Overexpression of estrogen receptor β (ERβ) in endometrium contributes to endometriosis (EM) pathogenesis. Trimethylation of the H3 lysine (K) 4 (H3K4me3) in promoters is strongly correlated with gene expression. This study aimed to explore the effects of bisphenol A (BPA) exposure on EM development from the perspective of the regulation of ERβ expression in eutopic endometrium via the H3K4me3-related epigenetic pathway. A mouse EM model was established to investigate the effects of BPA. Immortalized human normal endometrial stromal cells (iESCs) were cultured and treated with BPA to explore the underlying mechanism. Eutopic endometria from patients with or without EM were collected and analyzed. Results showed that BPA elevated ERβ expression in mouse eutopic endometrium and promoted lesion growth. BPA also promoted WD repeat domain 5 (WDR5) expression and upregulated H3K4me3 levels in the ERβ promoter and Exon 1. Further research indicated that WDR5 interacted with tet methylcytosine dioxygenase 2 (TET2), while BPA exposure enhanced the interaction between these two proteins, promoted the recruitment of the WDR5/TET2 complex to the ERβ promoter and Exon 1, and inhibited DNA methylation of CpG islands. The WDR5/TET2 interaction was essential for BPA-induced ERβ overexpression. Enhanced WDR5/TET2 interaction was also observed in eutopic endometria from EM patients. Further results showed that BPA upregulated WDR5 expression through the G protein-coupled estrogen receptor (GPER)-mediated PI3K/mTOR signaling pathway. In conclusion, our study suggests that BPA exposure promotes EM development by upregulating ERβ expression in eutopic endometrium via the WDR5/TET2-mediated epigenetic pathway.

Fipronil, a phenyl-pyrazole insecticide, has a wide range of uses, from agriculture to veterinary medicine. Due to its large-scale applications, the risk of environmental and occupational exposure and bioaccumulation raises concerns. Moreover, relatively little is known about the intracellular mechanisms of fipronil in trophoblasts and the endometrium involved in implantation. Here, we demonstrated that fipronil reduced the viability of porcine trophectoderm and luminal epithelial cells. Fipronil induced cell cycle arrest at the sub-G1 phase and apoptotic cell death through DNA fragmentation and inhibition of DNA replication. These reactions were accompanied by homeostatic changes, including mitochondrial depolarization and cytosolic calcium depletion. In addition, we found that exposure to fipronil compromised the migration and implantation ability of pTr and pLE cells. Moreover, alterations in PI3K-AKT and MAPK-ERK1/2 signal transduction were observed in fipronil-treated pTr and pLE cells. Finally, the antiproliferative and apoptotic effects of fipronil were also demonstrated in 3D cell culture conditions. In summary, our results suggest that fipronil impairs implantation potentials in fetal trophectoderm and maternal endometrial cells during early pregnancy.

Tris (4-hydroxyphenyl)ethane (THPE), a trisphenol compound widely used as a branching agent and raw material in plastics, adhesives, and coatings is rarely regarded with concern. However, inspection of in vitro data suggests that THPE is an antagonist of estrogen receptors (ERs). Accordingly, we aimed to evaluate the antiestrogenicity of THPE in vivo and tested its effect via oral gavage on pubertal development in female CD-1 mice. Using uterotrophic assays, we found that THPE either singly, or combined with 17β-estradiol (E₂) (400 μg/kg bw/day) suppressed the uterine weights at low doses (0.1, 0.3, and 1 mg/kg bw/day) in 3-day treatment of weaning mice. When mice were treated with THPE during adolescence (for 10 days beginning on postnatal day 24), their uterine development was significantly retarded at doses of at least 0.1 mg/kg bw/day, manifest as decreased uterine weight, atrophic endometrial stromal cells and thinner columnar epithelial cells. Transcriptome analyses of uteri demonstrated that estrogen-responsive genes were significantly downregulated by THPE. Molecular docking shows that THPE fits well into the antagonist pocket of human ERα. These results indicate that THPE possesses strong antiestrogenicity in vivo and can disrupt normal female development in mice at very low dosages.

