Comparison of selected cryoprotective agents to stabilize meiotic spindles of human oocytes during cooling

BACKGROUND This study examined the primary effect of selected cryoprotective agents (CPAs) on the meiotic spindles of human oocytes during cooling. METHODS Fresh metaphase II oocytes (n=26) donated from patients undergoing IVF treatment were analyzed via Polscope. In experiment one, 16 oocytes with visible spindle at 37°C were cooled to 20°C and rewarmed to 37°C to test the spindle response to cooling. They were then cooled to 20°C, 10°C, 0°C and rewarmed to 37°C after having been equilibrated with 1.5 M 1,2-propanediol (PROH), 1.5 M dimethyl sulfoxide (DMSO), 1.5 M ethylene glycol (EG) or 10 μM taxol at 37°C. In experiment two, 10 oocytes without visible spindles at 37°C were cooled to 20°C and then equilibrated with PROH, EG and taxol at 20°C. Spindle images were recorded at each temperature. RESULTS Meiotic spindles remained visible or became more distinct during cooling to 20°C, 10°C and 0°C when equilibrated with PROH, EG, DMSO and Taxol. Without these agents, meiotic spindles of the same oocytes disappeared after cooling to 20°C. CONCLUSION The primary effect of PROH, EG and DMSO on the meiotic spindle is to stabilize and protect it against low temperature disassembly. A higher equilibration temperature (≥33°C) for oocyte freezing is recommended.