122. Hormone-responsive Porcine Endometrial Epithelial Organoids: Establishment, Validation, and Transcriptomic Characterization

Abstract The endometrium, composed of luminal and glandular epithelial cells (EECs) and stromal cells (ESCs), plays a central role in integrating maternal hormonal signals and conceptus-derived cues to establish and sustain pregnancy in mammals. While endometrial epithelial organoids (EEOs) have advanced mechanistic studies in humans and mice. a comparable model in pigs has been lacking, limiting opportunities to investigate peri-implantation events in this agriculturally and biomedically relevant species. Here, we report the first establishment of porcine endometrial epithelial organoids (pEEOs) from postnatal day-10 uteri. Epithelial sheets were isolated, dissociated, embedded in growth factor-reduced Matrigel, and maintained in stem cell-supportive medium. The pEEOs demonstrated long-term viability ( > 10 passages) and maintained epithelial identity, confirmed by cytokeratin 8 expression and lack of mesenchymal contamination (vimentin-negative). Importantly, pEEOs displayed robust hormone responsiveness: estradiol (E2) alone or in combination with progesterone analog plus cAMP (EPC treatment) significantly induced expression of steroid-responsive genes critical for endometrial receptivity (PGR, HSD17B2, OLFM4, ESR2, SPP1; P < 0.05). Bulk RNA-seq analysis revealed that EPC-treated pEEOs exhibited a distinct transcriptomic landscape, with 1,078 genes upregulated and 1,257 genes downregulated relative to vehicle controls. Gene ontology enrichment highlighted pathways central to early pregnancy establishment, including transmembrane transport, cilium assembly, lipid transport, and regulation of cell communication and receptor signaling pathway (FDR < 0.05). Notably, transcriptomic profiles of hormone-treated pEEOs showed strong similarity to in vivo uterine luminal epithelial transcriptomes during early pregnancy, demonstrating their physiological relevance. Collectively, this work provides the first validated porcine endometrial epithelial organoid system, offering a powerful new platform for investigating endometrial biology conceptus-uterus interactions, and mechanisms regulating early pregnancy in pigs. This model holds broad potential for advancing both reproductive biology research and the improvement of swine reproductive efficiency.