Endocrine-disrupting chemicals (EDCs) are widely used in various consumer goods. Consequently, humans are constantly exposed to EDCs, which is associated with a variety of endocrine-related diseases. In this study, we demonstrated the effects of bisphenol A (BPA), benzyl butyl phthalate (BBP), and di(2-ethylhexyl) phthalate (DEHP) on estrogen receptor alpha (ERα) expression under normoxia and hypoxia. First, we confirmed the effects of EDCs on ER activity using OECD Test Guideline 455. Compared to the 100% activity induced by 1 nM 17-β-estradiol (positive control), BPA and BBP exhibited 50% ERα activation at concentrations of 1.31 μM and 4.8 μM, respectively. In contrast, and consistent with previous reports, DEHP did not activate ERα. ERα is activated and degraded by hypoxia in breast cancer cells. BPA, BBP, and DEHP enhanced ERα-mediated transcriptional activity under hypoxia. All three EDCs decreased ERα protein levels under hypoxia in MCF-7 cells. The transcriptional activity of hypoxia-inducible factor-1 was decreased and secretion of vascular endothelial growth factor (VEGF) was increased by BPA and BBP under hypoxia in MCF-7 cells, but not by DEHP. All three EDCs decreased the ERα protein expression level in Ishikawa human endometrial adenocarcinoma cells, and DEHP caused a weak decrease in VEGF secretion under hypoxia. These results demonstrate down-regulation of ERα by EDCs may influence the pathological state associated with hypoxia.

Etoxazole is an organofluorine insecticide widely used in agriculture. Exposure to insecticides is a serious environmental problem owing to their cytotoxic effects in humans and animals. Reproductive toxicity of various organofluorine insecticides have been shown in previous studies. However, few studies have evaluated the toxicity of etoxazole in mammals. We aimed to examine the toxic effects of etoxazole in porcine trophectoderm (pTr) and uterine luminal epithelial (pLE) cells. To estimate the effects of etoxazole, we conducted assays after treatment with multiple concentration of etoxazole (0, 2, 4, 6 and 9 μM) to pTr and pLE cells for 0–72 h. Etoxazole decreased the cell proliferation, viability, and migration of pTr and pLE cells. Further, etoxazole induced apoptosis via cell cycle arrest and disruption of mitochondrial membrane potential. We also found that pro-apoptotic proteins and endoplasmic reticulum (ER) stress-response proteins were activated in response to etoxazole. Finally, we observed that etoxazole altered the PI3K/AKT and MAPK signaling pathways and the mRNA expression of genes associated with implantation. Collectively, these results suggest that etoxazole disrupts normal cellular physiology and might cause early implantation failure.

It has been suggested that the toxic effects of cadmium (Cd) may disrupt ovarian and uterine functions in adults. However, Cd exposure during gestation and lactation and its effects on the reproductive development in female offspring is still not clear, and the mechanisms underlying exposure toxicology remain mostly unexplored. To investigate how Cd exposure of female rats (F0) during gestation and lactation affects the reproductive development of their female offspring, we studied the steroidogenesis, folliculogenesis, puberty onset, and litter size of the first (F1) and second (F2) filial generations following F0 female rats which had been exposed to CdCl2. The mechanisms related to the early onset of puberty induced by such exposure in female offspring were explored. Maternal exposure to Cd dramatically increased the biosynthesis of steroid hormones in F1 female offspring by the activation of cAMP/PKA pathway and up-regulated expression of steroidogenesis related proteins such as StAR, CYP11A1, 3β-HSD and CYP19A1. The high levels of steroid hormones contributed to an early puberty onset, promoted the differentiation and maturation of follicles, and led to the proliferation of endometrium that resulted in a uterus weight gain. The increased number of antral follicles eventually caused a big litter size. Despite of being free from additional Cd exposure, the levels of CYP11A1 and CYP19A1 in the ovaries of F2 female rats were also high, which resulted in a high concentration of serum progesterone. These results suggested that hormonal changes induced by exposure to Cd in utero might have a lasting effect beyond the first generation. These findings may help to better understand the origin of female sexual dysfunction in the developmental stages in general.

Benzo[a]pyrene (BaP) is a ubiquitous environmental persistent organic pollutant and a well-known endocrine disruptor. BaP exposure could alter the steroid balance in females. Endometrium decidualization and decidual angiogenesis are critical events for embryo implantation and pregnancy maintenance during early pregnancy and are modulated by steroids. However, the effect of BaP on decidualization is not clear. This study aimed to explore the effects of BaP on decidualization and decidual angiogenesis in pregnant mice. The result showed that the uteri in the BaP-treated groups were smaller and exhibited an uneven size compared with those in the control group. Artificial decidualization was detected in the uteri of the controls, but weakened decidualization response was observed in the BaP-treated groups. BaP significantly reduced the levels of estradiol, progesterone, and their cognate receptors ER and PR, respectively. The expression of several decidualization-related factors, including FOXO1, HoxA10, and BMP2, were altered after BaP treatment. BaP reduced the expression of cluster designation 34 (CD34), which indicated that the decidual angiogenesis was inhibited by BaP treatment. In addition, BaP induced the downregulation of vascular endothelial growth factor A. These data suggest that oral BaP ingestion compromised decidualization and decidual angiogenesis. Our results provide experimental data for the maternal reproductive toxicity of BaP during early pregnancy, which is very important for a comprehensive risk assessment of BaP on human reproductive health.

