Prenatal polychlorinated biphenyl exposure promotes invasion of progeny ectopic endometrial stromal cells via epigenetic modification of EZH2

Prenatal polychlorinated biphenyl exposure promotes invasion of progeny ectopic endometrial stromal cells via epigenetic modification of EZH2 | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Prenatal polychlorinated biphenyl exposure promotes invasion of progeny ectopic endometrial stromal cells via epigenetic modification of EZH2 Yinfeng Wang, Chaoshuang Ye, Changchang Hu, Pei Chen, Chaolu Chen, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-328521/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Polychlorinated biphenyls(PCBs) are persistent environmental endocrine disruptor. This study aim to investigate the changes of the ectopic endometrium invasive ability and the possible mechanism after exposure to PCBs during pregnancy. In total, 12 female Sprague Dawley rats were intraperitoneally injected with Aroclor 1221 (1 mg/kg) or dimethyl sulfoxide (1 mg/kg) at 16 and 18 days of gestation. The endometriosis model was established by autogenous uterine abdominal wall implantation 2 months after birth. The degree of adhesion between the endometriosis and the greater omentum, as well as cell morphology and Transwell invasion patterns were used to evaluate the invasive ability of progeny ectopic endometrial stromal cells (ESCs). Moreover, the effect of the enhancer of zeste 2 polycomb repressive complex 2 subunit (EZH2)/ trimethylation of Histone 3 lysine 27 (H3K27me3) axis was examined using a highly selective EZH2 inhibitor, GSK126. After gestational PCBs exposure, the adhesion between the endometrium and the greater omentum was enhanced (P<0.05), and the length and number of microvilli were significantly increased (P<0.01). Exposure to PCBs during pregnancy increased the invasive ability of the ectopic ESCs (P<0.01), with upregulated expression levels of EZH2 and H3k27me3, which were abrogated by the EZH2 inhibitor GSK126. In conclusion, exposure to PCBs during pregnancy increased the invasive ability of the ectopic endometrium, which may be mediated by EZH2. Environmental Engineering Environmental Policy polychlorinated biphenyls pregnancy endometriosis invasion epigenetics EZH2 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Endometriosis is a chronic benign gynecologic disease in which the growing endometrium appears outside the uterus, causing chronic pelvic pain, dysmenorrhea, infertility and dysmenorrhea (Altayyeb et al. 2020). There are various theories regarding the pathogenesis of endometriosis, such as retrograde menstruation, genetic factors, blood lymphatic spread, cell apoptosis, immune factors and environmental toxins (Lagana et al. 2017; Parazzini et al. 2013; Sofo et al. 2015). Although the classic ‘Retrograde menstruation’ theory is widely accepted, it does not account for all cases of endometriosis(Giudice and Kao 2004; McKinnon et al. 2018). With the development of industrialization, environmental problems are prominent, and the influence of environmental endocrine disruptors on the incidence of endometriosis has attracted increased attention (Gerkowicz et al. 2020; Martinez-Zamora et al. 2015; Matta et al. 2020; Wen et al. 2020). Accumulating evidence has suggested that exposure to certain environmental endocrine chemicals, including dioxins, bisphenol A, phthalates and polychlorinated biphenyls (PCBs), may increase the risk of endometriosis (Simonelli et al. 2017; Smarr et al. 2016). Furthermore, our previous study has confirmed that PCBs are associated with the migration and invasion of ectopic endometrial stromal cells through in vitro culture of ectopic endometrial stromal cells (ESCs) (Hu et al. 2018). PCBs are synthetic organic compounds, which contain 209 homologs. Due to their physicochemistry stability, non-flammability and favorable electrical insulation properties, PCBs were widely used in capacitors, lubricants, adhesives, turbines and plasticizers (Andersen et al. 2020; Ravenscroft et al. 2018), Although PCBs have been banned globally since the 1970s, they are still released from industrial incinerators and landfills, or leaked from old capacitors and transformers (Adams et al. 2019; Ravenscroft et al. 2018). With characteristics such as the resistance to environmental degradation, dissolving in liquid and bioaccumulation, PCBs can circulate in the environment via the food chain, water, soil and air (Klocke et al. 2019; Warenik-Bany et al. 2019). They also transfer from the mother to fetus via the placenta and lactation (Abraham et al. 1998). According to the hypothesis of the Developmental Origins of Health and diseases (DOHaD), gestation is considered to be a particularly sensitive time to external environment. Intrauterine exposure to an adverse environment will not only affect fetal development and lead to unfavorable pregnancy outcomes, but it will also have long-term impacts on disease susceptibility into late adulthood (Barker 1997; Godfrey and Barker 2001), including endometriosis (Wei et al. 2016). Previous studies have reported that human diseases are associated with epigenetic regulation caused by early life exposure to PCBs (Casati et al. 2013; Caspersen et al. 2016; Gladen et al. 2000; Su et al. 2015; Tang-Peronard et al. 2011). Histone methylation is one of the important epigenetic mechanisms, and is mediated by interacting with steroid receptors or non-genomic signaling pathways (Casati et al. 2012; Casati et al. 2013; Casati et al. 2015). Histone methyltransferase enhancer of zeste homolog 2 (EZH2) is an enzyme-catalyzed subunit of polycomb repressive complex 2 (PRC2) that alters gene expression by trimethylation of Histone 3 lysine 27 (H3K27me3)(Jones et al. 2018), which inhibits chromatin structure remodeling by switch/sucrose non-fermentable (SWI/SNF), resulting in gene inactivation. EZH2 is associated with the invasion and progression of breast cancer, lymphoma, ovarian cancer and other malignant tumors, and is consider to be one of the new targeted therapeutic factors for malignant tumors (Booth et al. 2018; Chang et al. 2011; Italiano et al. 2018; McCabe et al. 2012). Although endometriosis is a benign disease, it has biological characteristics of invasion (Zhang et al. 2017). The invasion indices of peritoneal endometriosis cells is similar to that of metastatic bladder cancer cells, while normal endometrial cells are non-invasive (Gaetje et al. 1995). Based on previous research, we suppose that gestational exposure to PCBs increases the invasive ability of the ectopic endometrium, thus leading to the occurrence and development of endometriosis via the EZH2/ H3K27me3 pathway. Material And Methods Animal model . The present study was performed in the Experimental Research Laboratory of Zhejiang Chinese Medical University, and complied with the approval of the ethic committee (approval no. 2018102904). In total, 12 non-pregnant female and 12 male Sprague Dawley (SD) rats (age, 10–12 weeks; weight, 180–220 g) were purchased from SLAC Laboratory Animal Co., Ltd. Rats were housed in a single cage with an environmentally controlled temperature (10–24˚C) and humidity (50–70%) under a 12/12 h (light/dark) schedule. They were feed with standard pellet food and water. The experiment began ≥ 1 week after the rats arrived to acclimatize them to the new housing conditions. The female rats were randomly assigned into either a PCB group (n = 6) or a control group (n = 6). Any female rat was housed with a random male rat. The successful mating (indicated by morning sperm positive vaginal smear) was termed embryonic day (E)0. On E16 and E18, the rats in PCB group and control group were intraperitoneal injected with 1 mg/kg Aroclor1221 (cat. no. C221N; AccuStandard; New Haven, CT) or dimethyl sulfoxide (DMSO, cat. no. D4540; Sigma-Aldrich; Merck KGaA), respectively, as previously reported (Bell et al. 2016 ; Reilly et al. 2015 ; Steinberg et al. 2007 ; Steinberg et al. 2008 ; Walker et al. 2013 ). Male pups were removed after delivery. The endometriosis model was performed 2 months postpartum as described by Saltan et al (Saltan et al. 2016 ). The rats were fixed in the supine position after general anesthesia with intraperitoneally injected 3% chloral hydrate (350 mg/kg). A 3-4-cm longitudinal incision was made in the middle of the lower abdomen. The left side of uterine cornu was excised and cut into 5x5 mm pieces. The endometrium layer was stripped from myometrium and autotransplanted at the abdominal walls with abundant blood supply (Fig. 1 ). In total, two pieces of endometrium were transplanted on each side. The abdominal cavity was closed with absorbable suture. A single intramuscular injection of 80,000 U penicillin was administered to prevent infection. The rats were anesthetized, as previously mentioned, 1 month after the first operation and were surgically examined to visualize the adhesion score of endometriotic foci. The adhesion scored was blindly evaluated by two doctors as previously reported by Belluco et al (Belluco et al. 2001 ), which involved the extent of site involvement (0–4), type of adhesion (0–4) and the tenacity of adhesion (0–3). The endometriotic foci were cultured or immobilized by 10% formalin for further experiment. Then, rats were euthanized by carbon dioxide inhalation. Cell culture and treatment . The ectopic endometrium was cut into pieces and then digested with 0.2% type I collagenase (Sigma-Aldrich; Merck KGaA) for 1 h on a