Phthalates Impair Estrogenic Regulation of HIF2α and Extracellular Vesicle Secretion by Human Endometrial Stromal Cells

High levels of exposure to Di(2-ethylhexyl) phthalate (DEHP), a known endocrine disruptor, have been linked to adverse pregnancy outcomes, yet the mechanisms by which it impacts human uterine functions remain unclear. Here, we report that exposure of differentiating primary human endometrial stromal cells (HESC) to an environmentally relevant concentration of DEHP or its primary metabolite MEHP markedly reduces the expression of the estrogen-regulated transcription factor hypoxia-inducible factor 2 alpha (HIF2α). We also noticed a simultaneous decrease in RAB27B expression, which is crucial for the trafficking and secretion of extracellular vesicles (EVs). EVs enhance communication among various cell types within the pregnant uterus, thereby ensuring reproductive success. We found that estrogen receptor alpha (ERα) could no longer bind to the HIF2α regulatory region following phthalate treatment, and epigenetic analysis suggested that this may be due to hypermethylation of nearby CpG islands. Further investigation revealed a potential interaction between ERα and the transcription factor Sp1 within the HIF2α regulatory region, which is affected by the inhibition of Sp1 binding to the phthalate-induced hypermethylated DNA. Additionally, our results suggest that the abnormal DNA methylation is likely due to increased expression of the DNA methyltransferase 1 (DNMT1) gene in response to phthalate exposure. Overall, this study provides valuable mechanistic insights into how phthalate-induced differential DNA methylation disrupts estrogenic regulation of the HIF2α gene and, consequently, EV secretion during HESC differentiation. This knowledge is essential for understanding how phthalates may lead to adverse reproductive outcomes by disrupting hormonal regulation of cell-to-cell communication in the uterus.