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 2 2 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.

Analysis of Fertility Preservation by Ovarian Tissue Cryopreservation in Pediatric Children in China

“…At present, the low temperature (4°C to 8°C) is a widely used standard tissue transportation method. Low temperatures decrease cellular metabolism in ovarian tissue, reduce cellular oxygen demand and consumption, and preserve tissue without vascular perfusion ( 36 , 37 ). Because of the high density of follicles in children, we generally prepared ovarian cortical slices with the size of 6 mm × 3 mm × 1 mm, which is consistent with the international community ( 38 ).…”

Effects of hypoxia-preconditioned HucMSCs on neovascularization and follicle survival in frozen/thawed human ovarian cortex transplanted to immunodeficient mice

“…Apoptosis was evaluated in the three non-grafted tissues and the 4 grafts of each group and each time point. The details were the same as in our previous publication [ 34 ]. The tissue information from 4 slides from each non-grafted tissue and grafts of each group of each time point were scanned using a panoramic section scanner (PANORAMIC DESK/MIDI/250/1000; 3DHISTECH, Hungary).…”

“…VEGFA , HIF1a , growth differentiation factor 9 ( GDF9 ), phosphor-protein kinase B ( p-Akt ), protein kinase B ( Akt ) protein expression in 2 or 3 grafts of each time point of each group and two non-grafted fragments were separately evaluated by western blot. The procedures were performed according to the previous description [ 34 ]. Ovarian tissue lysates were obtained using RIPA lysis, and extraction buffer (catalogue #89900; Thermo Fisher, USA) supplemented with protease inhibitor (catalogue #04693159001; Roche, Switzerland) and phosphatase inhibitor (catalogue #04906837001; Roche).…”