Single-cell RNA Sequencing of Endometrial Assembloid Cultures

Decidual remodelling of midluteal endometrium leads to a short implantation window after which the uterine mucosa either breaks down or is transformed into a robust matrix that accommodates the placenta throughout pregnancy. To gain insights into the underlying mechanisms, we established and characterised endometrial assembloids, consisting of gland organoids and primary stromal cells. Single-cell transcriptomics revealed that decidualized assembloids closely resemble midluteal endometrium, harbouring differentiated and senescent subpopulations in both glands and stroma. We show that acute senescence in glandular epithelium drives secretion of multiple canonical implantation factors, whereas in the stroma it calibrates the emergence of anti-inflammatory decidual cells and pro-inflammatory senescent decidual cells. Pharmacological inhibition of stress responses in pre-decidual cells accelerated decidualization by inhibiting senescence and mesenchymal-epithelial transition, processes involved in endometrial breakdown and regeneration, respectively. Accelerated decidualization resulted in entrapment of co-cultured human blastocysts in a largely static decidual matrix. By contrast, the presence of senescent decidual cells created a dynamic implantation environment, enabling embryo expansion and attachment, although their persistence led to gradual disintegration of assembloids. Together, our findings demonstrate that senescence controls endometrial fate decisions at implantation and highlight how endometrial assembloids may accelerate the discovery of new treatments to prevent reproductive failure. Overall design: Endometrial assembloids were prepared from cultured endometrial stromal cells and epithelial gland organoids. Assembloid cultures were grown for four days in Expansion Medium (ExM)+E2 , then either harvested (Day 0) or differentiated in the presence of 8-bromo-cAMP (cAMP), medroxyprogesterone acetate (MPA) and estradiol (E2) with or without the senolytic drug, dasatinib. Cells were recovered and subjected to single-cell analysis using nanoliter droplet barcoding and high-throughput sequencing of RNA (Drop-Seq).