Designing and Optimizing a Protocol for Whole-Ovary Vitrification

Ovarian tissue cryopreservation (OTC), a process to preserve human ovarian tissue by cooling to subzero temperature without ice formation, has been increasingly studied within the last 15 years. This is due to the growing scientific capabilities as well as more women who want the procedure for medical or elective reasons. For example, women at or below reproductive age who have to undergo radiological treatments might have to forfeit their fertility. However, if they cryopreserve part or all of their ovary, the ovaries can be reimplanted or in vitro fertilization (IVF) can occur to allow fertility options in the future. Other women have been undergoing OTC for more elective reasons such as delaying menopause so that they don’t have to live a majority of their life as postmenopausal. There are types of OTC; slow freezing and vitrification. Both require coupled mass and heat transfer. Slow freezing consists of low concentrations of cryoprotective agents (CPAs) which displace water to prevent freezing and a slow cooling rate of about 4∘C min−1. While this is the more thoroughly researched cryopreservation method, research suggests that vitrification is better in the long run, on cellular organelles. Vitirification consists of a higher amount of CPAs present and a cooling rate close to 150∘C min−1.While vitrificiation has many advantages, there is not a universally agreed-upon standard protocol for vitrification, especially not for the whole ovary. The advantages of OTC on the whole organ would be that upon reimplantation, the hormonal health is assumed to be easily maintained. In order to create a standard vitrification protocol, the criteria for vitirification have to be met (namely the ovary has 6M CPA and then is cooled to -150∘C), while then also maximizing the cellular viability. With higher concentration of CPAs especially in vitrification, there is an increase in cytotoxicity, so a cytotoxicity cost function was implemented in aiding the optimization. While creating a 3D computational model of the ovary, with a branched artery and six capillaries, we found that the mesh converged for both heat and mass transfer physics at around 200,000 elements. Using the optimization function, we were able to ascertain that the optimal conditions for the protocol were to submerge the ovary in 9.5 M dimethylsulfoxide (DMSO) for 6 hours and then placed in liquid nitrogen for around 60 seconds. This resulted in a cellular viability prediction of 29.07%, on par with current methods where only parts of the ovaries are cryopreserved. To test the robustness of our model, we varied some of the parameters to see the effect on the protocol. Future directions are analyzed to further improve our model