horizontal concussion meter at 37˚C and 180 rpm. After centrifugation at 1,000 rpm for 5 min, 3–5 ml PBS was added and the cell suspension was filtered with 100 and 40 µm strainers. Cells were centrifuged at 1,000 rpm for 5 min at 37˚C, then an appropriate amount of DMEM/F12 medium was added and incubated in 5% CO2 at 37˚C in an incubator (Forma 3111; Thermo Fisher Scientific, Inc.). The culture medium was replaced at 24 h after primary culture and then every 2–3 days. In PCB group, GSK126 (cat. no.5005800001, Sigma-Aldrich; Merck KGaA), a highly selective inhibitor of EZH2 via competitive combination with S-adenosine methionine (McCabe et al. 2012 ), was added to evaluate the role of EZH2. ESCs were inoculated into a 6-well plate with a density of 3.0x10 5 per well, and GSK126 was added at the doses of 0, 2.5, 5, 10 and 20 µM for 48 h, where 5µM was the optimal inhibitory amount and was used in subsequent experiments. GSK126 was not added to the control group. The role of EZH2 in ESCs invasion was further determined by observing microvilli under transmission electron microscopy and transwell invasion assay. Immunofluorescence. The purity of ESCs was determined via immunofluorescence, using Cytokeratin 7(cat. no. ab181598, Abcam) and Vimentin(cat. no. ab92547, Abcam) as markers of epithelial and stromal cells respectively. The primary cells were fixed with 4% paraformaldehyde at room temperature for 30 min, followed by incubation with 0.5% tritox-100 at room temperature for 15 min. Cells were then washed three times with PBS and sealed with 5% BSA(Amresco, USA) solution at 37˚C for 1 h. Subsequently, anti-vimentin antibody or Cytokeratin 7 were added, incubated at 4˚C overnight and washed three times with PBS. FITC-labeled IgG(Amresco, USA) was added at room temperature for 1 h and cells were washed three times with PBS. Finally, 2 mg/l DAPI solution was added to re-stain the nucleus at room temperature for 10 min. Cells were washed twice with PBS and sealed with neutral resin, and the fluorescence signals of the cells were observed under a fluorescence microscope (Axiovert200; Zeiss AG). Transmission electron microscopy . Transmission electron microscopy was performed as described previously (Chen et al. 2019 ). ESCs cells were fixed overnight with 2.5% glutaraldehyde at 4˚C and then fixed with 1% osmium acid for 1 h at room temperature. Cells were rinsed with water 2–3 times for 10 min. Then, 50, 70, 90 and 100% ethanol for 15 min and 100% acetone for 15–20 min were used for dehydration at room temperature. Cells were embedded at room temperature for 4 h and ultrathin sections at 70-nm were sliced. The sections were placed in copper grids and stained with uranyl acetate and lead citrate. A transmission electron microscope (FEI Tecnai T10, USA) was used to observe the number and length of microvilli. In total, five fields were randomly selected from each section. The length of microvilli was measured using Image J software (V1.51, National Institute of Health). The number of microvilli was counted as number/circumference (n/µm). Immunohistochemistry . All tissues were fixed with 10% neutral formalin, paraffin-embedded and prepared into slices of 4-µm thickness. The process and scoring of immunohistochemistry were based on a previous report (Kumari et al. 2018 ). According to the antibody instructions, each slice was incubated with 50–100 µM primary antibody and the tissue solution (EZH2, 1:8,000, cat. no. ab191080; H3K27me3, 1:40, cat. no. ab6002; Abcam) overnight at 4˚C. Then, 50–100 µl Envision +/horseradish peroxidase (HRP) secondary antibody (DAKO; Agilent Technologies, Inc.) was added to each slice and incubated at room temperature for 30–60 min. The immunoreactivity was detected using diaminobenzidine. The slices were subsequently stained with hematoxylin, and observed under a light microscope (DM500; Leica Microsystems GmbH), and images were captured at a magnification of x40 or x200. The staining intensity of ESCs was scored as follows: 1 for absent or weak; 2 for moderate; and 3 for strong. The percentage positive cells was scored as: Percent of cells < 1, 0 point; percent of cells between 1–10, 1 point; percent of cells between 11–33, 2 points; percent of cells between 34–66, 3 points; and percent of cells between 67–100, 4 points. The Q score of all the slices was calculated by multiplying the staining intensity by the percentage positive of ECSs cells (Kumari et al. 2018 ). Western blotting . Western blotting was performed as described previously (Chen et al. 2019 ). ESCs were washed in PBS prior to being lysed in RIPA lysis buffer (Beyotime Institute of Biotechnology). The proteins were electrophoresed via 12% SDS-PAGE and transferred to PVDF membranes(Millipore, Billerica, MA, USA). Membranes were blocked in 3% bovine serum albumin (BSA, Sigma-Aldrich; Merck KGaA) at 37˚C for 2 h and then incubated with primary antibodies (EZH2, 1:100; H3K27me3, 1:80) overnight at 4˚C. Then, membranes were incubated with horseradish peroxidase-conjugated secondary antibody (1:10,000, cat.no. ab236555, Abcam) for 1.5 h at room temperature. Enhanced chemiluminescence detection reagent (Sigma-Aldrich, St Louis, MO, USA) was used to detect the signal. Relative protein expression levels were semi-quantified with β-actin expression as the reference gene using ImageJ software (V1.51, National Institute of Health). Reverse transcription-quantitative PCR (RT-qPCR) . EZH2 gene expression was analyzed using RT-qPCR with the following sequences (Sangon Biotech Co., Ltd.) as previously reported (Chen et al. 2019 ): EZH2 forward, 5’-CTTACTGCTGGCACCGTCTG-3’ and reverse, 5’-CTTTCCCTCTTCTGTCTGCTTCA-3’; and GAPDH forward, 5’- CTGGAGAAACCTGCCAAGTATG-3’ and reverse, 5’- GGTGGAAGAATGGGAGTTGCT-3’. Total mRNA was extracted from ESCs using RNAiso Plus reagent TaKaRa RNAiso Plus (Takara Bio, Inc.) according to the manufacturer’s instructions. Total RNA was reverse transcribed to cDNA using the PrimeScript™ RT reagent (Takara Bio, Inc.). qPCR was performed using a SYBR Premix Ex Taq™ Ⅱ kit (Takara Bio, Inc.) according to the manufacturer’s instructions. The following thermocycling conditions were used: Amplification of 48 cycles of 95˚C for 10 sec, 60˚C for 30 sec. The cyclic threshold (Cq) value was calculated using the average of three wells for each sample. The relative expression levels of target genes were normalized against actin using the 2 −ΔΔCq method(Livak and Schmittgen 2001 ). Cell migration and invasion assays . The migration and invasion of ESCs were evaluated as previously reported (Hu et al. 2018 ). Transwell chambers were 6.5-mm in diameter with an 8-µm pore size (Corning, Inc.). A density of 2x10 5 cells with 200 µl serum-free DMEM/F12 was seeded on the upper chamber. The lower chamber was filled with 500 µl DMEM/F12 containing 10% FBS. After incubation for 24 h, the cells on the underside of the membrane were fixed and then stained with crystal violet solution. For the invasion assay, the upper chamber was pre-coated with 25 µl Matrigel (BD Biosciences), and incubated for 48 h. A total of five fields were randomly selected to count the number of cells under a light microscope (x100). Statistical analysis. Statistical analyses were performed using SPSS 23.0 software (IBM Corp.). Data are presented as the mean ± SD. Student’s t-test was used to analyze differences for paired data and one-way ANOVA was used to analyze multiple comparisons. P 0.05). One month after the endometrium transplantation, the endometriotic implants formed a cystic nodule with vascularization. Hematoxylin and eosin staining showed that the ectopic lesions formed one or several cysts, and the cyst wall was composed of epithelial cell layer, stromal cell layer and a small amount of fibrous connective tissue from the inside to the outside. After 2–4 generations of purification, the purity of ESCs was > 95%. It had a long spindle shape with an oval nucleus, and immunofluorescence detection identified that Vimentin was positive and Cytokeratin7 was negative (Fig. 2 ).The adhesion score of the control group was 4.6 ± 1.3. After PCB exposure during pregnancy, the adhesion score of ectopic endometrial lesions in the offspring was significantly increased to 7.1 ± 0.9 (P < 0.05). PCB promotes the invasion of offspring ectopic ESCs and GSK126 abrogates the effect . There was no difference in the migratory ability of the ESCs from rat offspring. However, the invasive potential was significantly increased in PCB group compared with the control group. To investigate the effect of EZH2 on cell invasion, ESCs were treated with GSK126 for 48 h. In addition, after treatment with GSK126, it was proved that the down-regulation of EZH2 significantly reduced the invasion ability of ESCs(Fig. 3 ). PCB exposure increases the length and number of microvilli via EZH2 . Transmission electron microscope was used to assess the microvilli on the surface of ESCs. The microvilli were mainly slender cylindrical and interwoven with the surrounding microvilli, and their bases were connected to the cell membrane. The number of microvilli in the PCB exposed group was 1.07 ± 0.21 per perimeter(µm), and that in the control group was 0.67 ± 0.26 per perimeter(µm) (P < 0.01). Moreover, the microvilli length was 1.14 ± 0.58 µm in PCB group and 0.82 ± 0.32 µm in control group, which demonstrated a significant difference (P < 0.01). After GSK126 treatment, the microvilli were smooth and the length was shortened. The number of microvilli was 0.43 ± 