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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 mM. 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 0 :5 0 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 (ERb) and P receptor (PR) localization were evaluated by immunofluorescence. Result(s): The ESC exposed to 0, 19, 20, 50, and 100 mM of genistein, daidzein, and genistein þ daidzein showed a dose-dependent proliferation decrease. After 48-96 hours of culture, this reduction was significant in the presence of 50 mM of phytoestrogens versus 10 mM 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 ERb and PR as the control dESC. Conclusion(s):This study reveals that high doses of phytoestrogens could affect the in vitro decidualization process.

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Context Non-classical membrane progesterone receptor (mPRs) and PGRMC1 expression have been detected in endometrium, but their role in decidualization was not yet investigated. We previously demonstrated PGRMC1 downregulation in receptive endometrium and that its overexpression inhibits decidualization. Furthermore, during decidualization, PGRMC1 mainly interacts with proteins involved in biosynthesis, intracellular transport and mitochondrial activity. Objective To determine PGRMC1 and mPRs signaling role during decidualization. Design and Interventions Isolated primary endometrial stromal cells (EnSC) were in vitro decidualized in presence of classic stimuli (E2+P4), PGRMC1 inhibitor (AG205), or membrane-impermeable P4 (P4-BSA). Setting and Participants Endometrial biopsies from 19 fertile oocyte donors attending IVI-Valencia IVF clinic. Main Outcome Measure(s) EnSC decidualization was evaluated by prolactin ELISA and F-actin immunostaining. Progesterone receptor localization was evaluated by immunofluorescence. EnSC transcriptomic profiles were analyzed by microarray technology. Result(s) PGRMC1 inhibition during EnSC decidualization (AG205dEnSC) does not interfere with EnSC cytoskeletal rearrangements and prolactin secretion. However, global transcriptional profiling revealed more differentially expressed genes in AG205dEnSC than in dEnSC, compared with non-decidualized EnSC (ndEnSC). In silico analysis showed that PGRMC1 inhibition upregulated more genes related to metabolism, molecular transport, and hormonal biosynthesis compared to control dEnSC. EnSC decidualized in the presence of P4-BSA showed a similar behavior as ndEnSC in terms of morphological features, absence of prolactin secretion, and transcriptomic pattern. Conclusion(s) Our findings associate PGRMC1 to hormonal biosynthesis, metabolism, and vesicular transport—important cellular functions for dEnSC supporting pregnancy. Activation of membrane P4 receptor signaling alone was unable to induce downstream effects needed for proper decidualization.

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