Comparative transcriptomic profiling of human 3D endometrial stromal organoids under distinct decidualization stimuli

Endometrial stromal decidualization is a hormone-regulated process essential for establishing uterine receptivity and supporting embryo implantation. Three-dimensional (3D) endometrial organoid systems provide a physiologically relevant platform to study this process; however, how different stimulatory conditions influence decidualization responses in 3D culture remains incompletely characterized. In this study, we generated 3D human endometrial stromal organoids and subjected them to four experimental conditions: vehicle control, cyclic adenosine monophosphate (cAMP) stimulation, combined estrogen–progesterone–cAMP (EPC) treatment, and co-culture with in vitro–grown follicles derived from mouse ovary. Organoids were cultured for 6 days under each condition, followed by bulk RNA sequencing. Principal component analysis (PCA) revealed clear separation between control and stimulated groups, with cAMP, EPC, and follicle co-culture conditions showing distinct but partially overlapping transcriptional profiles. Differential gene expression analysis identified both shared and condition-specific transcriptional changes across the four treatments. Pathway-level analysis using gene set variation analysis (GSVA) further demonstrated common decidualization-associated biological processes alongside stimulus-specific functional responses. This dataset provides a comprehensive resource for investigating how distinct biochemical and paracrine stimuli regulate decidualization in 3D endometrial organoid systems. It enables comparative analysis of canonical cAMP-driven responses, hormone-mediated signaling, and follicle-derived paracrine effects on endometrial stromal decidualization.