Effect of varying equilibration time in a two-step vitrification method on the post-warming DNA integrity of mouse blastocysts

Objective: To assess the effect of equilibration time on the DNA integrity of vitrified-warmed mouse blastocysts. Design: Prospective in vitro study. Setting: Embryology research laboratory. Intervention(s): Mouse nonexpanded blastocysts (NEB) (n = 54) and expanded blastocysts (EB) (n = 56) were vitrified using cryotips. Blastocysts were vitrified using a two-step media protocol, with a first (equilibration) step of 4 minutes [NEB (n = 20), EB (n = 24)], 8 minutes [NEB (n = 17), EB (n = 16)], or 15 minutes: [NEB (n = 17), EB (n = 16)]. Control samples were fresh NEB (n = 17) and EB (n = 18) blastocysts. Main Outcome Measure(s): DNA integrity index (DII) using terminal deoxynucleotide transferase-mediated dUTP nick-end labeling, confocal imaging, and viability. Result(s): 1) The DII of the vitrified warmed NEB and EB improved significantly with 8-minute equilibration protocol compared with 4 minutes. 2) The DII of the EB significantly decreased with 15-minute equilibration protocol compared with 4 or 8 minutes. 3) The DII in the NEB significantly improved with 8- and 15-minute equilibration protocols compared with 4 minutes, with no significant difference between 8 or 15 minutes. Conclusion(s): Vitrification with an 8-minute equilibration step improved DII of the NEB and EB after warming (Fertil Steril (R) 2010; 93: 2640-5. (C) 2010 by American Society for Reproductive Medicine.)