PROJECT SUMMARY/ABSTRACT Trophoblast cell derivatives of the early embryo contribute to restructuring the uterine environment, an event required for the establishment of a successful pregnancy. The human and rat each possess a uterine- trophoblast interface that is characterized by deep intrauterine infiltration of trophoblast cells, which contributes to dynamic changes in uterine immune cell, endothelial cell, smooth muscle cell, and stromal cell constituents. This important developmental process is directed by specialized trophoblast cells possessing invasive properties referred extravillous trophoblast (EVT) in the human or by the generic term invasive trophoblast cells in other species, including the rat. Differentiation into these specialized trophoblast cell types requires suppression of pathways essential for maintaining the trophoblast cell stem state and activation of the invasive/EVT cell differentiation program. We hypothesize that repression of the trophoblast cell stem state is an event essential for the establishment of pregnancy. In this project, we investigate roles for activin-follistatin like 3 and WNT-WNT antagonist regulatory networks controlling development of the invasive/EVT cell lineage. Roles for the activin-follistatin like 3 and WNT-WNT antagonist regulatory networks are the focus of Aims Nos. 1 and 2, respectively. In Aim No. 3, we examine FSTL3 and the WNT antagonist, NOTUM, in patient-specific human trophoblast stem cells. Experimentation includes genome-wide analyses of the transcriptome and the chromatin landscape. Execution of this research project will facilitate elucidation of elements of the regulation of the invasive/EVT cell lineage and will create a platform for understanding the pathogenesis of early pregnancy loss.