Project Summary Decidualization is defined as the transformation of endometrial stromal fibroblasts into a specialized secretory decidual cell. This transformation is essential for embryo implantation and placental development. Prior to the onset of decidualization, the endometrium must transition into a receptive state and implantation requires a precise synchronization between the embryo and the receptive uterus. In primates decidualization requires both progesterone (P) and increasing levels of cAMP. Although many of the cellular events that occur during decidualization have been described, the precise genes as well as the cellular and molecular pathways that regulate this process remain to be elucidated. Our studies have provided an essential role for NOTCH1 in the initiation of the decidualization. NOTCH1 is a highly conserved arbiter of cell fate and regulates processes that are required for decidualization. During decidualization, NOTCH1 interacts with FOXO1 to initiate this critical process and that the transcriptional activation of NOTCH1 signaling pathways requires P. When Notch signaling is compromised in the context of endometriosis, stromal cells are unable to decidualize. Alternatively, the overexpression of NOTCH1 in the ectopic endometrium may contribute to lesion development. In this application we propose three specific aims to understand the cellular and molecular mechanisms associated with NOTCH1 signaling during decidualization and the impact of aberrant NOTCH1 expression in endometriosis associated pathologies. Aim 1, will focus studying the critical interactions between NOTCH1 and FOXO1 which we hypothesize are critical for initiating decidualization and the process by which NOTCH1 is suppressed to allow CREB-mediated cAMP- activation to transform the stromal cells into the fully differentiated decidual phenotype. In Aim 2, we will test the hypothesis that P regulates NOTCH1 cleavage in vivo and in vitro which is critical for the nuclear translocation and transcriptional activation of target genes by the intracellular domain of NOTCH1 (N1ICD). Aim 3, we will test the hypothesis that overexpression of NOTCH1 contributes to the pathophysiology of endometriosis by promoting methylation of Pgr via PU.1 mediated recruitment of DNMT3b and inducing epithelial to mesenchymal transition (EMT). This aim will also use a PRCre mouse model that overexpresses N1ICD. These studies we will further elucidate the NOTCH1- regulated pathways that are critical for decidualization and the implications of its altered expression in contributing to endometriosis associated infertility. These studies have translational significance because if the initiation or progression of decidualization is compromised the result is early pregnancy failure.