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-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.

Results: In 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.

Conclusion: Ovarian 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.

Keywords: Apoptosis; Fertility preservation; Low temperature; Ovarian tissue cryopreservation; Ovarian tissue transportation; Oxidative stress.