Long-time low-temperature transportation of human ovarian tissue before cryopreservation

Abstract Research question : Can the low-temperature transport time of removed human ovarian tissue be prolonged until cryopreservation? Design Fresh ovarian cortex from nine premenopausal patients was either slow-programmed frozen immediately or stored at 4℃ for 24h or 48h before slow-freezing. The fresh and frozen-thawed biopsies were evaluated by follicle counting via calcein-staining, histologic analyses via HE, and apoptosis via TUNEL. The fresh cortex was assessed by ROS and TAC assay for oxidative stress detection. The frozen-thawed cortex biopsies were also evaluated by qPCR for mRNA expression of BCL-2, BAX, TNFa, HIF-1a, BMP15, and GDF9, western-blot for detection of BCL-2, BMP15, GDF9, and CASPASE3. The frozen-thawed cortex was cultured in-vitro for four-days, AMH and glucose were assessed in the supernatant, and ROS and TAC assay detected cortex's oxidative stress. Results :In the fresh cortex, no significant differences between the three groups. In the frozen-thawed cortex, no significant differences between the three groups regarding follicle viability, TUNEL, mRNA expression of TNFa, HIF-1a, and BMP15. GDF9 mRNA and BAX/BCL-2 were lower and higher at 48h than at 0h, respectively. However, the protein expression of BCL-2, CASPASE3, GDF9, and BMP15 were not different. In the cultured cortex, ROS, TAC, and glucose uptake were not different in the three groups. Conclusion : Ovarian tissue transportation was validated for up to 24h in the practical procedure. This study showed that 4-8℃ transportation for 24h or 48h does not seem to damage the ovarian tissue. However, ovarian tissue transportation above 48h still needs to be further studied for conclusions to be made.