Summary
The fate of the human endometrium is determined during the mid-luteal window of implantation, coinciding with differentiation of endometrial stromal cells (EnSCs) into specialized decidual cells. In response to successful embryo implantation, differentiating EnSCs transform the endometrium into a decidua that maintains the placenta throughout gestation; whereas falling progesterone levels in the absence of pregnancy lead to tissue destruction and menstrual shedding. We used single-cell RNA sequencing (scRNA-seq) to map the temporal transcriptomic changes in cultured EnSCs along a decidual time-course and in response to withdrawal of differentiation signals. We demonstrate that decidual transformation of EnSCs first involves a continuous and largely synchronous transition through intermediate states before emerging as divergent subpopulations, representing mature decidual cells and stressed cells. Subsequent withdrawal of decidual signals imposes a second branching event, driven primarily by de-differentiation of a subset of cells. Further, scRNA-seq analysis of timed biopsies highlighted endometrial cellular complexity, confirmed the preponderance of different EnSC subpopulations upon progression from mid-to late-luteal phase, and uncovered genes with conserved branching dynamics in vivo and in vitro. Taken together, our single-cell analysis indicates that multiple EnSC states and dynamic subpopulations of decidual cells underpin endometrial fate decisions during the window of implantation.