Hybrid endometrial-derived hydrogel and human endometrial organoids synergize for uterine regeneration in an immunocompetent murine model.

BACKGROUND: The human endometrium is a regenerative tissue essential for fertility, but pathological conditions like Asherman syndrome, endometrial atrophy, and thin endometrium can impair its function. Current therapies lack efficacy, driving demand for innovative regenerative therapies. In this context, endometrial-derived hydrogels and organoids have shown promise individually for tissue regeneration, but their combined therapeutic potential has not been previously evaluated in vivo. This study explores a dual regenerative strategy combining a hybrid hydrogel - composed of synthetic PuraMatrix(®) and endometrial extracellular matrix hydrogel - with human endometrial organoids in an immunocompetent murine model with uterine damage. METHODS: Endometrial damage model was established in female C57BL/6 mice (n = 46) via uterine injury using 70° ethanol. After 4 days of endometrial damage, human endometrial organoids were co-injected with the hybrid hydrogel into the uterine horns. Two weeks post-injection, a subset of mice (n = 25) was sacrificed for biocompatibility, histological, and transcriptomic analyses. Functional recovery of the endometrium was assessed in the remaining animals (n = 21) through fertility outcome evaluation. For endometrial regeneration analyses, normally distributed data were analyzed by one-way ANOVA and Tukey's multiple comparisons, while non-normally distributed data were analyzed by the Kruskal-Wallis test with Dunn's multiple comparisons. For fertility outcomes, t-test or Mann-Whitney U tests for 2-by-2 comparisons were performed. RESULTS: Histological and molecular analyses revealed that the therapy improved endometrial thickness, gland density, and vascularization, and reduced fibrosis and ferroptosis, aligning tissue characteristics closer to healthy controls. However, fertility outcomes were not fully restored, potentially due to the persistence of the synthetic component of the hybrid hydrogel. Thus, further studies are needed to confirm complete hydrogel resorption and its impact on fertility restoration. CONCLUSIONS: In conclusion, this study demonstrates the biocompatibility and regenerative potential of human endometrial organoids delivered within the hybrid hydrogel, highlighting a promising strategy for endometrial regeneration.