controls. The OR to have combined moderate-severe endometriosis was 3.01 (95% CI ; P = 0.03) for women with higher concentrations of DL-compounds compared with women with lower concentrations (Table 2). The OR to have deep infiltrating endometriosis only or to have peritoneal endometriosis only was respectively 3.55 (95% CI )

5 Endometriosis and increased exposure to dioxin-like compounds 685 Figure 1 Dot plot illustrating median concentrations of dioxin-like compounds in controls and in women with endometriosis stages I IV. White box indicates median; bars indicate cent quartiles. or 2.93 (95% CI for women with higher compared with lower concentrations. Discussion The results indicate, for the first time in a statistically significant way, that women with higher exposure to DLC (i.e. concentration in plasma lipids above the 75th centile of the study population 25 pg CALUX-TEQ/g lipid) are 2.5 times more likely to have endometriosis and five times more likely to have severe endometriosis than women with lower concentrations of DL-compounds (concentration below the 25th centile of the study population 14 pg CALUX-TEQ/g lipid). Crude OR were similar to age-adjusted OR indicating that age had no correlation with dioxin contamination in women, which can possibly be explained by the narrow age range (20 46 years) of the study participants. These findings are in line with the results from the Italian Seveso study (Heilier et al., 2005) reporting a trend towards a positive association between DL exposure and endometriosis: the relative risk ratios to have endometriosis for women with serum TCDD concentrations of ppt and >100 ppt were 1.20 (90% CI ) and 2.10 (90% CI ), respectively, when compared with women with TCDD concentrations below or equal to 20 ppt. This association was statistically significant in this study but not in the Seveso study, possibly because endometriosis was not laparoscopically excluded in the control group (Heilier et al., 2005). This study s results are in disagreement with a Japanese study showing a negative association between DLC concentrations and endometriosis, but this study can be criticized since the control group included both women with a normal pelvis and women with minimal endometriosis (Tsukino et al., 2005). The data from the present study also did not confirm that DL exposure is associated with an increased risk for deeply infiltrative endometriosis, as reported in another study describing that an increase of serum DLC with 10 pg/g lipids was associated with an increased risk (OR = 3.31, 95% CI ) for deeply infiltrative endometriotic (adenomyotic) nodules, but not for peritoneal endometriosis (Heilier et al., 2005). A first strength of the present study is the large number of patients (n = 202) studied. All previous case control studies included a lower number (range ) of subjects (Boyd et al., 1995; De Felip et al., 2004; Heilier et al., 2005; Lebel et al., 1998; Mayani et al., 1997; Pauwels et al., 2001; Tsukino et al., 2005). A second strength is that, as in the pilot study, the patient/control population was very well defined: both cases and controls were infertile women undergoing a laparoscopic investigation with definitive diagnosis regarding the presence (with histological confirmation) or absence of endometriosis (Pauwels et al., 2001). Controls were also well defined (laparoscopic absence of endometriosis) in only two other publications, evaluating a much lower number of subjects (De Felip et al., 2004; Mayani et al., 1997). However, in all other case control or population-based studies, the control group did not systematically receive a laparoscopy to rule out endometriosis (Boyd et al., 1995; Eskenazi et al., 2002; Gerhard and Runnebaum, 1992; Heilier et al., 2005; Lebel et al., 1998) or included both women with a normal pelvis and women with minimal endometriosis (Tsukino et al., 2005). Furthermore, in some studies, not all cases with endometriosis received a laparoscopy to confirm the presence of the disease (Eskenazi et al., 2002). In the present study, the median DLC concentration was higher in the subset of women with moderate to severe endometriosis (rafs) than in controls, suggesting also that dioxin exposure may be positively related with the severity of endometriosis, as had been observed previously in rhesus monkeys (Rier et al., 1993). In other studies, plasma DLC have been reported to be higher (Heilier et al., 2005; Mayani et al., 1997), lower (Tsukino et al., 2005) or similar (Boyd et al., 1995; De Felip et al., 2004; Fierens et al., 2003; Lebel et al., 1998; Pauwels et al., 2001), in women with endometriosis when compared with controls. All of these studies were marked by important methodological limitations with respect to sample size and definition of control groups, as mentioned above. The current study had been designed based on the pilot study, in which women with DLC concentrations above 100 pg CALUX-TEQ/g plasma lipids were 4.6 times as likely to have endometriosis than women with a lower concentration of DLC, but this difference was not significant (OR = 4.60, 95% CI ), possibly due to the lack of power (Pauwels et al., 2001). The current study was not able to measure concentrations of 100 pg CALUX-TEQ/g lipid or higher. Indeed, the median concentration of DLC in the current study was significantly lower both in endometriosis cases (median 21.2, range pg CALUX-TEQ/g lipid) and in controls (median 19.4, range pg CALUX-TEQ/g lipid) than the median concentration in the previous study (cases: median 29, range pg CALUX-TEQ/g lipid; controls: median 27, range pg CALUX-TEQ/g lipid, respectively). This evolution is part of a worldwide and Belgian trend showing decreasing dioxin/ furan concentrations in the environment and in human blood or breast milk (Hays and Aylward, 2003). In Belgium, the plasma concentration of dioxins/furans (PCDD/F) has been reported to decrease from 38.1 to 36.4 pg TEQ/g plasma lipids in an unselected group of donor participants between 1998 and 2000 (Van Wouwe et al., 2004). Also in Belgium, the breast milk concentration of PCDD/Fs dropped between 1988 and 2006 (pg TEQ/g lipid: 37 in 1988, over 25

