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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 (ERβ) 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 ERβ and PR as the control dESC. CONCLUSION(S) This study reveals that high doses of phytoestrogens could affect the in vitro decidualization process.