Towards the best enzymatic treatment prior to ovarian tissue cryopreservation. A statistical and model-based analysis

The objective was to study an innovative approach based on a mathematical model to analyze the potential benefits of using enzymatic pre-treatment of ovarian tissue to increase its permeability. Our approach aims to elucidate whether enzymatic pre-treatment would enhance the penetration of cryoprotectants in the ovarian tissue, leading to better preservation and giving best results for obtaining primordial and primary follicles without damage. The effect of five enzymatic treatments (TrypLE, collagenase, dispase, accutase, hyaluronidase) over time on sheep ovarian tissue was evaluated, focusing on penetration depth and tissue damage. First results showed that up to 45 min of incubation, all enzymes penetrated similarly, but longer treatments revealed differences. Dispase showed the greatest penetration at 180 min but was highly aggressive, causing rapid tissue lysis. TrypLE and hyaluronidase were found to be the most effective in minimizing follicular damage while ensuring sufficient tissue penetration, particularly when applied for 10 min. TrypLE caused significant edema after 10 min, but this was reduced after 15 min. Hyaluronidase caused minimal edema and had excellent penetration, making it ideal for enhancing ovarian tissue permeability. The present study showed that pre-treatments with hyaluronidase and TrypLe for 10 min are potential candidates for enhancing ovarian tissue permeabilization, offering the best balance between penetration and minimal tissue damage. These findings could improve fertility preservation techniques and future cryopreservation strategies.