Optimization of freezing and thawing protocols for human ovarian tissue cryopreservation through thermophysical characterisation of freezing medium by differential scanning calorimetry

Fertility preservation should be offered to patients facing gonadotoxic therapy. The method for preserving prepubescent girls' fertility, which is also suitable for women, is ovarian tissue cryopreservation (OTC). Although 200 births have been reported worldwide with this approach, significant improvements are needed. The literature indeed reports numerous protocols for freezing and thawing ovarian tissue, with no clear rationale for selection criteria. This study aims to optimize human OTC protocols by characterizing the thermodynamic properties of freezing medium. The freezing medium associated with most live births after autograft (Leibovitz L-15 medium with 4 mg/mL human serum albumin (HSA), 1.5M DMSO, and 0.1M sucrose) was characterized using differential scanning calorimetry. We obtained -120.49 °C for glass transition temperature (Tg'), -20 °C for crystallization temperature when cooling at 2.5 °C/min (Tc) and -4.11 °C for melting temperature (Tm). With these parameters, we optimized a freezing protocol in a programmable freezer (Nano-Digitcool, Cryo Bio System) and a thawing protocol. The freezing curve was as follows: 5 min at 4 °C, 1 °C/min to -7 °C, seeding: 60 °C/min to -32 °C, and 10 °C/min to -15 °C, 0.3 °C/min to -40 °C, 10 °C/min to -140 °C. The thawing protocol consisted in a 3.5-min step in a cold chamber to reach slowly Tg', limiting thermal and mechanical shocks, and then a 2-min incubation at 37 °C to quickly reach Tm. Ovarian tissue frozen-thawed according to these protocols had a similar quality to that of fresh tissue and could resume folliculogenesis during organotypic culture. Our study will contribute to improve human OTC and optimize women fertility preservation.