Xenoestrogen concentration in women with endometriosis or leiomyomas: A case-control study

Endocrine-disrupting chemicals (EDCs) are exogenous substances that alter the functions of the endocrine system, resulting in adverse health effects in an intact organism, its progeny or subpopulations [ 1 ]. Xenoestrogens, including phytoestrogens, phthalates, polychlorinated byphenils, isoflavonoids, parabens, bisphenol A, and other exogenous estrogens, can act as EDCs. Environmental exposure to EDCs is widespread [ 1 ]. Due to their lipophilic characteristics and slow metabolism and detoxification, they accumulate in adipose tissue, where they are released into the bloodstream [ 1 ]. The pathogenic mechanisms of xenobiotics are multiple and complex; they can affect the synthesis, release, or transport of natural hormones or modulate the response of target tissues. They can also exert stimulant or inhibitory effects depending on their concentration. Interaction with other EDCs can result in agonistic or antagonistic effects, depending on the composition of the mixture and the concentration of each substance [ 2 ]. Although EDCs can affect the endocrine system at any stage of life, embryologic or fetal stages are critical because they can cause epigenetic modifications that lead to morbidity in adult life [ 2 ]. Epidemiological studies and experimental models have reported a potential link between xenoestrogens and hormone-dependent pathologies, such as leiomyomas or endometriosis [ 3 – 12 ]. However, a direct causal relationship between these entities has yet to be established. Most publications have focused on individual or small groups of EDCs and their effects on health outcomes [ 3 – 5 ], failing to consider the agonistic, antagonistic, or synergistic interactions between multiple xenobiotics that can modify the final estrogenic effects of a mixture [ 2 ]. Furthermore, the potential association between individual xenoestrogens and certain diseases should be assessed by considering their interactions with other EDCs [ 13 ]. In accordance with this idea, the assessment of the total concentration of xenoestrogens has emerged as a reliable biomarker for the cumulative effect of xenoestrogen mixtures on organisms [ 1 ]. Additionally, most publications have reported the use of blood, urine, or even subcutaneous fatty tissue to quantify xenoestrogens [ 6 – 8 ]. Adipose tissue has been regarded as reliable for evaluating the long-term bioaccumulation of xenoestrogens [ 2 ]. However, visceral fat has shown a higher deleterious metabolic effect than subcutaneous fat [ 14 ] and seems to be a more appropriate tissue for this purpose. The XENOBEM (XENOBiotics, Endometriosis and Myomas) study aimed to determine whether there is an association between xenoestrogens and the presence of leiomyomas or endometriosis, and to assess the xenoestrogen concentration in omental fat. We conducted a case-control pilot study to compare women who underwent surgery and had leiomyomas or endometriosis with women who went through surgery for other benign indications.