0.21 per perimeter(µm) and the length of microvilli was 0.67 ± 0.37 µm, which were significantly reduced compared with those of the PCB group (Fig. 4 ). The expression of EZH2 and H3K27me3 increased in offspring ectopic ESCs . After exposure to PCBs during pregnancy, the expression levels of EZH2 and H3k27me3 in the ectopic ESCs of the offspring were significantly increased (P < 0.01). In the cell culture of the PCB group, after adding the inhibitor GSK126, Western blotting detected that the expression levels of EZH2 and its target molecule H3K27me3 were significantly decreased(Fig. 5 ). Discussion Endometriosis is a common refractory gynecological disease. Although it is estrogen-dependent benign disease, its biological behavior is similar to that of malignant tumors (Zhang et al. 2017 ). The migration, adhesion and invasion of retrograde endometrial cells are important steps in the formation of ectopic lesions (Gaetje et al. 1995 ; Meng et al. 2019 ; Witz et al. 2001 ). Previous studies have reported that PCBs increase the invasion of ESCs, as well as the incidence of endometriosis (Hu et al. 2018 ; Ploteau et al. 2016 ; Ploteau et al. 2017 ; Roy et al. 2015 ). However, little is known regarding the relationship between gestational PCBs in offspring endometriosis, and the role of histone methylation is not fully understood. In the present study, it was found that prenatal exposure to PCBs significantly increase the invasive ability of offspring ectopic ESCs with high expression levels of EZH2 and H3K27me3, which serve an important role in promoting this proses. An increase in the number and length of microvilli was the main morphological change observe in the present study. The relationship between cell morphology and cell invasiveness has been widely studied in tumors (Antonescu and Baren 2004 ; Aznavoorian et al. 1993 ). The ultrastructure of the cell surface can predict the contact, movement and adhesion of cells to other cells and the cell matrix, which serves an important role in the occurrence and development of tumors (Black 1980 ; Nicolson and Poste 1976 ). A decrease of tumor cell invasiveness is often accompanied by the loss of microvilli (Priester et al. 2013 ), while more microvilli and pseudopodia on the cell surface indicates a higher invasive ability of tumor cells (Yan et al. 2013 ). In patients with endometriosis, the morphology of endometrial gland epithelial cells (EGECs) is irregular, the number and length of microvilli is increased, the organelles are abundant and EGECs possess proliferative, migratory and invasive abilities, which are associated with the formation of endometriosis cysts (Yu et al. 2018 ). It has been reported that Aroclor1221 is a mildly chlorinated PCBs mixture, and its level is difficult to determine directly in the offspring after gestational exposure (Thomas et al. 1998 ). According to previous literature reports (Bell et al. 2016 ; Reilly et al. 2015 ; Steinberg et al. 2007 ; Steinberg et al. 2008 ; Walker et al. 2013 ), the dose of 1 mg/kg intraperitoneal injection of Aroclor1221 at E16 and E18 is close to the observed exposure of humans and wild animals in PCBs contaminated areas. PCBs may inhibit the activity of human estrogen sulfotransferase, an estrogen-degrading enzyme (Kester et al. 2000 ), thereby increasing the secretion of estrogen and the risk of hormone-dependent diseases. Accumulating evidence had suggested that higher PCBs exposure may increase the risk of endometriosis (Hu et al. 2018 ; Martinez-Zamora et al. 2015 ; Porpora et al. 2006 ; Wei et al. 2016 ). A reduced responsiveness to progesterone and overexpression of matrix metalloproteinases may be a key element of endometrial dysfunction associated with endometriosis (Osteen et al. 2005 ). With similar effects to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), dioxin-like-PCBs can combine with aryl hydrocarbon receptor(AhR) and form an activated heterodimerization complex with the aromatic hydrocarbon receptor nuclear translocator protein, leading to the overexpression of AhR in endometrioid cells (Okino and Whitlock 2000 ; Sherr and Monti 2013 ). Over activated AhR induced by PCBs regulates multiple downstream biological events, including the epigenetic silencer activity of histone methyltransferase EZH2 (Lee et al. 2020 ). Epigenetics refers to the heritable non-gene sequence changes, including DNA methylation, post-translational histone modification and microRNA. However, these are dynamic and may be affected by environmental exposures (Holland 2017 ). Pregnancy is a critical period of fetal epigenetic remodeling, which is sensitive to environmental endocrine disruptors (Casati et al. 2015 ; Pozharny et al. 2010 ). Maternal health can determine susceptibility to certain diseases in adulthood (Kanherkar et al. 2014 ). Moreover, gestational PCBs exposure not only induces an aberrant DNA methylation pattern, but also affect the histone modification balance, leading to long term and transgenerational effects (Casati et al. 2015 ). Histone methylation has been shown to serve an important role in the etiology of endometriosis, and EZH2 and H3K27me3 are epigenetic marks of endometriosis (Colon-Caraballo et al. 2015 ). Zhang et al (Zhang et al. 2017 ) revealed that EZH2 was associated with the invasion of the ectopic endometrium, and that the levels of EZH2 and H3K27me3 in the ectopic endometrium were significantly higher compared with those in normal endometrium. EZH2 and H3K27me3 directly induce epithelial-mesenchymal transition, thereby promoting the occurrence and development of endometriosis (Cao et al. 2008 ; Colon-Caraballo et al. 2015 ; Zhang et al. 2017 ). The present study also identified similar findings; after PCBs exposure during pregnancy, the expression levels of EZH2 and H3K27me3 in ectopic ESCs were significantly increased and the cell invasive ability was enhanced. However, after inhibition of EZH2 using GSK126, the expression levels of EZH2 and H3K27me3 were decreased, accompanied by a decreased cell invasive ability, suggesting that EZH2 serves an important role in ectopic endometrial invasion caused by PCBs exposure during pregnancy. In conclusion, PCBs exposure during pregnancy serves a significant role in the development and progression of endometriosis in offspring. As an early stage of life development, women should consider the effects of environmental pollution during pregnancy with regards to potential harm to next generations. EZH2-mediated epigenetic regulation may serve an important role in this process. However, further research is required to identify the underlying molecular mechanisms activated by EZH2 and H3K27me3. Declarations Ethics approval and consent to participate This study was approved by the Ethics Committee of Zhejiang Chinese Medical University (approval no. 2018102904). Patient consent for publication Not applicable. Availability of data and materials All data generated or analyzed during this study are included in this published article. Competing interests The authors declare that they have no competing interests. Funding This study was funded by a grant from the Zhejiang Provincial Key Research & Development Project Grants (grant no.2021C03095) and Education Department of Zhejiang Province (grant no. 2019150). Authors' contributions WYF performed the experiments，HCC wrote the manuscript, YCS performed the experiments, CP and CCLanalyzed the data. WRJ designed the study. All authors read and approved the final manuscript. Acknowledgements Not applicable. 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(2000) Potent inhibition of estrogen sulfotransferase by hydroxylated PCB metabolites: a novel pathway explaining the estrogenic activity of PCBs Endocrinology 141:1897-1900 doi:10.1210/endo.141.5.7530 Klocke C, Sethi S, Lein PJ (2019) The developmental neurotoxicity of legacy vs. contemporary polychlorinated biphenyls (PCBs): similarities and differences Environ Sci Pollut Res Int doi:10.1007/s11356-019-06723-5 Kumari K, Das B, Adhya A, Chaudhary S, Senapati S, Mishra SK (2018) Nicotine associated breast cancer in smokers is mediated through high level of EZH2 expression which can be reversed by methyltransferase inhibitor DZNepA Cell Death Dis 9:152 doi:10.1038/s41419-017-0224-z Lagana AS et al. (2017) Unus pro omnibus, omnes pro uno: A novel, evidence-based, unifying theory for the pathogenesis of endometriosis Med Hypotheses 103:10-20 doi:10.1016/j.mehy.2017.03.032 Lee JE, Cho SG, Ko SG, Ahrmad SA, Puga A, Kim K (2020) Regulation of a long noncoding RNA MALAT1 by aryl hydrocarbon receptor in pancreatic cancer cells and tissues Biochem Biophys Res Commun 532:563-569 doi:10.1016/j.bbrc.2020.08.053 Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method Methods 25:402-408 doi:10.1006/meth.2001.1262 Martinez-Zamora MA et al. (2015) Increased levels of dioxin-like substances in adipose tissue in patients with deep infiltrating endometriosis Hum Reprod 30:1059-1068 doi:10.1093/humrep/dev026 Matta K et al. (2020) Associations between persistent organic pollutants and endometriosis: A multipollutant assessment using machine learning algorithms Environ Pollut 260:114066 doi:10.1016/j.envpol.2020.114066 McCabe MT et al. (2012) EZH2 inhibition as a therapeutic strategy for lymphoma with EZH2-activating mutations Nature 492:108-112 doi:10.1038/nature11606 McKinnon B, Mueller M, Montgomery G (2018) Progesterone Resistance in Endometriosis: an Acquired Property? Trends Endocrinol Metab 29:535-548 