6 686 P Simsa et al. in 1991, 17 in 2002, in 2006) (Colles et al., 2008; Liem et al., 1996; Van Cleuvenbergen et al., 1994; Van Leeuwen and Malisch, 2002). Since the original hypothesis could not be tested, another validated method was used to analyse the data. In studies evaluating the effects of environmental agents on human health, the comparison of subjects with exposure concentrations below the 25th centile with subjects having exposure concentrations above the 75th centile of the study population is among other categorisation methods a quite commonly used approach. Indeed, 75th to 25th centile OR have been calculated to assess the risk of peripheral arterial disease or myocardial infarction in relation to internal metal concentrations (Kardinaal et al., 1997; Navas-Acien et al., 2005), to assess growth outcomes of children in relation to maternal exposure to organochlorines (Jusko et al., 2006) or to study insulin resistance in case of exposure to persistent pollutants (Lee et al., 2007). In the present study, using logistic regression, women with DLC concentrations above the 75th centile of the study population were compared with those below the 25th centile in order to calculate the age-adjusted OR. A limitation of this study is that it only took into account patient age as a confounding variable when calculating the age-adjusted OR to have endometriosis in women with higher exposure (>75th centile) versus women with lower exposure (<25th centile), as shown in Table 2. Statistical analysis of the log-transformed TEQ data with ANOVA and ANCOVA including age as covariable revealed no difference between the groups. The study did not include other potentially important confounders like body mass index, history of cigarette smoking, age at menarche, reproductive history, duration of disease or infertility and infertility diagnosis. It was not possible to include these data since blood samples were obtained from the biobank, which included full clinical information related to the stage of endometriosis but only partial information regarding other variables mentioned above. However, it is also not clear to what extent each of these variables are associated with a significant risk for the development of endometriosis and/or for increased exposure to dioxin. Relevant clinical data are now being collected prospectively linked to tissues stored in the biobank and will be useful for future research. Also, the present study could not compare the relationship between DLC exposure and the type of endometriotic lesion since not all endometriotic lesions were individually listed or described in surgical reports and most women had peritoneal lesions with a variety of phenotypes (black-puckered, white, red). Furthermore, numbers become far too limited for an additional analysis on this level, since the study had not been powered to test this hypothesis. Obviously, it is possible that the increase in OR is because the mix of lesions in this study s subjects is different from that in patients in other studies. However, the hypothesis that DLC exposure is more strongly related to deeply infiltrating or peritoneal endometriosis is not supported by the observation that the OR to have deep infiltrating endometriosis only or to have peritoneal endometriosis only were not significantly higher for women with higher dioxin concentrations when compared with women with lower dioxin concentrations. In this study, it was not possible to assess the relationship between exposure to DLC and pain perception, since the samples were collected and analysed retrospectively. The data of this study do not rule out the possibility of reverse causation. Suffering with a chronic illness like endometriosis can cause people to seek not only more medical attention and receive more medication, but also prompt them to do other things, such as eat different diets that they think may help their condition and avoid exercise, and all of these lifestyle changes could contribute to differences in residue concentrations for target contaminants like dioxins. However, there is no reason to anticipate that women in the control population behaved differently in terms of diet or exercise to account for lower exposure concentrations than women with endometriosis. In contrast, clinical impressions are that women with endometriosis are generally more highly focused on healthy diet and lifestyle than the general population. As far as is known, this and the previous study are the only ones using the CALUX assay to determine exposure to DLC (dioxins and coplanar PCB) in women with endometriosis (Pauwels et al., 2001). In other studies, chemical methods based on gas chromatography mass spectrometry (GC-MS) technologies were used to measure dioxins in plasma Boyd et al., 1995; De Felip et al., 2004; Eskenazi et al., 2002; Gerhard and Runnebaum, 1992; Heilier et al., 2005; Lebel et al., 1998; Mayani et al., 1997; Tsukino et al., 2005). Although a good correlation has been demonstrated between CALUX-TEQ concentrations in plasma and chemically determined PCDD/F TEQ concentrations, other chemicals could also have contributed to the effects observed (Van Wouwe et al., 2004). Indeed, in the CALUX bioassay, direct activation of the aryl hydrocarbon receptor by a mixture of persistent compounds present in plasma lipid extracts is measured, which represents a more direct estimate of DLC toxic activity than can be assessed during chemical analysis of dioxins and furans. In fact, the hypothesis was that the risk for endometriosis is positively associated with the presence of compounds that activate the aryl hydrocarbon receptor. This activation is typical for dioxins, furans and DLC PCB, but it cannot be excluded that other compounds present in the plasma lipid extract also bind to the aryl hydrocarbon receptor. In conclusion, these results show for the first time with adequate statistical strength that, at current environmental exposure, women with higher concentrations of dioxinlike compounds are at a higher risk of having endometriosis than women with lower exposure. Acknowledgements This study was financially supported by the Endometriosis Association, the FWO (Fonds voor Wetenschappelijk Onderzoek/Foundation for Scientific Research, Vlaanderen) and the Onderzoeksraad KU Leuven (Leuven University Research Foundation). References AFS Revised American Fertility Society classification of endometriosis, Fertil. Steril. 43, Agency for Toxic Substances and Disease Registry ATSDR, Comprehensive Environmental Response, Compensation, and Liability

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