PROJECT SUMMARY Dilation & curettage (D&C) is one of the most common surgical procedures performed on women throughout the world. Intrauterine adhesions (iUA), or Asherman, develop in about 40% of women who undergo D&C in the postpartum (6 week period after birth), but is very rare after D&C in nonpregnant women (<1%) for reasons that are not well understood. It is a debilitating condition characterized by intrauterine fibrosis and scarring. Patients with iUA suffer from infertility, recurrent pregnancy loss and a broad range of dangerous pregnancy complications (e.g. preterm birth). While endometrial mesenchymal stem/progenitor cells (eMPCs) are crucial for endometrial repair, the role of these cells and underlying molecular mechanisms in this postpartum susceptibility of the endometrium to fibrosis and abnormal repair are unknown. This application is specifically focused on defining the role that eMPCs and their stromal fibroblast progeny play in abnormal uterine repair. The central hypothesis is that eMPCs of the recently postpartum uterus are more senescent and inherently different in their response to uterine injury compared to eMPCs of the nonpregnant uterus, leading to fibrosis and scar formation. The approach is to use our novel postpartum mouse uterine injury model which recapitulates the susceptibility of the human postpartum uterus to injury. Using it, we will define the dynamic changes in eMPCs and their differential response to uterine injury in the postpartum vs. nonpregnant, identify using lineage tracing the eMPC subsets that become the profibrotic fibroblast cells, and conditionally ablate each of these eMPC subsets to define their functional role in endometrial fibrosis (Aim 1). We will obtain fresh human endometrial tissue from women undergoing postpartum D&C and compare it to nonpregnant tissue using innovative single cell technology and functional in vitro studies to gain detailed insights into the cellular and molecular differences that predispose the human endometrium to form iUA (Aim 2). In Aim 3, we will obtain archived endometrial specimens from the time of inciting D&C event from women who developed Asherman vs. non-Asherman. We will use the innovative deterministic barcoding in tissue spatial multi-omics sequencing (DBiT-seq) platform and integrate it with immunofluorescence to gain detailed molecular insights regarding eMPCs and their cell interactions within the tissue, identifying novel therapeutic targets for iUA prevention. The proposed aims are conceptually and technically innovative and together will have a broad impact on the field by filling a substantial gap in our fundamental knowledge of endometrial biology and infertility pathogenesis using Omics approach, which are major research priorities of the Fertility and Infertility Branch of the NICHD. Ultimately, the knowledge gained from this proposal will not only be invaluable to our understanding of many more subtle conditions of abnormal endometrial repair, ...