Egg freezing remains inefficient largely because of the detrimental effects of cooling on meiotic spindles. Even slight cooling for brief periods of time triggers spindle disassembly. Spindle disassembly increases aneuploidy risk and decreases post-thaw survival, fertilization, embryo development, and pregnancy rates. Over four decades ago, deuterium oxide (D2O) was shown to promote polymerization of microtubules and stabilize spindles in marine species (Inoue et al. Biological Bulletin, 1963;125: 380-381). The objective of this current study was to test the hypothesis that D2O stabilizes meiotic spindles in mammalian eggs, and thus limits cooling-related spindle disassembly. Prospective study of the effects of D2O on spindle stability in living mouse eggs and on embryo development after parthenogenetic activation of mouse eggs treated with D2O, using the PolScope and confocal microscopy. Mouse eggs were exposed to solutions containing 0%, 25%, or 50% D2O and spindle integrity was assessed by polarized light and confocal immunostaining. Living oocytes were imaged noninvasively with an orientation independent polarized light microscope (PolScope, Cambridge Research and Instrumentation) before and after cooling. D2O treated eggs and control eggs were cooled to room temperature or 4°C. Each egg was imaged for 80 minutes after the heating stage was turned off. Spindle birefringence was quantified using Metamorph software (Universal Imaging Corporation). Spindle integrity and chromosome alignment were confirmed by immunostaining with antitubulin antibody and imaging by confocal microscopy. In a follow-up experiment, mouse eggs were exposed to 25% D2O at room temperature or 50% D2O at 4°C for one hour, and subsequent embryo development following parthenogenetic activation by 10 mM strontium was assessed. Birefringent spindles were visualized in all eggs at the start of the experiment. In the 0% D2O solution, all birefringent spindles depolymerized at room temperature after one hour. In the 25% and 50% D2O solutions, all spindles remained assembled and actually exhibited increased microtubule accumulation, as evidenced by both increased birefringence and increased size at room temperature. Additionally, D2O influenced spindle assembly in a dose-dependent fashion, so 50% D2O maintained spindle integrity better than 25% D2O. The findings were similar after cooling to 4°C. Meiotic spindles in 0% D2O were disassembled within one hour at 4°C. Disassembly was also accompanied by slight chromosomal misalignment. In the presence of D2O (25% and 50% solutions), however, meiotic spindles remained stable and showed no evidence of chromosomal misalignment at 4°C. Finally, control eggs cooled without D2O showed poor development, while eggs preserved in 25% D2O at room temperature or cooled to 4°C in 50% D2O for one hour developed normally to blastocysts following parthenogenetic activation. D2O stabilizes mammalian meiotic spindles and eliminates cooling-related microtubule disassembly. D2O added to the media during handling of eggs in the IVF laboratory and during egg cryopreservation promises to greatly improve the efficiency of egg cryopreservation by eliminating cooling-associated disassembly. Moreover, D2O is an isotope of H2O, so it does not need to be injected because it permeates freely into eggs, and its effects are completely reversible after replacement of D2O by H2O.