Treatment with trypLE before freezing improves thawing integrity and functionality of sheep ovarian tissue

Objective : To study innovative approaches to ovarian tissue cryopreservation, a critical issue for fertility preservation in pediatric cancer patients. Despite historical attempts, recent advances in cancer treatment have underscored the urgent need for more effective and reliable ovarian tissue cryopreservation methods. Our research aims to evaluate if decreasing the rigidity of stroma before cryopreservation by investigating pre-treatments with enzymes can enhance the quality of ovarian tissue post-thawing. Design : Our research evaluated the use of five commonly used enzymes to disaggregate tissue (trypLE, collagenase, dispase, accutase and hyaluronidase) before freezing ovarian tissue to decrease rigidity and facilitate cryopreservation. Sheep ovaries, with high similarity to human ovaries, were used as an animal model. Tissue structure, cell proliferation, apoptosis and viability were assessed before and after thawing. Results : Our findings showed that enzymatic treatment with trypLE before freezing offered immediate benefits post-thawing with the highest viability values and percentage of intact follicles. However, 2 and 7 days after thawing and culture, dispase showed the highest viability but with loci of necrosis at the edges and more damaged follicles compared to trypLE. A decrease in viability was observed after thawing and culturing the samples. The pretreatment with accutase damaged the tissue severely with also the lowest viability values. Ki67-positive follicles and stromal cells were observed in fresh samples, but only trypLE and hyaluronidase maintained Ki67-positive antral follicles after 2 days culture. Besides, only trypLE maintained all follicles negative to caspase-3 after thawing, and 7 days after culture primordial follicles were apoptotic in all treatments apart from trypLE. Conclusion : our findings suggest that trypLE pretreatment could provide a beneficial approach for maintaining the functions and viability of cryopreserved ovaries after thawing. Further research is needed to fully understand their impact and optimize cryopreservation protocols in this important clinical context.