RESUMEN
Mercury (Hg) cytotoxicity, which is largely mediated through oxidative stress (OS), can be relieved with antioxidants. Thus, we aimed to study the effects of Hg alone or in combination with 5 nM N-Acetyl-L-cysteine (NAC) on the primary endometrial cells' viability and function. Primary human endometrial epithelial cells (hEnEC) and stromal cells (hEnSC) were isolated from 44 endometrial biopsies obtained from healthy donors. The viability of treated endometrial and JEG-3 trophoblast cells was evaluated via tetrazolium salt metabolism. Cell death and DNA integrity were quantified following annexin V and TUNEL staining, while the reactive oxygen species (ROS) levels were quantified following DCFDA staining. Decidualization was assessed through secreted prolactin and the insulin-like growth factor-binding protein 1 (IGFBP1) in cultured media. JEG-3 spheroids were co-cultured with the hEnEC and decidual hEnSC to assess trophoblast adhesion and outgrowth on the decidual stroma, respectively. Hg compromised cell viability and amplified ROS production in trophoblast and endometrial cells and exacerbated cell death and DNA damage in trophoblast cells, impairing trophoblast adhesion and outgrowth. NAC supplementation significantly restored cell viability, trophoblast adhesion, and outgrowth. As these effects were accompanied by the significant decline in ROS production, our findings originally describe how implantation-related endometrial cell functions are restored in Hg-treated primary human endometrial co-cultures by antioxidant supplementation.
Asunto(s)
Antioxidantes , Endometrio , Femenino , Humanos , Antioxidantes/farmacología , Antioxidantes/metabolismo , Línea Celular Tumoral , Especies Reactivas de Oxígeno/metabolismo , Endometrio/metabolismo , Implantación del Embrión/fisiología , Trofoblastos/metabolismo , Suplementos Dietéticos , Células del Estroma/metabolismo , Decidua , Células CultivadasRESUMEN
OBJECTIVE: To investigate whether phytoestrogens (genistein and daidzein) alter in vitro decidualization of human endometrial stromal cells (ESCs). DESIGN: Isolated primary ESCs were exposed to phytoestrogens and decidualized in vitro. SETTING: Academic fertility center. PATIENT(S): Twenty fertile oocyte donors attending the IVI Valencia clinic. INTERVENTION(S): Treatment of ESC with phytoestrogens at 0, 10, 20, 50, and 100 µM. MAIN OUTCOME MEASURE(S): The ESC proliferation was analyzed by MTS assay. In vitro decidualization was induced in the presence of phytoestrogens by medroxyprogesterone acetate/cyclic adenosine 3':5' monophosphate and evaluated by prolactin (PRL) ELISA and F-actin immunostaining. The Ki67 proliferative marker was analyzed by immunofluorescence. The ESC apoptosis was assessed by annexin V/propidium iodide detection using flow cytometry. Estrogen (ERß) and P receptor (PR) localization were evaluated by immunofluorescence. RESULT(S): The ESC exposed to 0, 19, 20, 50, and 100 µM of genistein, daidzein, and genistein + daidzein showed a dose-dependent proliferation decrease. After 48-96 hours of culture, this reduction was significant in the presence of 50 µM of phytoestrogens versus 10 µM untreated ESC. The ESC decidualized in the presence of phytoestrogens did not rearrange their cytoskeletons and showed a significant decrease in PRL secretion compared with untreated decidualized ESCs (dESCs). However, phytoestrogens did not alter proliferative status or the percentage of viable/apoptotic cells in dESC compared with untreated dESC. During decidualization, phytoestrogens induced the same nuclear translocation of ERß and PR as the control dESC. CONCLUSION(S): This study reveals that high doses of phytoestrogens could affect the in vitro decidualization process.