doi:10.1016/j.tem.2018.05.006 Meng X, Liu J, Wang H, Chen P, Wang D (2019) MicroRNA-126-5p downregulates BCAR3 expression to promote cell migration and invasion in endometriosis Mol Cell Endocrinol 494:110486 doi:10.1016/j.mce.2019.110486 Nicolson GL, Poste G (1976) The cancer cell: dynamic aspects and modifications in cell-surface organization (first of two parts) N Engl J Med 295:197-203 doi:10.1056/NEJM197607222950405 Okino ST, Whitlock JP, Jr. (2000) The aromatic hydrocarbon receptor, transcription, and endocrine aspects of dioxin action Vitam Horm 59:241-264 doi:10.1016/s0083-6729(00)59009-8 Osteen KG, Bruner-Tran KL, Eisenberg E (2005) Reduced progesterone action during endometrial maturation: a potential risk factor for the development of endometriosis Fertil Steril 83:529-537 doi:10.1016/j.fertnstert.2004.11.026 Parazzini F, Vigano P, Candiani M, Fedele L (2013) Diet and endometriosis risk: a literature review Reprod Biomed Online 26:323-336 doi:10.1016/j.rbmo.2012.12.011 Ploteau S, Antignac JP, Volteau C, Marchand P, Venisseau A, Vacher V, Le Bizec B (2016) Distribution of persistent organic pollutants in serum, omental, and parietal adipose tissue of French women with deep infiltrating endometriosis and circulating versus stored ratio as new marker of exposure Environ Int 97:125-136 doi:10.1016/j.envint.2016.08.011 Ploteau S et al. (2017) Associations between internal exposure levels of persistent organic pollutants in adipose tissue and deep infiltrating endometriosis with or without concurrent ovarian endometrioma Environ Int 108:195-203 doi:10.1016/j.envint.2017.08.019 Porpora MG et al. (2006) Increased levels of polychlorobiphenyls in Italian women with endometriosis Chemosphere 63:1361-1367 doi:10.1016/j.chemosphere.2005.09.022 Pozharny Y et al. (2010) Genomic loss of imprinting in first-trimester human placenta Am J Obstet Gynecol 202:391 e391-398 doi:10.1016/j.ajog.2010.01.039 Priester M et al. (2013) STAT3 silencing inhibits glioma single cell infiltration and tumor growth Neuro Oncol 15:840-852 doi:10.1093/neuonc/not025 Ravenscroft J, Schell LM, Akwesasne Task Force on the E (2018) Patterns of PCB exposure among Akwesasne adolescents: The role of dietary and inhalation pathways Environ Int 121:963-972 doi:10.1016/j.envint.2018.05.005 Reilly MP, Weeks CD, Topper VY, Thompson LM, Crews D, Gore AC (2015) The effects of prenatal PCBs on adult social behavior in rats Horm Behav 73:47-55 doi:10.1016/j.yhbeh.2015.06.002 Roy D et al. (2015) Integrated Bioinformatics, Environmental Epidemiologic and Genomic Approaches to Identify Environmental and Molecular Links between Endometriosis and Breast Cancer Int J Mol Sci 16:25285-25322 doi:10.3390/ijms161025285 Saltan G et al. (2016) Viburnum opulus L.: A remedy for the treatment of endometriosis demonstrated by rat model of surgically-induced endometriosis J Ethnopharmacol 193:450-455 doi:10.1016/j.jep.2016.09.029 Sherr DH, Monti S (2013) The role of the aryl hydrocarbon receptor in normal and malignant B cell development Semin Immunopathol 35:705-716 doi:10.1007/s00281-013-0390-8 Simonelli A et al. (2017) Environmental and occupational exposure to bisphenol A and endometriosis: urinary and peritoneal fluid concentration levels Int Arch Occup Environ Health 90:49-61 doi:10.1007/s00420-016-1171-1 Smarr MM, Kannan K, Buck Louis GM (2016) Endocrine disrupting chemicals and endometriosis Fertil Steril 106:959-966 doi:10.1016/j.fertnstert.2016.06.034 Sofo V et al. (2015) Correlation between dioxin and endometriosis: an epigenetic route to unravel the pathogenesis of the disease Arch Gynecol Obstet 292:973-986 doi:10.1007/s00404-015-3739-5 Steinberg RM, Juenger TE, Gore AC (2007) The effects of prenatal PCBs on adult female paced mating reproductive behaviors in rats Horm Behav 51:364-372 doi:10.1016/j.yhbeh.2006.12.004 Steinberg RM, Walker DM, Juenger TE, Woller MJ, Gore AC (2008) Effects of perinatal polychlorinated biphenyls on adult female rat reproduction: development, reproductive physiology, and second generational effects Biol Reprod 78:1091-1101 doi:10.1095/biolreprod.107.067249 Su PH, Chen HY, Chen SJ, Chen JY, Liou SH, Wang SL (2015) Thyroid and growth hormone concentrations in 8-year-old children exposed in utero to dioxins and polychlorinated biphenyls J Toxicol Sci 40:309-319 doi:10.2131/jts.40.309 Tang-Peronard JL, Andersen HR, Jensen TK, Heitmann BL (2011) Endocrine-disrupting chemicals and obesity development in humans: a review Obes Rev 12:622-636 doi:10.1111/j.1467-789X.2011.00871.x Thomas GO, Sweetman AJ, Parker CA, Kreibich H, Jones KC (1998) Development and validation of methods for the trace determination of PCBs in biological matrices Chemosphere 36:2447-2459 doi:10.1016/s0045-6535(97)10211-9 Walker DM, Kermath BA, Woller MJ, Gore AC (2013) Disruption of reproductive aging in female and male rats by gestational exposure to estrogenic endocrine disruptors Endocrinology 154:2129-2143 doi:10.1210/en.2012-2123 Warenik-Bany M, Maszewski S, Mikolajczyk S, Piskorska-Pliszczynska J (2019) Impact of environmental pollution on PCDD/F and PCB bioaccumulation in game animals Environ Pollut 255:113159 doi:10.1016/j.envpol.2019.113159 Wei M, Chen X, Zhao Y, Cao B, Zhao W (2016) Effects of Prenatal Environmental Exposures on the Development of Endometriosis in Female Offspring Reprod Sci 23:1129-1138 doi:10.1177/1933719116630418 Wen X et al. (2020) Bisphenol A Exposure Enhances Endometrial Stromal Cell Invasion and Has a Positive Association with Peritoneal Endometriosis Reprod Sci doi:10.1007/s43032-019-00076-7 Witz CA, Thomas MR, Montoya-Rodriguez IA, Nair AS, Centonze VE, Schenken RS (2001) Short-term culture of peritoneum explants confirms attachment of endometrium to intact peritoneal mesothelium Fertil Steril 75:385-390 doi:10.1016/s0015-0282(00)01699-x Yan W, Chen Y, Yao Y, Zhang H, Wang T (2013) Increased invasion and tumorigenicity capacity of CD44+/CD24- breast cancer MCF7 cells in vitro and in nude mice Cancer Cell Int 13:62 doi:10.1186/1475-2867-13-62 Yu CX et al. (2018) Correlation between replicative senescence of endometrial gland epithelial cells in shedding and non-shedding endometria and endometriosis cyst during menstruation Gynecol Endocrinol 34:981-986 doi:10.1080/09513590.2018.1480709 Zhang Q, Dong P, Liu X, Sakuragi N, Guo SW (2017) Enhancer of Zeste homolog 2 (EZH2) induces epithelial-mesenchymal transition in endometriosis Sci Rep 7:6804 doi:10.1038/s41598-017-06920-7 Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-328521","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":19336077,"identity":"da51375d-20e1-4eb1-8d53-afad99b9bb36","order_by":0,"name":"Yinfeng Wang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA8klEQVRIiWNgGAWjYNACGwkeNobDBxgkQJwDRGlJs5DjZzyWQJKWCmPJ5jMGEA4hLQbHzx5+zZMgkbjh2JnPHyzbGOT4biQwfi7Ap+VMXpo1WMuZsxsMJNsYjCVvJDBLz8CjxexAjpkx7w+glhtnNyQAtQAZCWzMPPi0nH9jZgy25f6bBweAWuoJa7mRY/wYqMVYsuEMYwNQS4IBIS32N96YMc5JkJDjZzhmzCBxTsJw5pmHzdL4tEj25xh/eJNQB4rKx58lymzk+Y4nH/yMTwsQsEnBFDBLgCOTsQG/BqDCjz+gLMYPhNSOglEwCkbBiAQAQFdQkN2XL3kAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0002-4489-9658","institution":"Zhejiang University School of Medicine Women's Hospital","correspondingAuthor":true,"prefix":"","firstName":"Yinfeng","middleName":"","lastName":"Wang","suffix":""},{"id":19336078,"identity":"1f2737f9-c93a-48ed-a997-02712eaec052","order_by":1,"name":"Chaoshuang Ye","email":"","orcid":"","institution":"Zhejiang University School of Medicine Women's Hospital","correspondingAuthor":false,"prefix":"","firstName":"Chaoshuang","middleName":"","lastName":"Ye","suffix":""},{"id":19336079,"identity":"dba637b5-ca8e-4c4e-9e91-7aad2552363f","order_by":2,"name":"Changchang Hu","email":"","orcid":"","institution":"Zhejiang University School of Medicine Women's Hospital","correspondingAuthor":false,"prefix":"","firstName":"Changchang","middleName":"","lastName":"Hu","suffix":""},{"id":19336080,"identity":"fbfa9c25-8c7e-47b3-9352-1657475147f1","order_by":3,"name":"Pei Chen","email":"","orcid":"","institution":"Zhejiang University School of Medicine Women's Hospital","correspondingAuthor":false,"prefix":"","firstName":"Pei","middleName":"","lastName":"Chen","suffix":""},{"id":19336081,"identity":"36bf4c00-1ad7-4e44-87e0-c9b0129a9846","order_by":4,"name":"Chaolu Chen","email":"","orcid":"","institution":"Zhejiang University School of Medicine Women's Hospital","correspondingAuthor":false,"prefix":"","firstName":"Chaolu","middleName":"","lastName":"Chen","suffix":""},{"id":19336082,"identity":"7ab45980-2ae8-4217-9e1a-d56ad337129b","order_by":5,"name":"Ruijin Wu","email":"","orcid":"","institution":"Zhejiang University School of Medicine Women's Hospital","correspondingAuthor":false,"prefix":"","firstName":"Ruijin","middleName":"","lastName":"Wu","suffix":""}],"badges":[],"createdAt":"2021-03-14 09:05:41","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-328521/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-328521/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":7585175,"identity":"1da0f517-168f-4feb-8f55-e52f0a682a9e","added_by":"auto","created_at":"2021-04-01 20:41:09","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":267983,"visible":true,"origin":"","legend":"Rat model construction. (a) Adult female Sprague Dawley rats were fixed in supine position after anesthesia. (b) The left side of the uterine body was removed (white arrow), (c) cut into a size of ~5x5 mm and stripped of the myometrium. (d) Autologous transplantation was performed in the