Abstract
Study question
To investigate whether adipose tissue-derived stem cells (ASCs) modulate hypoxia and oxidative stress in human ovarian tissue transplants to reduce early follicle loss.
Summary answer
ASCs protect the follicle pool by mitigating the hypoxia-related response through HIF1↑ signaling in human xenografts and enhancing revascularization by ensuring faster tissue reperfusion.
What is known already
ASCs are known for their angiogenic potential and capacity to boost angiogenesis by secreting growth factors and differentiating into vessels in numerous models of wound healing in regenerative medicine. In a 2-step ovarian tissue xenotransplantation involving grafting inside a fibrin scaffold two weeks prior to transplantation, ASCs reduced follicle loss after short- and long-term grafting, as well as abnormal follicle activation, by increasing reoxygenation and revascularization in human xenografts.
Study design, size, duration
Prospective experimental study. Cryopreserved ovarian cortex from five adult women was transplanted to 30 nude mice, with or without ASCs (ASC group; OT group). Ovarian grafts were retrieved on days 3 (n = 5), 10 (n = 5) and 21 (n = 5). One piece of ovarian tissue per patient was fixed for analysis after thawing to serve as non-grafted controls.
Participants/materials, setting, methods
The 10 animals grafted for 21 days underwent in vivo microdialysis evaluation to investigate direct reactive oxygen species (ROS) kinetics. Analyses of ovarian grafts at all time points and non-grafted controls included immunolabeling for double CD34 (revascularization by host and graft components), immunofluorescence for HIF1α (hypoxia-related response), Nrf2 (oxidative stress-related response) and 8OHdG (oxidative stress-related DNA damage), and gene expression (RT-qPCR) for VEGF-A (neoangiogenesis), SOD2 (antioxidant activity) and Nrf1 (mitochondrial biogenesis).
Main results and the role of chance
ROS peaked sooner in the ASC group (day 2, p < 0.0001) than the OT group (day 10, p = 0.01) after grafting, indicating earlier tissue reperfusion. Total vascularization was stable in the ASC group at all time points, but lower in the OT group 3 days after grafting (p = 0.01) due to a drop in both host and graft vascular components. HIF1α expression, detected mainly in follicles, was significantly lower in primordial follicles in the ASC group than the OT group on days 3 (p = 0.008) and 10 (p = 0.01). VEGF gene expression rose significantly (around 40x) in both groups on day 3 and persisted significantly longer in the ASC group (10 days) than the OT group (3 days) (p = 0.04), emphasizing the role of ASCs as enhancers of proangiogenic factors. There was no upturn in the oxidative stress-related response (Nrf2 pathway) nor DNA damage (8OHdG) to follicles in any of the grafted groups over time, while a modest increase in both markers was observed only in the stroma after 21 days. Neither was there any major increase in SOD2 and Nrf1 gene expression, suggesting no significant activation of the Nrf2 pathway for cytoprotection from oxidative stress.
Limitations, reasons for caution
Although Nrf2 signaling activation was detected in human granulosa cell cultures in increasing ROS concentrations, our findings did not confirm its role in tissue damage modulation after ovarian tissue transplantation. Further studies may evidence the involvement of other pathways that modulate oxidative stress after transplantation.
Wider implications of the findings
The role of ASCs in protecting the follicle pool appears to be related to a decrease in hypoxia and faster ovarian graft revascularization and reperfusion, sustained by an increase in VEGF for a longer period after grafting. There was no evidence of oxidative stress-related damage, irrespective of the transplantation strategy.
Trial registration number
Correction to: Characteristics of a decellularized human ovarian tissue created by combined protocols and its interaction with human endometrial mesenchymal cells