Establishment of a cell line-based 3-D coculture 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 three-dimensional (3-D) coculture 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 cells (St-T1b) separated by Matrigel and cultured in different media [such as growth factor-reduced, serum-containing standard, and cytokine- and epidermal growth factor (EGF)-supplemented] or exposed to hormonal stimuli [estradiol (E2), progesterone (P4), and 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 hairy/enhancer-of-split related with YRPW motif protein 1 (HEY1) in both, but for HEY2 solely in the Ishikawa model and endothelin receptor type B (EDNRB) only in the RL95-2 model, cytokine-supplemented media for leukemia inhibitory factor (LIF) and transgelin (TAGLN) in both models, whereas heparanase (HPSE) only in RL95-2 models and dipeptidylpeptidase 4 (DPP4) and HEY1 only in Ishikawa models. Notably, the long-term hormonal treatment triggered a transcriptomic signature, especially in the RL95-2 model, which 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.NEW & NOTEWORTHY A receptive endometrium is essential for successful embryo implantation, yet its dynamic cellular and molecular features remain poorly understood. Here, we establish and characterize a three-dimensional (3-D) model of the endometrium combining epithelial and stromal cell lines. The model reflects the phenotypic and transcriptional dynamics of the in vivo endometrium and responds distinctly to external stimuli, serving as a robust platform to study endometrial receptivity mechanisms.