area with abundant blood vessels in the abdominal wall (black arrow) to establish the endometriosis model.","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-328521/v1/a8cbf23e82b42da9a2e0cbde.jpg"},{"id":7584504,"identity":"e7ba2818-0c9a-4724-9277-3a964d61a4f0","added_by":"auto","created_at":"2021-04-01 20:35:09","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":586809,"visible":true,"origin":"","legend":"Identification of ESCs. (a) General observation of ectopic endometrial lesions. (b) Hematoxylin and eosin stain of ectopic lesion (magnification, x200). (c) ESCs were negative for Cytokeratin7. (d) ESCs were positive for Vimentin. Magnification, x100; Scale bar, 200 μm. ESCs, endometrial stromal cells.","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-328521/v1/1a88bb73ef52c21aff77948a.jpg"},{"id":7584506,"identity":"2476ebff-277e-4cf3-9e50-2818457bc0e2","added_by":"auto","created_at":"2021-04-01 20:35:09","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":4479604,"visible":true,"origin":"","legend":"Effects of gestational PCBs exposure on ectopic ESC migration and invasion. (a) No difference was found in the migratory ability of the ESCs of the offspring. (b) Prenatal PCBs exposure promoted the invasion of offspring ectopic ESCs. Furthermore, GSK126, an inhibitor of enhancer of zeste 2 polycomb repressive complex 2 subunit, significantly decreased the invasive ability induced by PCBs. Magnification, x100. **P\u003c0.01. ESCs, endometrial stromal cells; PCBs, polychlorinated biphenyls.","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-328521/v1/b13bf2c4831cd3594afac632.jpg"},{"id":7585039,"identity":"da01e321-b5ef-4320-84d4-6c9703643de4","added_by":"auto","created_at":"2021-04-01 20:38:09","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":335869,"visible":true,"origin":"","legend":"Ultrastructure of cell surface under transmission electron microscope. (a) Cell surface of ectopic ESCs from control group, PCB group and ectopic ESC in PCB group after EZH2 was inhibited by GSK126. (b) Analysis of microvilli number. (c) Analysis of microvilli length. a(left): Magnification, x4,800; a(right): Magnification, x18,500. **P\u003c0.01. ESCs, endometrial stromal cells; PCBs, polychlorinated biphenyls;EZH2, enhancer of zeste 2 polycomb repressive complex 2 subunit.","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-328521/v1/7da61b10706a78c53ac6aa34.jpg"},{"id":7585041,"identity":"e01749b1-afd1-4ac2-9bf6-d76c83c0072e","added_by":"auto","created_at":"2021-04-01 20:38:09","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2764486,"visible":true,"origin":"","legend":"Expression levels of EZH2 and H3k27me3 in offspring ectopic ESCs. (a) Immunohistochemistry (magnification, x200; scale bar, 100 μm ) in tissue sections shows EZH2 and H3k27me3 expression levels were increased after gestational PCBs exposure. (b) Graph representing the Q score for EZH2 and H3H27me3 expression levels in offspring ectopic ESCs. (c) Graph depicting the mRNA expression of EZH2 in offspring ectopic ESCs. (d) Western blotting results demonstrated that (e) EZH2 and H3k27me3 expression levels were abrogated by GSK126. *P\u003c0.05, **P\u003c0.01. ESCs, endometrial stromal cells; PCBs, polychlorinated biphenyls; EZH2, enhancer of zeste 2 polycomb repressive complex 2 subunit; H3k27me3, trimethylation of Histone 3 lysine 27.","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-328521/v1/970f2796e34f56ed568ff50b.jpg"},{"id":13684528,"identity":"7648a754-1af8-4513-bc34-a364c93089e6","added_by":"auto","created_at":"2021-09-17 12:08:31","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":830399,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-328521/v1/8a136565-e884-4e36-a678-d0444acd2491.pdf"}],"financialInterests":"","formattedTitle":"Prenatal polychlorinated biphenyl exposure promotes invasion of progeny ectopic endometrial stromal cells via epigenetic modification of EZH2","fulltext":[{"header":"Introduction","content":"\u003cp\u003eEndometriosis is a chronic benign gynecologic disease in which the growing endometrium appears outside the uterus, causing chronic pelvic pain, dysmenorrhea, infertility and dysmenorrhea (Altayyeb et al. 2020). There are various theories regarding the pathogenesis of endometriosis, such as retrograde menstruation, genetic factors, blood lymphatic spread, cell apoptosis, immune factors and environmental toxins (Lagana et al. 2017; Parazzini et al. 2013; Sofo et al. 2015). Although the classic \u0026lsquo;Retrograde menstruation\u0026rsquo; theory is widely accepted, it does not account for all cases of endometriosis(Giudice and Kao 2004; McKinnon et al. 2018). With the development of industrialization, environmental problems are prominent, and the influence of environmental endocrine disruptors on the incidence of endometriosis has attracted increased attention (Gerkowicz et al. 2020; Martinez-Zamora et al. 2015; Matta et al. 2020; Wen et al. 2020). Accumulating evidence has suggested that exposure to certain environmental endocrine chemicals, including dioxins, bisphenol A, phthalates and polychlorinated biphenyls (PCBs), may increase the risk of endometriosis (Simonelli et al. 2017; Smarr et al. 2016). Furthermore, our previous study has confirmed that PCBs are associated with the migration and invasion of ectopic endometrial stromal cells through \u003cem\u003ein vitro\u003c/em\u003e culture of ectopic endometrial stromal cells (ESCs) (Hu et al. 2018).\u003c/p\u003e\n\u003cp\u003ePCBs are synthetic organic compounds, which contain 209 homologs. Due to their physicochemistry stability, non-flammability and favorable electrical insulation properties, PCBs were widely used in capacitors, lubricants, adhesives, turbines and plasticizers (Andersen et al. 2020; Ravenscroft et al. 2018), Although PCBs have been banned globally since the 1970s, they are still released from industrial incinerators and landfills, or leaked from old capacitors and transformers (Adams et al. 2019; Ravenscroft et al. 2018). With characteristics such as the resistance to environmental degradation, dissolving in liquid and bioaccumulation, PCBs can circulate in the environment via the food chain, water, soil and air (Klocke et al. 2019; Warenik-Bany et al. 2019). They also transfer from the mother to fetus via the placenta and lactation (Abraham et al. 1998). According to the hypothesis of the Developmental Origins of Health and diseases (DOHaD), gestation is considered to be a particularly sensitive time to external environment. Intrauterine exposure to an adverse environment will not only affect fetal development and lead to unfavorable pregnancy outcomes, but it will also have long-term impacts on disease susceptibility into late adulthood (Barker 1997; Godfrey and Barker 2001), including endometriosis (Wei et al. 2016).\u003c/p\u003e\n\u003cp\u003ePrevious studies have reported that human diseases are associated with epigenetic regulation caused by early life exposure to PCBs (Casati et al. 2013; Caspersen et al. 2016; Gladen et al. 2000; Su et al. 2015; Tang-Peronard et al. 2011). Histone methylation is one of the important epigenetic mechanisms, and is mediated by interacting with steroid receptors or non-genomic signaling pathways (Casati et al. 2012; Casati et al. 2013; Casati et al. 2015). Histone methyltransferase enhancer of zeste homolog 2 (EZH2) is an enzyme-catalyzed subunit of polycomb repressive complex 2 (PRC2) that alters gene expression by trimethylation of Histone 3 lysine 27 (H3K27me3)(Jones et al. 2018), which inhibits chromatin structure remodeling by switch/sucrose non-fermentable (SWI/SNF), resulting in gene inactivation. EZH2 is associated with the invasion and progression of breast cancer, lymphoma, ovarian cancer and other malignant tumors, and is consider to be one of the new targeted therapeutic factors for malignant tumors (Booth et al. 2018; Chang et al. 2011; Italiano et al. 2018; McCabe et al. 2012). Although endometriosis is a benign disease, it has biological characteristics of invasion (Zhang et al. 2017). The invasion indices of peritoneal endometriosis cells is similar to that of metastatic bladder cancer cells, while normal endometrial cells are non-invasive (Gaetje et al. 1995).\u003c/p\u003e\n\u003cp\u003eBased on previous research, we suppose that gestational exposure to PCBs\u0026nbsp; increases the invasive ability of the ectopic endometrium, thus leading to the occurrence and development of endometriosis via the EZH2/ H3K27me3 pathway.\u003c/p\u003e"},{"header":"Material And Methods","content":"\u003cp\u003e\u003cem\u003eAnimal model\u003c/em\u003e. The present study was performed in the Experimental Research Laboratory of Zhejiang Chinese Medical University, and complied with the approval of the ethic committee (approval no.\u003c/p\u003e\n\u003cp\u003e2018102904). In total, 12 non-pregnant female and 12 male Sprague Dawley (SD) rats (age, 10\u0026ndash;12 weeks; weight, 180\u0026ndash;220 g) were purchased from SLAC Laboratory Animal Co., Ltd. Rats were housed in a single cage with an environmentally controlled temperature (10\u0026ndash;24˚C) and humidity (50\u0026ndash;70%) under a 12/12 h (light/dark) schedule. They were feed with standard pellet food and water. The experiment began\u0026thinsp;\u0026ge;\u0026thinsp;1 week after the rats arrived to acclimatize them to the new housing conditions.