Establishment of a cell line-based 3D co-culture model of the endometrium for studying endometrial receptivity

A receptive endometrium is essential for successful embryo implantation, yet its dynamic cellular and molecular features remain poorly understood due to limitations in current in vitro models. We aimed to establish a 3D co-culture endometrial model mimicking physiological conditions, characterizing its response to varied culture conditions using receptivity-associated gene expression profiling. Epithelial Ishikawa or RL95-2 cells were layered above stromal St-T1b cells separated by Matrigel and cultured in different media (growth factor-reduced, serum-containing standard, cytokine-supplemented and EGF-supplemented) or exposed to hormonal stimuli (E2, P4, cAMP) over a 10-day differentiation period. Our model effectively recapitulates the layered structure of the endometrium, as confirmed by scanning electron microscopy and immunofluorescence. Transcriptional analysis of fertility-associated markers revealed media- and cell line-dependent alterations of key receptivity markers; In standard media gene expression alterations occurred for HEY1 in both, but for HEY2 solely in the Ishikawa model and EDNRB only in the RL95-2 model, in cytokine-supplemented media for LIF and TAGLN in both models, while HPSE only in RL95-2 models and DPP4 and HEY1 only in Ishikawa models. Notably, the long-term hormonal treatment triggered a transcriptomic signature, especially in the RL95-2 model that closely correlates to the clinically determined one at the window of implantation (WOI). We conclude that our in vitro model reflects the phenotypic and transcriptional dynamics of the in vivo endometrium and responds distinctly to external stimuli. It serves as a robust platform to study endometrial receptivity mechanisms and holds potential for future applications in infertility and implantation research.