P-372 Optimization of freezing and thawing protocols for human ovarian tissue cryopreservation through thermophysical characterisation of freezing medium by Differential Scanning Calorimetry

Abstract Study question What is the most suitable protocol to freeze and thaw human ovarian tissue? Summary answer Determination of thermodynamic properties of the most successfully used freezing medium allowed us to define optimal freezing and thawing protocols for human ovarian tissue. What is known already 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 more than 200 births have been reported worldwide with this approach, significant improvements are needed. Indeed the literature reports numerous protocols for freezing and thawing ovarian tissue. Notably, a great diversity in the freezing medium composition (e.g. type and concentration of cryoprotectants) and in the temperature reduction ramps are described without any clear justification of the selection’s criteria. Study design, size, duration The thermodynamic properties of the freezing medium associated with most live births after autograft were evaluated by Differential Scanning Calorimetry. These properties were used to develop a freezing and a thawing protocol for OTC. To evaluate the post-thawing quality and functionality of human ovarian tissue, we used a pericystic ovarian tissue model (three patients, <37 years, no ovulatory disorder). Fresh and frozen-thawed tissues were evaluated immediately (D0), and after 7 days of organotypic culture (D7). Participants/materials, setting, methods Thermodynamic properties of freezing medium (Leibovitz medium with 4 mg/mL HSA, 1.5M DMSO and 0.1M sucrose) were measured by Differential Scanning Calorimeter (DiamondDSC, PerkinElmer). The crystallization temperature (Tc), end-of-melting temperature (Tm), and transition temperature (Tg’) were measured. An adapted slow freezing protocol, performed in a programmable freezer (NanoDigit-Cool, Cryo Bio System) and a thawing protocol were developed from these parameters. The post-thawing quality and functionality of ovarian tissue were determined by histochemical analysis (HES staining). Main results and the role of chance The measured thermodynamic properties of the freezing medium were: -20 °C (±3 °C) for Tc, -4.11 °C (±0.49 °C) for Tm and -120.49 °C (±0.41 °C) for Tg'. The optimized freezing protocol consisted in the following ramps: sample introduction at + 4 °C for 5 min, cooling at 1 °C/min to -7 °C, seeding at 60 °C/min to -32 °C (reaching Tc), followed by 10 °C/min to -15 °C, 0.3 °C/min to -40 °C, and 10 °C/min to -140 °C. The developed thawing protocol was a two-stage process with a first slow step (3.5 min in a cold chamber) to gradually reach Tg', minimizing thermal and mechanical shocks, and a second ultra-fast step (2 min at 37 °C) to quickly reach Tm. To validate these protocols, 782 and 392 follicles from fresh and frozen/thawed tissues were analysed, respectively. At D0, follicles stage distribution was similar between fresh and frozen/thawed tissues. A clear decrease in the percentage of reserve follicles was observed between D0 and D7 in both fresh (14.20% vs 2.93%, p = 0.000 for primordial follicles and 69.67% vs 31.49%, p = 0.002 for intermediate follicles) and frozen-thawed tissue (16.85% vs 2.91%, p = 0.021 for primordial follicles), with a concomitant increase in the percentage of growing follicles in fresh (8.92% vs 57.61%, p = 0.000) and frozen-thawed tissue (22.45% vs 47.65% p = 0.052). Limitations, reasons for caution These results should be validated in vivo to assess the functionality and quality of the transplanted tissues, as well as clinical outcomes such as live birth rates. Wider implications of the findings This project will help to reach a better understanding of the impact of freezing and thawing on ovarian tissue functionality and to implement an optimal protocol for OTC to optimize cancer patient fertility preservation. Trial registration number Yes