Butylated hydroxytoluene (BHT), one of the most widely used synthetic phenolic antioxidants, is a popular food additive. Previous studies have reported the possible health hazards of BHT. However, BHT effects on female reproduction, especially on endometrial decidualization, are still unknown. During early pregnancy, decidualization plays important roles for embryo implantation and pregnancy establishment. This study aimed to explore the effects of BHT on endometrial decidualization in pregnant mice. The pregnant mice received BHT via intraperitoneal injection at doses of 0, 200, and 400 mg/kg/day from day 1 (D1) of pregnancy until sacrifice. Under BHT exposure, maternal body weight was significantly decreased during early pregnancy. Compared with the control group, the number of implantation sites and uterine weight were significantly reduced in the BHT groups. The uterine lumen failed to close after BHT exposure, and the decidual morphology of endometrial stromal cells was inhibited by BHT. Furthermore, BHT significantly decreased the expression of endometrial decidual markers including COX2, HOXA10, and MMP9. Notably, the levels of serum estrogen (E2) and progesterone (P4) and expression levels of uterus estrogen receptor α (ERα) and progesterone receptor (PR) during early pregnancy were significantly upregulated following BHT exposure. In conclusion, these results demonstrated that gestational BHT exposure could inhibit decidualization of mouse endometrium during early pregnancy. The disorders of reproductive hormones and changes of hormone receptor signals could be responsible for the impaired decidualization. This study provided new evidence for the deleterious effects of BHT on female reproduction and revealed the potential reproductive toxicity of synthetic phenolic antioxidants.

Fluorene-9-bisphenol (BHPF), a new derivative of bisphenol A (BPA), has been introduced for treatment with estrogen-related tumors, such as endometrial cancer. This study investigated the potential mechanism underlying the action of BHPF against endometrial cancer in vitro. We used the cell counting kit-8 (CCK8) method on Ishikawa cells to screen sub-lethal doses of BHPF and established the optimal concentration at which BHPF influenced the proliferation of Ishikawa cells. Effect of BHPF on cell migration and invasion was investigated by cell scratch assay and transwell assay, respectively. Expression levels of epithelial-mesenchymal transition (EMT)–related proteins were detected by Western blot analysis. BHPF was found to inhibit the proliferation of Ishikawa cells, whose migration and invasion abilities were also reduced. Western blot indicated that BHPF can significantly inhibit the EMT process of Ishikawa cells by blocking transforming growth factor-β (TGF-β) signaling pathway. This is the first report of the effect of BHPF on the biological behavior of endometrial cancer cells and its inhibition of endometrial cancer progression by repressing both endometrial cell proliferation and epithelial-mesenchymal transition, hence suggesting it as a novel anti-cancer drug. Graphical abstract Schematic representation of the molecular basis underlying BHPF treatment. BHPF repressed the EMT process by regulating EMT-related genes, such as E-cadherin, N-cadherin, and vimentin as well as the TGF-β signaling pathway–related genes, including p-Smad2/3 and slug, in a BHPF-dependent manner.

Chlorpyrifos (CPF), as a worldwide pesticide, can effect on the integrins αv and β3 which play a main role in the implantation window. Therefore, the aim of this study was to consider CPF effects on integrin alpha v and beta 3 in implantation window phase. Thirty female NMRI mice were separated into groups of CPF, sham, and control. After 6 weeks, each group was mated, and on the 5th day of gestation, all mice were euthanized. Estradiol and progesterone levels were detected by the enzyme-linked immunosorbent assay (ELISA) test; two subunits of integrins (αv and β3) genes and proteins of endometrium were analyzed by real-time polymerase chain reaction (RT-PCR) and immunohistochemistry method, respectively. Fibrosis of the liver which evaluated by Masson’s trichrome stain was increased in the CPF group compared with the others. But estradiol and progesterone levels were significantly decreased in CPF groups. Based on the findings, the proportion of genes’ expressions of integrin subunits declined by the effect of CPF, while there was not any notable consequence on mice in the sham group. Alpha v and beta 3 integrin proteins expressed in all groups, but the concentration of these proteins in CPF groups was lower than in other groups. This study has shown that the decline of estradiol and progesterone downregulates the expression of αv and β3 integrins which were influenced by CPF exposure. Changing these patterns of proteins could have numerous influences on unsuccessful implantation. Therefore, this experimental study recommends that inclusive consideration of the effects of insecticides may be crucial to women’s unrecognized cause of infertility.