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Updated: Jun 20, 2026
Morphometric Protocol for the Objective Assessment of Blastocyst Behavior During Vitrification and Warming Steps
Published on: February 28, 2019
Effect of varying equilibration time in a two-step vitrification method on the post-warming DNA integrity of mouse
Amr Kader1, Audrey Choi, Rakesh K Sharma
1Center for Reproductive Medicine, Glickman Urological and Kidney Institute, and Obstetrics and Gynecology and Women's Health Institute, Cleveland Clinic, Cleveland, Ohio 44195, USA.
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.
