Optimization of cryoprotectant loading into murine and human oocytes

Area of Science:

• Reproductive biology
• Biophysics
• Cryobiology

Background:

• Cryopreservation of oocytes is crucial for fertility preservation.
• Loading cryoprotectants can cause osmotic stress and chemical toxicity, damaging oocytes.
• Current methods may lead to suboptimal fertilization and development rates.

Purpose of the Study:

• To optimize cryoprotectant loading methods for mouse and human oocytes using physics-based mathematical modeling.
• To investigate the causes of oocyte damage during cryoprotectant loading.
• To develop improved strategies for oocyte cryopreservation.

Main Methods:

• Applied physics-based mathematical optimization to design cryoprotectant loading protocols.
• Compared conventional one-step loading with optimized two-step loading for mouse oocytes at different temperatures.
• Conducted experiments to decouple the effects of cell shrinkage and cryoprotectant exposure time.
• Utilized hypotonic diluents to reduce cryoprotectant exposure time.

Main Results:

• Optimized two-step loading significantly increased fertilization (85%) and development (87%) rates in mouse oocytes compared to one-step loading (34% fertilization, 60% development).
• Oocyte damage resulted from the interaction of osmotic stress and dimethyl sulfoxide (Me(2)SO) toxicity, not solely from shrinkage or exposure time.
• At 30°C, one-step loading yielded low fertilization rates (8%), while optimized loading achieved 86%.
• Reduced Me(2)SO exposure time to 2.5 minutes using hypotonic diluents, maintaining 92% fertilizability.

Conclusions:

• Physics-based mathematical optimization is effective for designing superior cryoprotectant loading protocols.
• The optimized methods significantly improve oocyte cryopreservation outcomes, particularly for human oocytes.
• Understanding the interplay between osmotic stress and chemical toxicity is key to preventing cryopreservation-induced damage.