Evaluation of post-thaw DNA integrity of mouse blastocysts after ultrarapid and slow freezing

Objective: To evaluate the effect of vitrification and two other methods of slow cryopreservation on DNA integrity in expanded and nonexpanded blastocysts. Design: Prospective in vitro study. Setting: Tertiary care academic hospital. Intervention(s): 1) Twenty-two expanded blastocysts (EB) and 17 nonexpanded blastocysts (NEB) vitrified in cryotips; 2) 15 EB and 16 NEB by slow freezing using propanediol; 3) 11 EB and 16 NEB by slow cryopreservation using glycerol; and 4) 14 EB and 13 NEB as fresh control samples. Main Outcome Measure(s): DNA fragmentation by TUNEL and confocal imaging. Result(s): Blastocysts slowly cryopreserved with glycerol showed DNA integrity of 94.76 ± 4.70% and 90.87 ± 6.16% for NEB and EB, respectively. Propanediol cryopreservation showed values of 72.63 ± 13.44% and 56.19 ± 25.49% and vitrification 84.36 ± 8.7%6 and 77.61 ± 16.65%, respectively, for the same groups. The NEB showed less DNA fragmentation than EB in all cryopreservation techniques, but this was significant only with slow freezing using propanediol. Conclusion(s): All cryopreservation techniques induce DNA damage to blastocysts. Damage is maximal with propanediol and minimal with slow freezing using glycerol. The more expanded the blastocyst, the greater is the susceptibility to DNA damage during cryopreservation.