Out of the 240 omental fat samples initially collected, 11 were not analyzed because of anatomopathological findings other than fibroids, endometriosis, or incorrect labelling or preservation. Of the remaining 229 samples, 117 corresponded to the case group: 74 patients with leiomyomas, 21 with endometriosis, and 22 with both leiomyomas and endometriosis. Of these 117 patients, eight (6.8%) were pregnant women with miomas/endometriosis undergoing cesarean section. Within the control group, we excluded eight patients due to a past medical history of one of these diseases, resulting in a total of 104 patients ( Fig 1 ). In the control group, 93 (86.5%) of the 104 patients were pregnant women who underwent cesarean sections for obstetric indications. Table 1 presents the main characteristics of the groups. A family history of fibroids was more common in the case group than in the control group (49 vs. 24 cases, p = 0,006). Both groups had a higher percentage of patients living in the Madrid Community: 85 women (83.3%) in the control group and 78 in the case group (67.2%) (p = 0.006). In addition, 87 women in the control group (85.3%) and 79 women among the cases (68.1%) had workplaces within the Madrid Community (p = 0.003). Regarding the clinical features of the case group, we should point out that a great number of patients had multiple fibroids and in different uterine locations. In addition, the majority of patients with endometriosis had a combination of ovarian and deep endometriosis. The main indications for surgery in patients with leiomyomas were abnormal uterine bleeding, and pelvic pain for women with endometriosis, both unresponsive to medical treatment. The detection frequency of TEXB-α for the samples was 68.8%, with no difference between the groups: 76 of the 104 control women (73.1%) and 76 of the 117 cases (65.0%) had xenoestrogen levels over the LD of 0.1 Eeq pM/g (p = 0.194). There were also no differences in xenoestrogen detection frequency among the different subgroups: 49 of 74 patients with leiomyomas (66.2%), 13 of 21 women with endometriosis (61.9%), and 14 of 22 patients with both pathologies (63.6%) had detectable xenoestrogen levels (p = 0.603). Considering only those patients with xenoestrogen concentrations over the LD, the median level for controls was 4.91 [5.81], vs 4.62 [4.34] Eeq pM/g in cases (p = 0.078). We observed a significant dispersion of the values for both groups, as shown in Fig 2 . We found no differences in the TEXB-α levels between controls and patients with leiomyomas, endometriosis, or both: patients with fibroids showed TEXB-α levels of 4.63 [3.77] Eeq pM/g; for those with endometriosis, the median was 4.63 [7.32] Eeq pM/g, while for patients with both endometriosis and leiomyomas, the median concentration was 3.35 [7.28] Eeq pM/g. The solid and dashed lines show the median and quartile values, respectively. Comparisons made after the logarithmic conversion of xenoestrogen concentrations showed no statistically significant differences between the two groups. Finally, we compared xenoestrogen levels after homogenization of the groups. Thus, the control group, consisting of 93 pregnant women, was compared with 109 non-pregnant cases. This additional analysis confirmed the absence of statistically significant differences in the detectability and TEXB-α levels between the groups as well as the dispersion of the values. We performed a comparative analysis that included only patients with TEXB-α levels above the limit of detection. The results were similar to those obtained from the comparative study that included all case and control groups ( S1 Table ). The controls were significantly younger (36.0 [6.0] vs 43.0 [10.0] years). In addition, cases were more likely to be ever-users of levonorgestrel-releasing intrauterine devices (LNG-IUDs) and reported more years of smoking; the former smokers in the group of cases had been smoke-free for more years than controls. Both the case and control groups had a higher percentage of women living within the Madrid Community: 61 patients (82.4%) in the control group and 50 (65.8%) in the case group (p = 0.020). In addition, 62 women in the control group (83.8%) and 52 among the cases (68.4%) had workplaces in the Madrid Community (p = 0.028). None of the study variables showed to have an influence on the xenoestrogen concentration. Nevertheless, when we analyzed the case and control groups separately, we found that in the group of cases, the use of hormonal contraceptives and having a residence or workplace within the Madrid Community were associated with higher TEXB-α levels (Figs 3 and 4 ). Interestingly, patients who reported living or working mainly in Madrid during the last 10 years had, on average, 3.12 Eeq pM/g TEXB-α higher levels than those working outside the area (p = 0.030). Women who reported having ever used hormonal contraceptives had, on average, 3.02 Eeq pM/g higher levels than those who had never used these drugs (p = 0.022). The regression coefficients and 95% confidence intervals are shown in the right-hand panel. BMI, body mass index; FH, Family history; HC, Hormonal contraceptives; LNG, Levonorgestrel; IUD, intrauterine device. * p<0.05. The regression coefficients and 95% confidence intervals are shown in the right-hand panel. BMI, body mass index; FH, Family history; HC, Hormonal contraceptives; LNG, Levonorgestrel; IUD, intrauterine device. Notably, the time between stopping contraceptives and undergoing surgery averaged 8.0 (8.0) years. When investigating the type of contraceptive used, this difference remained for all estrogen-containing drugs but did not apply to progesterone-releasing devices, such as IUDs or contraceptive implants. We confirmed these findings in the tertile comparison ( Fig 5 ): while there were no differences in the control group (p = 0.299), we found a significant difference in the distribution of the cases, where oral contraceptive users were mainly in T2 (2.62–6.51) and T3 (>6.51 Eeq pM/g), (p = 0.049). The results of the case group were confirmed by multivariate analysis after adjusting for age and BMI. A relationship between the use of hormonal contraceptives and having a workplace or residence within the Madrid Community was discarded ( Fig 6 ). The regression coefficients, 95% confidence intervals (CI) and p-value are shown in the right-hand panel. BMI, body mass index. Xenoestrogen concentration showed no relationship with the clinical presentation of leiomyomas or endometriosis, such as symptomatology, fibroid size or location, endometriosis type, or bowel involvement.

The results of the XENOBEM study confirmed the high exposure of individuals to xenoestrogens in our population. Although TEXB-α concentrations in the omentum do not seem to be associated with leiomyomas or endometriosis, the finding of detectable levels of xenoestrogens in most patients highlights the importance of regulating the use of these substances, which have proven to have harmful effects on humans and animals. The association between xenoestrogen concentrations and working or living in highly polluted areas confirms the remarkable effect of environmental pollution on the absorption of xenobiotics. The unexpected association between TEXB-α levels and estrogen-containing contraceptives warrants further investigation.