\u003c/p\u003e\n\u003cp\u003eThe female rats were randomly assigned into either a PCB group (n\u0026thinsp;=\u0026thinsp;6) or a control group (n\u0026thinsp;=\u0026thinsp;6). Any female rat was housed with a random male rat. The successful mating (indicated by morning sperm positive vaginal smear) was termed embryonic day (E)0. On E16 and E18, the rats in PCB group and control group were intraperitoneal injected with 1 mg/kg Aroclor1221 (cat. no. C221N; AccuStandard; New Haven, CT) or dimethyl sulfoxide (DMSO, cat. no. D4540; Sigma-Aldrich; Merck KGaA), respectively, as previously reported (Bell et al. \u003cspan class=\"CitationRef\"\u003e2016\u003c/span\u003e; Reilly et al. \u003cspan class=\"CitationRef\"\u003e2015\u003c/span\u003e; Steinberg et al. \u003cspan class=\"CitationRef\"\u003e2007\u003c/span\u003e; Steinberg et al. \u003cspan class=\"CitationRef\"\u003e2008\u003c/span\u003e; Walker et al. \u003cspan class=\"CitationRef\"\u003e2013\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003eMale pups were removed after delivery. The endometriosis model was performed 2 months postpartum as described by Saltan \u003cem\u003eet al\u003c/em\u003e (Saltan et al. \u003cspan class=\"CitationRef\"\u003e2016\u003c/span\u003e). The rats were fixed in the supine position after general anesthesia with intraperitoneally injected 3% chloral hydrate (350 mg/kg). A 3-4-cm longitudinal incision was made in the middle of the lower abdomen. The left side of uterine cornu was excised and cut into 5x5 mm pieces. The endometrium layer was stripped from myometrium and autotransplanted at the abdominal walls with abundant blood supply (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). In total, two pieces of endometrium were transplanted on each side. The abdominal cavity was closed with absorbable suture. A single intramuscular injection of 80,000 U penicillin was administered to prevent infection.\u003c/p\u003e\n\u003cp\u003eThe rats were anesthetized, as previously mentioned, 1 month after the first operation and were surgically examined to visualize the adhesion score of endometriotic foci. The adhesion scored was blindly evaluated by two doctors as previously reported by Belluco \u003cem\u003eet al\u003c/em\u003e (Belluco et al. \u003cspan class=\"CitationRef\"\u003e2001\u003c/span\u003e), which involved the extent of site involvement (0\u0026ndash;4), type of adhesion (0\u0026ndash;4) and the tenacity of adhesion (0\u0026ndash;3). The endometriotic foci were cultured or immobilized by 10% formalin for further experiment. Then, rats were euthanized by carbon dioxide inhalation.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eCell culture and treatment\u003c/em\u003e. The ectopic endometrium was cut into pieces and then digested with 0.2% type I collagenase (Sigma-Aldrich; Merck KGaA) for 1 h on a horizontal concussion meter at 37˚C and 180 rpm. After centrifugation at 1,000 rpm for 5 min, 3\u0026ndash;5 ml PBS was added and the cell suspension was filtered with 100 and 40 \u0026micro;m strainers. Cells were centrifuged at 1,000 rpm for 5 min at 37˚C, then an appropriate amount of DMEM/F12 medium was added and incubated in 5% CO2 at 37˚C in an incubator (Forma 3111; Thermo Fisher Scientific, Inc.). The culture medium was replaced at 24 h after primary culture and then every 2\u0026ndash;3 days.\u003c/p\u003e\n\u003cp\u003eIn PCB group, GSK126 (cat. no.5005800001, Sigma-Aldrich; Merck KGaA), a highly selective inhibitor of EZH2 via competitive combination with S-adenosine methionine (McCabe et al. \u003cspan class=\"CitationRef\"\u003e2012\u003c/span\u003e), was added to evaluate the role of EZH2. ESCs were inoculated into a 6-well plate with a density of 3.0x10\u003csup\u003e5\u003c/sup\u003e per well, and GSK126 was added at the doses of 0, 2.5, 5, 10 and 20 \u0026micro;M for 48 h, where 5\u0026micro;M was the optimal inhibitory amount and was used in subsequent experiments. GSK126 was not added to the control group. The role of EZH2 in ESCs invasion was further determined by observing microvilli under transmission electron microscopy and transwell invasion assay.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eImmunofluorescence.\u003c/em\u003e The purity of ESCs was determined via immunofluorescence, using Cytokeratin 7(cat. no. ab181598, Abcam) and Vimentin(cat. no. ab92547, Abcam) as markers of epithelial and stromal cells respectively. The primary cells were fixed with 4% paraformaldehyde at room temperature for 30 min, followed by incubation with 0.5% tritox-100 at room temperature for 15 min. Cells were then washed three times with PBS and sealed with 5% BSA(Amresco, USA) solution at 37˚C for 1 h. Subsequently, anti-vimentin antibody or Cytokeratin 7 were added, incubated at 4˚C overnight and washed three times with PBS. FITC-labeled IgG(Amresco, USA) was added at room temperature for 1 h and cells were washed three times with PBS. Finally, 2 mg/l DAPI solution was added to re-stain the nucleus at room temperature for 10 min. Cells were washed twice with PBS and sealed with neutral resin, and the fluorescence signals of the cells were observed under a fluorescence microscope (Axiovert200; Zeiss AG).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eTransmission electron microscopy\u003c/em\u003e. Transmission electron microscopy was performed as described previously (Chen et al. \u003cspan class=\"CitationRef\"\u003e2019\u003c/span\u003e). ESCs cells were fixed overnight with 2.5% glutaraldehyde at 4˚C and then fixed with 1% osmium acid for 1 h at room temperature. Cells were rinsed with water 2\u0026ndash;3 times for 10 min. Then, 50, 70, 90 and 100% ethanol for 15 min and 100% acetone for 15\u0026ndash;20 min were used for dehydration at room temperature. Cells were embedded at room temperature for 4 h and ultrathin sections at 70-nm were sliced. The sections were placed in copper grids and stained with uranyl acetate and lead citrate. A transmission electron microscope (FEI Tecnai T10, USA) was used to observe the number and length of microvilli. In total, five fields were randomly selected from each section. The length of microvilli was measured using Image J software (V1.51, National Institute of Health). The number of microvilli was counted as number/circumference (n/\u0026micro;m).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eImmunohistochemistry\u003c/em\u003e. All tissues were fixed with 10% neutral formalin, paraffin-embedded and prepared into slices of 4-\u0026micro;m thickness. The process and scoring of immunohistochemistry were based on a previous report (Kumari et al. \u003cspan class=\"CitationRef\"\u003e2018\u003c/span\u003e). According to the antibody instructions, each slice was incubated with 50\u0026ndash;100 \u0026micro;M primary antibody and the tissue solution (EZH2, 1:8,000, cat. no. ab191080; H3K27me3, 1:40, cat. no. ab6002; Abcam) overnight at 4˚C. Then, 50\u0026ndash;100 \u0026micro;l Envision +/horseradish peroxidase (HRP) secondary antibody (DAKO; Agilent Technologies, Inc.) was added to each slice and incubated at room temperature for 30\u0026ndash;60 min. The immunoreactivity was detected using diaminobenzidine. The slices were subsequently stained with hematoxylin, and observed under a light microscope (DM500; Leica Microsystems GmbH), and images were captured at a magnification of x40 or x200. The staining intensity of ESCs was scored as follows: 1 for absent or weak; 2 for moderate; and 3 for strong. The percentage positive cells was scored as: Percent of cells\u0026thinsp;\u0026lt;\u0026thinsp;1, 0 point; percent of cells between 1\u0026ndash;10, 1 point; percent of cells between 11\u0026ndash;33, 2 points; percent of cells between 34\u0026ndash;66, 3 points; and percent of cells between 67\u0026ndash;100, 4 points. The Q score of all the slices was calculated by multiplying the staining intensity by the percentage positive of ECSs cells (Kumari et al. \u003cspan class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eWestern blotting\u003c/em\u003e. Western blotting was performed as described previously (Chen et al. \u003cspan class=\"CitationRef\"\u003e2019\u003c/span\u003e). ESCs were washed in PBS prior to being lysed in RIPA lysis buffer (Beyotime Institute of Biotechnology). The proteins were electrophoresed via 12% SDS-PAGE and transferred to PVDF membranes(Millipore, Billerica, MA, USA). Membranes were blocked in 3% bovine serum albumin (BSA, Sigma-Aldrich; Merck KGaA) at 37˚C for 2 h and then incubated with primary antibodies (EZH2, 1:100; H3K27me3, 1:80) overnight at 4˚C. Then, membranes were incubated with horseradish peroxidase-conjugated secondary antibody (1:10,000, cat.no. ab236555, Abcam) for 1.5 h at room temperature. Enhanced chemiluminescence detection reagent (Sigma-Aldrich, St Louis, MO, USA) was used to detect the signal. Relative protein expression levels were semi-quantified with \u0026beta;-actin expression as the reference gene using ImageJ software (V1.51, National Institute of Health).