The regulatory role of endometrial gland on decidualization process

Abstract

The maternal-fetus interface consists of endometrial glands, stromal cells, immune cells, trophoblasts and endothelial cells, is essential for a successful pregnancy. Defective endometrial glandular development and/or functions is associated with infertility and pregnancy complications, though the pathogenesis is unknown. This project hypothesizes that endometrial glands modulate stromal cells and immune cell functions at the fetal-maternal interface. In this study, a human endometrial glandular organoid (EEO) system was established using primary endometrial gland tissue. The derived EEOs demonstrated the glandular epithelial origin by expressing the specific glandular markers. The resemblance between in vitro generated EEOs and the in vivo ones was supported by single-cell RNA-seq analysis. Moreover, we have cultured EEOs in hormonal conditions mimicking the proliferative phase (Estrogen, E2), secretory phase (E2+progesterone, P4) and early pregnancy (E2+P4+human chorionic gonadotropin, hCG). Our results showed that EEOs were hormone responsive and hormonal treatments can modulate their secretome. Decidualization is essential for the establishment/maintenance of pregnancy. It involves the differentiation of endometrial stromal cells (ESCs) and the recruitment of leukocytes. To investigate the role of endometrial glands on stromal cell decidualization, a co-culture model was established using EEO and primary/telomerase-transformed human ESCs (T-HESCs). The results showed that EEO secretome suppressed the decidualization of ESCs in a time and EEO amount dependent manner. The transcriptome of the primary ESCs before and after EEO co-culture were also compared. Based on the differentially expressed genes, protein association network analysis and literature search, 9 genes in ESCs were identified as potential genes associated with the suppressive effect of EEO on decidualization. We have then employed in vitro co-culture model and in vivo endometrial hyperplasia model to validate the results and identified CEBPA (CCAAT enhancer-binding protein alpha) and EDN1 (Endothelin 1) were closely related to the suppressive effect of EEO. Decidual macrophages (dMs) function to regulate maternal immune tolerance and remodel the placenta. However, the factors regulating the differentiation/functions of dMs remains unclear. Therefore, another objective of this project is to determine the regulatory role of EEO on macrophage functions. Human monocytes were isolated from female blood and were differentiated into macrophages using macrophage colony-stimulating factor (MCSF). The inclusion of the E2+P4-treated EEO secretome during the differentiation process increased indoleamine 2, 3-dioxygenase 1 (IDO-1) expression in the resulting macrophages. Interestingly, IDO-1 is an immunoregulatory enzyme marker of dMs. Cytokine array further demonstrated altered expressions of migration inhibitory factor (MIF), serpin family E member 1 (Serpin E1), granulocyte colony-stimulating factor (GCSF), interleukin-6 (IL-6) and macrophage inflammatory protein 1α (MIP-1α) in macrophage after EEO secretome treatment. All these cytokines are known to be related to immune tolerance and placenta development during pregnancy. Moreover, the phagocytosis of the MCSF-differentiated macrophages was induced by the secretome of hormones treated EEOs. In conclusion, the results of this study not only demonstrated that EEO could be a reliable model to study the endometrial gland functions in humans, but also provide confirmatory evidence on the maternal regulation of stromal cell decidualization and dM functions via the endometrial glandular secretion. (494 words)