Hypoxia-preconditioned hUCMSCs protect frozen-thawed human ovarian tissue by modulating the HIF-1α/VEGF pathway.

Abstract

Objective: To evaluate whether hypoxia-preconditioned human umbilical cord mesenchymal stem cells (hUCMSCs) can protect frozen-thawed human ovarian tissue via in vitro co-culture.

Methods: Frozen-thawed ovarian cortical pieces from 10 patients were randomly divided into a control group (no co-culture), N‑MSCs group (normoxia‑preconditioned hUCMSCs co-culture), and H‑MSCs group (hypoxia‑preconditioned hUCMSCs co-culture). Tissues in the co-culture groups were subjected to 48 h indirect Transwell co-culture. Apoptosis was assessed by TUNEL. Metabolic changes in the culture medium were measured, such as glucose consumption and lactate production, and AMH levels were determined. Oxidative stress in ovarian tissue was evaluated by measuring ROS and TAC. RNA-seq was performed, and key pathways were analyzed by GSEA. The protein expression of HIF-1α, VEGFA, GDF9, AKT, and p-AKT was examined by Western blot.

Results: Compared with both the control and N-MSCs groups, co-culture with H‑MSCs significantly reduced follicular atresia and apoptosis, while preserving a greater proportion of resting follicles. The H‑MSCs group presented lower glucose consumption and lactate production and elevated AMH levels in the culture medium. H‑MSCs markedly decreased reactive oxygen species (ROS) and enhanced total antioxidant capacity (TAC). Transcriptomic analysis showed that H‑MSCs induced a distinct gene expression profile characterized by upregulation of the HIF‑1 signaling pathway. H‑MSCs significantly upregulated HIF‑1α, VEGFA, and phosphorylated AKT at the protein level.

Conclusions: This in vitro study showed that co-culture of ovarian tissue with H-MSCs provides stronger protection than N-MSCs. This effect likely involves the HIF-1α/VEGFA pathway, with enhanced pro-angiogenic signaling, reduced apoptosis and oxidative stress, and preservation of the follicular reserve.