Optimization of cryoprotectant loading into murine and human oocytes

Loading of cryoprotectants into oocytes is an important step of the cryopreservation process, in which the cells are exposed to potentially damaging osmotic stresses and chemical toxicity. Thus, we investigated the use of physics-based mathematical optimization to guide design of cryoprotectant loading methods for mouse and human oocytes. We first examined loading of 1.5 M dimethyl sulfoxide (Me₂SO) into mouse oocytes at 23 °C. Conventional one-step loading resulted in rates of fertilization (34%) and embryonic development (60%) that were significantly lower than those of untreated controls (95% and 94%, respectively). In contrast, the mathematically optimized two-step method yielded much higher rates of fertilization (85%) and development (87%). To examine the causes for oocyte damage, we performed experiments to separate the effects of cell shrinkage and Me₂SO exposure time, revealing that neither shrinkage nor Me₂SO exposure single-handedly impairs the fertilization and development rates. Thus, damage during one-step Me₂SO addition appears to result from interactions between the effects of Me₂SO toxicity and osmotic stress. We also investigated Me₂SO loading into mouse oocytes at 30 °C. At this temperature, fertilization rates were again lower after one-step loading (8%) in comparison to mathematically optimized two-step loading (86%) and untreated controls (96%). Furthermore, our computer algorithm generated an effective strategy for reducing Me₂SO exposure time, using hypotonic diluents for cryoprotectant solutions. With this technique, 1.5 M Me₂SO was successfully loaded in only 2.5 min, with 92% fertilizability. Based on these promising results, we propose new methods to load cryoprotectants into human oocytes, designed using our mathematical optimization approach.