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eReverse transcription-quantitative PCR (RT-qPCR)\u003c/em\u003e. EZH2 gene expression was analyzed using RT-qPCR with the following sequences (Sangon Biotech Co., Ltd.) as previously reported (Chen et al. \u003cspan class=\"CitationRef\"\u003e2019\u003c/span\u003e): EZH2 forward, 5\u0026rsquo;-CTTACTGCTGGCACCGTCTG-3\u0026rsquo; and reverse, 5\u0026rsquo;-CTTTCCCTCTTCTGTCTGCTTCA-3\u0026rsquo;; and GAPDH forward, 5\u0026rsquo;- CTGGAGAAACCTGCCAAGTATG-3\u0026rsquo; and reverse, 5\u0026rsquo;- GGTGGAAGAATGGGAGTTGCT-3\u0026rsquo;. Total mRNA was extracted from ESCs using RNAiso Plus reagent TaKaRa RNAiso Plus (Takara Bio, Inc.) according to the manufacturer\u0026rsquo;s instructions. Total RNA was reverse transcribed to cDNA using the PrimeScript\u0026trade; RT reagent (Takara Bio, Inc.). qPCR was performed using a SYBR Premix Ex Taq\u0026trade; Ⅱ kit (Takara Bio, Inc.) according to the manufacturer\u0026rsquo;s instructions. The following thermocycling conditions were used: Amplification of 48 cycles of 95˚C for 10 sec, 60˚C for 30 sec. The cyclic threshold (Cq) value was calculated using the average of three wells for each sample. The relative expression levels of target genes were normalized against actin using the 2\u003csup\u003e\u0026minus;\u0026Delta;\u0026Delta;Cq\u003c/sup\u003e method(Livak and Schmittgen \u003cspan class=\"CitationRef\"\u003e2001\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eCell migration and invasion assays\u003c/em\u003e. The migration and invasion of ESCs were evaluated as previously reported (Hu et al. \u003cspan class=\"CitationRef\"\u003e2018\u003c/span\u003e). Transwell chambers were 6.5-mm in diameter with an 8-\u0026micro;m pore size (Corning, Inc.). A density of 2x10\u003csup\u003e5\u003c/sup\u003e cells with 200 \u0026micro;l serum-free DMEM/F12 was seeded on the upper chamber. The lower chamber was filled with 500 \u0026micro;l DMEM/F12 containing 10% FBS. After incubation for 24 h, the cells on the underside of the membrane were fixed and then stained with crystal violet solution. For the invasion assay, the upper chamber was pre-coated with 25 \u0026micro;l Matrigel (BD Biosciences), and incubated for 48 h. A total of five fields were randomly selected to count the number of cells under a light microscope (x100).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eStatistical analysis.\u003c/em\u003e Statistical analyses were performed using SPSS 23.0 software (IBM Corp.). Data are presented as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD. Student\u0026rsquo;s t-test was used to analyze differences for paired data and one-way ANOVA was used to analyze multiple comparisons. P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered to indicate a statistically significant difference.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cem\u003eIdentification of endometriosis and ESCs\u003c/em\u003e. The PCB group had 23 female offsprings, while the control group had 25(P\u0026thinsp;\u0026gt;\u0026thinsp;0.05). One month after the endometrium transplantation, the endometriotic implants formed a cystic nodule with vascularization. Hematoxylin and eosin staining showed that the ectopic lesions formed one or several cysts, and the cyst wall was composed of epithelial cell layer, stromal cell layer and a small amount of fibrous connective tissue from the inside to the outside. After 2\u0026ndash;4 generations of purification, the purity of ESCs was \u0026gt;\u0026thinsp;95%. It had a long spindle shape with an oval nucleus, and immunofluorescence detection identified that Vimentin was positive and Cytokeratin7 was negative (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e).The adhesion score of the control group was 4.6\u0026thinsp;\u0026plusmn;\u0026thinsp;1.3. After PCB exposure during pregnancy, the adhesion score of ectopic endometrial lesions in the offspring was significantly increased to 7.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9 (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003ePCB promotes the invasion of offspring ectopic ESCs and GSK126 abrogates the effect\u003c/em\u003e. There was no difference in the migratory ability of the ESCs from rat offspring. However, the invasive potential was significantly increased in PCB group compared with the control group. To investigate the effect of EZH2 on cell invasion, ESCs were treated with GSK126 for 48 h. In addition, after treatment with GSK126, it was proved that the down-regulation of EZH2 significantly reduced the invasion ability of ESCs(Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003ePCB exposure increases the length and number of microvilli via EZH2\u003c/em\u003e. Transmission electron microscope was used to assess the microvilli on the surface of ESCs. The microvilli were mainly slender cylindrical and interwoven with the surrounding microvilli, and their bases were connected to the cell membrane. The number of microvilli in the PCB exposed group was 1.07\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21 per perimeter(\u0026micro;m), and that in the control group was 0.67\u0026thinsp;\u0026plusmn;\u0026thinsp;0.26 per perimeter(\u0026micro;m) (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Moreover, the microvilli length was 1.14\u0026thinsp;\u0026plusmn;\u0026thinsp;0.58 \u0026micro;m in PCB group and 0.82\u0026thinsp;\u0026plusmn;\u0026thinsp;0.32 \u0026micro;m in control group, which demonstrated a significant difference (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01).\u003c/p\u003e\n\u003cp\u003eAfter GSK126 treatment, the microvilli were smooth and the length was shortened. The number of microvilli was 0.43\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21 per perimeter(\u0026micro;m) and the length of microvilli was 0.67\u0026thinsp;\u0026plusmn;\u0026thinsp;0.37 \u0026micro;m, which were significantly reduced compared with those of the PCB group (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eThe expression of EZH2 and H3K27me3 increased in offspring ectopic ESCs\u003c/em\u003e. After exposure to PCBs during pregnancy, the expression levels of EZH2 and H3k27me3 in the ectopic ESCs of the offspring were significantly increased (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). In the cell culture of the PCB group, after adding the inhibitor GSK126, Western blotting detected that the expression levels of EZH2 and its target molecule H3K27me3 were significantly decreased(Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eEndometriosis is a common refractory gynecological disease. Although it is estrogen-dependent benign disease, its biological behavior is similar to that of malignant tumors (Zhang et al. \u003cspan class=\"CitationRef\"\u003e2017\u003c/span\u003e). The migration, adhesion and invasion of retrograde endometrial cells are important steps in the formation of ectopic lesions (Gaetje et al. \u003cspan class=\"CitationRef\"\u003e1995\u003c/span\u003e; Meng et al. \u003cspan class=\"CitationRef\"\u003e2019\u003c/span\u003e; Witz et al. \u003cspan class=\"CitationRef\"\u003e2001\u003c/span\u003e). Previous studies have reported that PCBs increase the invasion of ESCs, as well as the incidence of endometriosis (Hu et al. \u003cspan class=\"CitationRef\"\u003e2018\u003c/span\u003e; Ploteau et al. \u003cspan class=\"CitationRef\"\u003e2016\u003c/span\u003e; Ploteau et al. \u003cspan class=\"CitationRef\"\u003e2017\u003c/span\u003e; Roy et al. \u003cspan class=\"CitationRef\"\u003e2015\u003c/span\u003e). However, little is known regarding the relationship between gestational PCBs in offspring endometriosis, and the role of histone methylation is not fully understood. In the present study, it was found that prenatal exposure to PCBs significantly increase the invasive ability of offspring ectopic ESCs with high expression levels of EZH2 and H3K27me3, which serve an important role in promoting this proses.\u003c/p\u003e\n\u003cp\u003eAn increase in the number and length of microvilli was the main morphological change observe in the present study. The relationship between cell morphology and cell invasiveness has been widely studied in tumors (Antonescu and Baren \u003cspan class=\"CitationRef\"\u003e2004\u003c/span\u003e; Aznavoorian et al. \u003cspan class=\"CitationRef\"\u003e1993\u003c/span\u003e). The ultrastructure of the cell surface can predict the contact, movement and adhesion of cells to other cells and the cell matrix, which serves an important role in the occurrence and development of tumors (Black \u003cspan class=\"CitationRef\"\u003e1980\u003c/span\u003e; Nicolson and Poste \u003cspan class=\"CitationRef\"\u003e1976\u003c/span\u003e). A decrease of tumor cell invasiveness is often accompanied by the loss of microvilli (Priester et al. \u003cspan class=\"CitationRef\"\u003e2013\u003c/span\u003e), while more microvilli and pseudopodia on the cell surface indicates a higher invasive ability of tumor cells (Yan et al. \u003cspan class=\"CitationRef\"\u003e2013\u003c/span\u003e). In patients with endometriosis, the morphology of endometrial gland epithelial cells (EGECs) is irregular, the number and length of microvilli is increased, the organelles are abundant and EGECs possess proliferative, migratory and invasive abilities, which are associated with the formation of endometriosis cysts (Yu et al. \u003cspan class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003eIt has been reported that Aroclor1221 is a mildly chlorinated PCBs mixture, and its level is difficult to determine directly in the offspring after gestational exposure (Thomas et al. \u003cspan class=\"CitationRef\"\u003e1998\u003c/span\u003e). According to previous literature reports (Bell et al. \u003cspan class=\"CitationRef\"\u003e2016\u003c/span\u003e; Reilly et al. \u003cspan class=\"CitationRef\"\u003e2015\u003c/span\u003e; Steinberg et al. \u003cspan class=\"CitationRef\"\u003e2007\u003c/span\u003e; Steinberg et al. \u003cspan class=\"CitationRef\"\u003e2008\u003c/span\u003e; Walker et al. \u003cspan