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.


METHODS
Prospective in vitro study.


METHODS
Embryology research laboratory.


METHODS
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.


METHODS
DNA integrity index (DII) using terminal deoxynucleotide transferase-mediated dUTP nick-end labeling, confocal imaging, and viability.


RESULTS
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.


CONCLUSIONS
Vitrification with an 8-minute equilibration step improved DII of the NEB and EB after warming.
Kader
Amr
A
Center for Reproductive Medicine, Glickman Urological and Kidney Institute, and Obstetrics and Gynecology and Women's Health Institute, Cleveland Clinic, Cleveland, Ohio 44195, USA.
Choi
Audrey
A
Sharma
Rakesh K
RK
Falcone
Tommaso
T
Agarwal
Ashok
A
D000818:Animals; D001755:Blastocyst / Q000187:drug effects / Q000502:physiology*; D002470:Cell Survival; D015925:Cryopreservation / Q000379:methods*; D003451:Cryoprotective Agents / Q000494:pharmacology; D004249:DNA Damage; D004355:Drug Stability; D046149:Embryo Culture Techniques / Q000379:methods; D005260:Female; D020287:In Situ Nick-End Labeling; D051379:Mice; D013997:Time Factors
D003451:Cryoprotective Agents