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

Abstract

Research questionCan the low-temperature transport time of removed human ovarian tissue be prolonged until cryopreservation? DesignFresh ovarian cortex from nine premenopausal patients was either slow-frozen immediately or stored at 4°C for 24 or 48 h before slow-freezing. The fresh and frozen–thawed biopsies were evaluated by follicle counting via calcein staining, histologic analyses via haematoxylin and eosin staining, and apoptosis via terminal deoxynucleotidyl transferase-mediated dUDP nick-end labelling (TUNEL). The fresh cortex was assessed by reactive oxygen species (ROS) and total antioxidant capacity (TAC) assay to detect oxidative stress. The frozen–thawed cortex biopsies were also evaluated by quantitative PCR for messenger RNA (mRNA) expression of BCL-2, BAX, TNFa, HIF-1a, BMP15 and GDF9, and Western blot for detection of BCL-2, BMP15, GDF9 and CASPASE-3. The frozen–thawed cortex was cultured in vitro for 4 days, anti-Müllerian hormone and glucose were assessed in the supernatant, and ROS and TAC assay detected any oxidative stress in the cortex. ResultsIn the fresh cortex, there were no significant differences between the three groups. In the frozen–thawed cortex, there were no significant differences between the three groups regarding follicle viability, TUNEL, mRNA expression of TNFa, HIF-1a or BMP15. GDF9 mRNA and BAX/BCL-2 were lower and higher at 48 h than at 0 h, respectively. However, the protein expression of BCL-2, CASPASE-3, GDF9 and BMP15 were no different. In the cultured cortex, ROS, TAC and glucose uptake were no different across the three groups. ConclusionOvarian tissue transportation was validated for 24 h in the procedure used in clinical practice. This study showed that 4–8°C transportation for 24 or 48 h does not seem to damage the ovarian tissue. However, ovarian tissue transportation beyond 48 h needs to be further studied for conclusions to be made.