class=\"CitationRef\"\u003e2013\u003c/span\u003e), the dose of 1 mg/kg intraperitoneal injection of Aroclor1221 at E16 and E18 is close to the observed exposure of humans and wild animals in PCBs contaminated areas. PCBs may inhibit the activity of human estrogen sulfotransferase, an estrogen-degrading enzyme (Kester et al. \u003cspan class=\"CitationRef\"\u003e2000\u003c/span\u003e), thereby increasing the secretion of estrogen and the risk of hormone-dependent diseases. Accumulating evidence had suggested that higher PCBs exposure may increase the risk of endometriosis (Hu et al. \u003cspan class=\"CitationRef\"\u003e2018\u003c/span\u003e; Martinez-Zamora et al. \u003cspan class=\"CitationRef\"\u003e2015\u003c/span\u003e; Porpora et al. \u003cspan class=\"CitationRef\"\u003e2006\u003c/span\u003e; Wei et al. \u003cspan class=\"CitationRef\"\u003e2016\u003c/span\u003e). A reduced responsiveness to progesterone and overexpression of matrix metalloproteinases may be a key element of endometrial dysfunction associated with endometriosis (Osteen et al. \u003cspan class=\"CitationRef\"\u003e2005\u003c/span\u003e). With similar effects to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), dioxin-like-PCBs can combine with aryl hydrocarbon receptor(AhR) and form an activated heterodimerization complex with the aromatic hydrocarbon receptor nuclear translocator protein, leading to the overexpression of AhR in endometrioid cells (Okino and Whitlock \u003cspan class=\"CitationRef\"\u003e2000\u003c/span\u003e; Sherr and Monti \u003cspan class=\"CitationRef\"\u003e2013\u003c/span\u003e). Over activated AhR induced by PCBs regulates multiple downstream biological events, including the epigenetic silencer activity of histone methyltransferase EZH2 (Lee et al. \u003cspan class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003eEpigenetics refers to the heritable non-gene sequence changes, including DNA methylation, post-translational histone modification and microRNA. However, these are dynamic and may be affected by environmental exposures (Holland \u003cspan class=\"CitationRef\"\u003e2017\u003c/span\u003e). Pregnancy is a critical period of fetal epigenetic remodeling, which is sensitive to environmental endocrine disruptors (Casati et al. \u003cspan class=\"CitationRef\"\u003e2015\u003c/span\u003e; Pozharny et al. \u003cspan class=\"CitationRef\"\u003e2010\u003c/span\u003e). Maternal health can determine susceptibility to certain diseases in adulthood (Kanherkar et al. \u003cspan class=\"CitationRef\"\u003e2014\u003c/span\u003e). Moreover, gestational PCBs exposure not only induces an aberrant DNA methylation pattern, but also affect the histone modification balance, leading to long term and transgenerational effects (Casati et al. \u003cspan class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003eHistone methylation has been shown to serve an important role in the etiology of endometriosis, and EZH2 and H3K27me3 are epigenetic marks of endometriosis (Colon-Caraballo et al. \u003cspan class=\"CitationRef\"\u003e2015\u003c/span\u003e). Zhang \u003cem\u003eet al\u003c/em\u003e (Zhang et al. \u003cspan class=\"CitationRef\"\u003e2017\u003c/span\u003e) revealed that EZH2 was associated with the invasion of the ectopic endometrium, and that the levels of EZH2 and H3K27me3 in the ectopic endometrium were significantly higher compared with those in normal endometrium. EZH2 and H3K27me3 directly induce epithelial-mesenchymal transition, thereby promoting the occurrence and development of endometriosis (Cao et al. \u003cspan class=\"CitationRef\"\u003e2008\u003c/span\u003e; Colon-Caraballo et al. \u003cspan class=\"CitationRef\"\u003e2015\u003c/span\u003e; Zhang et al. \u003cspan class=\"CitationRef\"\u003e2017\u003c/span\u003e). The present study also identified similar findings; after PCBs exposure during pregnancy, the expression levels of EZH2 and H3K27me3 in ectopic ESCs were significantly increased and the cell invasive ability was enhanced. However, after inhibition of EZH2 using GSK126, the expression levels of EZH2 and H3K27me3 were decreased, accompanied by a decreased cell invasive ability, suggesting that EZH2 serves an important role in ectopic endometrial invasion caused by PCBs exposure during pregnancy.\u003c/p\u003e\n\u003cp\u003eIn conclusion, PCBs exposure during pregnancy serves a significant role in the development and progression of endometriosis in offspring. As an early stage of life development, women should consider the effects of environmental pollution during pregnancy with regards to potential harm to next generations. EZH2-mediated epigenetic regulation may serve an important role in this process. However, further research is required to identify the underlying molecular mechanisms activated by EZH2 and H3K27me3.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was approved by the Ethics Committee of Zhejiang Chinese Medical\u0026nbsp;University (approval no. 2018102904).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePatient consent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analyzed during this study are included in this published article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was funded by a grant from the Zhejiang Provincial Key Research \u0026amp;\u0026nbsp;Development Project Grants (grant no.2021C03095) and Education Department of\u0026nbsp;Zhejiang Province (grant no. 2019150).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors' contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWYF performed the experiments，HCC wrote the manuscript, YCS performed the\u0026nbsp;experiments, CP and CCLanalyzed the data. WRJ designed the study. All authors read\u0026nbsp;and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e"},{"header":"References","content":"\u003cp\u003eAbraham K, Papke O, Gross A, Kordonouri O, Wiegand S, Wahn U, Helge H (1998) Time course of PCDD/PCDF/PCB concentrations in breast-feeding mothers and their infants Chemosphere 37:1731-1741\u003c/p\u003e\n\u003cp\u003eAdams EM, von Hippel FA, Hungate BA, Buck CL (2019) Polychlorinated biphenyl (PCB) contamination of subsistence species on Unalaska Island in the Aleutian Archipelago Heliyon 5:e02989 doi:10.1016/j.heliyon.2019.e02989\u003c/p\u003e\n\u003cp\u003eAltayyeb A et al. 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(2018) Correlation between replicative senescence of endometrial gland epithelial cells in shedding and non-shedding endometria and endometriosis cyst during menstruation Gynecol Endocrinol 34:981-986 doi:10.1080/09513590.2018.1480709\u003c/p\u003e\n\u003cp\u003eZhang Q, Dong P, Liu X, Sakuragi N, Guo SW (2017) Enhancer of Zeste homolog 2 (EZH2) induces epithelial-mesenchymal transition in endometriosis Sci Rep 7:6804 doi:10.1038/s41598-017-06920-7\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"polychlorinated biphenyls, pregnancy, endometriosis, invasion, epigenetics, EZH2","lastPublishedDoi":"10.21203/rs.3.rs-328521/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-328521/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePolychlorinated biphenyls(PCBs) are persistent environmental endocrine disruptor. This study aim to investigate the changes of the ectopic endometrium invasive ability and the possible mechanism after exposure to PCBs during pregnancy. In total, 12 female Sprague Dawley rats were intraperitoneally injected with Aroclor 1221 (1 mg/kg) or dimethyl sulfoxide (1 mg/kg) at 16 and 18 days of gestation. The endometriosis model was established by autogenous uterine abdominal wall implantation 2 months after birth. The degree of adhesion between the endometriosis and the greater omentum, as well as cell morphology and Transwell invasion patterns were used to evaluate the invasive ability of progeny ectopic endometrial stromal cells (ESCs). Moreover, the effect of the enhancer of zeste 2 polycomb repressive complex 2 subunit (EZH2)/ trimethylation of Histone 3 lysine 27 (H3K27me3) axis was examined using a highly selective EZH2 inhibitor, GSK126. After gestational PCBs exposure, the adhesion between the endometrium and the greater omentum was enhanced (P\u0026lt;0.05), and the length and number of microvilli were significantly increased (P\u0026lt;0.01). Exposure to PCBs during pregnancy increased the invasive ability of the ectopic ESCs (P\u0026lt;0.01), with upregulated expression levels of EZH2 and H3k27me3, which were abrogated by the EZH2 inhibitor GSK126. In conclusion, exposure to PCBs during pregnancy increased the invasive ability of the ectopic endometrium, which may be mediated by EZH2.\u003c/p\u003e","manuscriptTitle":"Prenatal polychlorinated biphenyl exposure promotes invasion of progeny ectopic endometrial stromal cells via epigenetic modification of EZH2","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2021-04-01 20:35:07","doi":"10.21203/rs.3.rs-328521/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"809f368c-192b-407d-afed-e854c6405937","owner":[],"postedDate":"April 1st, 2021","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":3370100,"name":"Environmental Engineering"},{"id":3370101,"name":"Environmental Policy"}],"tags":[],"updatedAt":"2021-07-11T23:21:33+00:00","versionOfRecord":[],"versionCreatedAt":"2021-04-01 20:35:07","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-328521","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-328521","identity":"rs-328521","version":["v1"]},"buildId":"WvIrzKhiLBfengagbw6Ux","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}