The present study was approved by the Institutional Ethics Review Board of Fundacion Jimenez Diaz Hospital (Madrid, Spain) on 12/20/2016, reference number 21/2016. The main objective of the XENOBEM study was to determine whether there is a positive association between the alpha fraction of the total xenoestrogen burden (TEXB-α) present in omental fat and the presence of endometriosis or uterine leiomyomas. Secondary endpoints included the relationship between the clinical and sociodemographic characteristics of these patients and their xenoestrogen levels. Xenoestrogen concentration (TEXB-α) in omental fat. The XENOBEM study is a case-control prospective study that included patients who underwent abdominal surgery at the University Hospital Quironsalud Madrid between February 24, 2017, and February 1, 2021. The inclusion criteria for the case group were women with benign leiomyomas or endometriosis, while the control group included women with other non-hormonal benign diseases (tubo-ovarian abscesses secondary to a pelvic inflammatory disease or hemorrhagic ovarian follicles that underwent surgery) or pregnant women who needed a cesarean section. The exclusion criteria for the case group included intraoperative findings or anatomopathological results indicating conditions other than leiomyomas or endometriosis. Women in the control group with a history of fibroids or endometriosis were excluded. All patients signed an informed consent form before participating in the study and completed a questionnaire that focused on clinical and sociodemographic data. We created a coded database including TEXB levels, age, body mass index (kg/m 2 ), medical and surgical history, parity, use of hormonal contraceptives (HC), menopausal status, use of hormonal therapy for menopause, smoking status, family history of fibroids, endometriosis, or gynecological cancer, and the main place of residence and work in the last 10 years. In the case group, we also analyzed the presence and type of symptoms, location, size, and number of fibroids, type of endometriosis, and bowel involvement. During surgery, we collected a 2–4 g omentum sample that was coded and stored at -80°C until chemical analysis. Bioaccumulative compounds were extracted according to the method described by Fernandez et al. [ 7 ] and separated by high-performance liquid chromatography (HPLC). This technique allows the separation of exogenous hormones (the so-called alpha fraction) from the beta fraction, composed of endogenous estrogens and more polar xenoestrogens that behave as natural hormones, being quickly metabolised and not acting as EDCs [ 6 ]. Subsequently, the alpha fraction was tested in the E-screen bioassay for estrogenicity, following the technique originally described by Soto et al. [ 9 ] with minor modifications [ 10 , 11 ]. Each sample was assayed in triplicate with a negative (steroid-free) control and a positive control, where 17-β-estradiol was added to a culture of MCF-7 breast cancer cells [ 7 ] at a concentration that would induce a maximum proliferative effect. The proliferative effect was transformed into estradiol equivalent units (Eeq) by reading the dose-response curve. The results were expressed as the alpha Total Effective Xenoestrogen Burden (TEXB-α) in Eeq per gram of lipid. TEXB-α is considered a reliable biomarker for the combined effect of mixtures of halogenated organic lipophilic xenoestrogens [ 6 ] and is also referred to in this article as the xenoestrogen concentration. The minimum concentration of TEXB-α required to elicit a significant proliferative effect on the MCF-7 cell culture, (as compared to the steroid-free control culture), was determined to be 0.1 Eeq pM/g, which represents the limit of detection (LD) for the bioassay [ 1 ]. The descriptive analysis included the clinical and sociodemographic variables of the study. We used absolute (n) and relative (%) frequencies for qualitative variables and mean ± standard deviation (SD) or median and interquartile range (IQR) for quantitative variables depending on their normality. Categorical variables were summarized using absolute and relative frequencies, and quantitative variables using means and standard deviations or medians and interquartile ranges based on the normality of the variable. To assess differences in xenoestrogen concentrations between the case and control groups, we used Student’s t-test or Mann-Whitney U test. We employed linear regression anaylsis to determine the influence of the study group and the impact of clinical and sociodemographic variables on xenoestrogen levels. We used chi-square tests to assess differences in the proportion of detectability between groups and other variables of interest. Furthermore, we conducted subgroup analyses to compare the detectability and xenoestrogen concentrations between controls and women with fibroids, endometriosis, or both. Additionally, we performed all comparisons between cases and controls regarding xenoestrogen levels after converting the concentration values to their natural logarithms. Following the methodology described in previous publications [ 7 ], we grouped participants into tertiles of serum TEXB-α based on TEXB-α distribution among controls and compared the variables of interest among tertiles. To ensure that the results obtained in the comparison between the case and control groups as a whole were not affected by the mixture of pregnant and non-pregnant individuals in both groups, we conducted a second comparison: in order to homogenize the groups, we compared xenoestrogen concentrations including only pregnant women in the control group. In the case group, pregnant women were excluded. In summary, we performed additional analyses comparing TEXB-α levels in a control group that included only pregnant women with those in a case group conformed by non-pregnant patients diagnosed with leiomyomas or endometriosis. Statistical significance was set at 95% (p≤0.05). SPSS software (version 29.0; IBM, Armonk, NY) was used for all statistical analyses.

(XLSX) (XLSX) (PDF)