Hybrid Endometrial-Derived Hydrogel and Human Endometrial Organoids Synergize for Uterine Regeneration in an Immunocompetent Murine Model

Study question: Can human endometrial organoids (hEOs) synergize with a hybrid endometrial-derived hydrogel (HG) to regenerate the endometrium in an immunocompetent murine model of Asherman's Syndrome/Endometrial Atrophy (AS/EA)? Summary answer: The combination of HG+hEOs was biocompatible in an immunocompetent murine model, restored endometrial thickness and glandular structure, reduced fibrosis and ferroptosis, promoted angiogenesis and regenerative immune pathways, but failed to fully restore fertility, likely due to the prolonged retention of the synthetic hydrogel component. Main results and the role of chance: No systemic immune alterations or hematological abnormalities were detected post-treatment, indicating that the combined treatment was compatible and safe in mice. Fourteen days after treatment, HG+hEOs treatment significantly restored endometrial thickness and gland density, reduced collagen deposition and iron accumulation (ferroptosis), and enhanced vascularization compared to the PBS condition (p < 0.05), reaching levels comparable to SHAM controls. RNA sequencing of the murine endometria confirmed restoration of transcriptomic profiles and upregulation of angiogenesis, cell proliferation, and regenerative immune pathways in the HG+hEOs group. However, fertility outcomes (pregnancy rate and embryo number) remained significantly impaired in all conditions, with no statistical improvement over the PBS group. Mice injected with PM confirmed that fertility impairment was linked to the presence of synthetic hydrogel in previously damaged uteri. Overall design: First, an AS/EA model was established in female C57BL/6 mice via uterine injury using 70° ethanol. After 4 days of endometrial damage, hEOs were co-injected with HG into the uterine horns. Animals (n = 46) were divided into four conditions: SHAM, PBS, HG and HG+hEOs. Two weeks post-injection, a subset of mice (n = 21) 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. In addition, two groups treated with PuraMatrix (PM) alone were also included (n = 12), with (n = 7, PM damaged) or without (n = 5, PM non-damaged) endometrial damage, to address inconsistencies observed in fertility outcomes.