THU633 Atonal BHLH Transcription Factor 8 (ATOH8) Functions In Decidualization

Abstract

Abstract Disclosure: R. Thapa: None. B. Bany: None. ATOH8 is a member of the vast basic helix-loop-helix (bHLH) protein family that is widely known to play a role in various physiological and pathological processes. However, the functional significance of ATOH8 expression in the endometrium during implantation and in the first trimester of pregnancy in women is still lacking. In the mid to late secretory phase of the menstrual cycle, elongated fibroblast-like mesenchymal cells in the uterine endometrium begin to trans-differentiate into polygonal epithelioid-like (decidual) cells. During pregnancy, this process continues more broadly and supports successful embryo implantation and early placental development. We employed an established in vitro model using uterine fibroblast (HuF) cells derived from the decidua parietalis of term patients to investigate the function of ATOH8 during in vitro decidualization. All experiments were carried out using cells isolated from at least six independent patients (N=6). We measured the expression of classical decidualization marker genes and the characteristic shape change from small fibroblast-like cells to large epitheloid-like decidual cells to assess decidualization. Treating cells with steroids plus a membrane-permeable cAMP analog (EPC) for 48h caused in vitro decidualization of these cells, while treatment with vehicle alone had no effect. We discovered that ATOH8 expression was dramatically upregulated during this decidualization at both the mRNA and protein levels, with the protein primarily localized to the nucleus. The functional role of ATOH8 during decidualization was investigated using lentivirus-mediated shRNA knockdown or overexpression of ATOH8. When ATOH8 expression was silenced, we discovered that the ability of the cells to undergo decidualization was greatly and significantly diminished. On the other hand, overexpression of ATOH8 enhanced some aspects of decidualization. The addition of recombinant BMP2 alone had little effect on ATOH8 expression. However, in the presence of EPC it significantly enhanced ATOH8 expression. This effect of BMP2 on ATOH8 expression was inhibited by preincubating the cells with 500 nM DMH-1 (an ALK2/3 inhibitor) for one hr prior to inducing decidualization. Steroids alone did not affect ATOH8 or BMP2 expression. However, the addition of cAMP alone represented the major effect of EPC on ATOH8 expression while having a slight impact on BMP2 expression. A robust increase in BMP2 expression required the presence of both steroids and cAMP together. Our results indicate that ATOH8 plays a crucial role in human endometrial stromal cell decidualization. Further, our results suggest that ALK2/3 receptors are most likely involved in mediating the effect of BMP2 on increasing ATOH8 expression. Keywords: ATOH8, Decidualization, Embryo Implantation Presentation: Thursday, June 15, 2023