External bending of cryodevice during vitrification leads to cryoprotectant cracks and damage to embryo blastomeres

Research Question Embryo blastomeres and the zona pellucida are occasionally damaged during vitrification. Is this a result of crack-induced mechanical damage in the glass state, which is caused by external bending of the device? Design A stereomicroscope was used to observe external bending-induced cracks in a cryoprotectant. Thereafter, 309 human cleavage-stage embryos derived from abnormally fertilised eggs were employed to assess embryo damage under two external bending conditions: forward bending (FB) and backward bending (BB), with three bending degrees applied. Three distinct embryo positions were used to examine the correlation between bending and embryo damage. Results A series of parallel cracks were identified in the cryoprotectant used for external bending; such cracks led to damage to the embryo blastomeres. The embryos were found to be easily damaged by BB, whereas embryo blastomeres were not impacted by FB. In addition, the degree of embryo damage increased as the degree of external forces increased. Notably, embryo position was correlated with the degree of embryo damage. Conclusions We identified a cause of embryo damage, namely cryoprotectant crack-induced damage, which is caused by mechanical damage to the glass state due to improper external bending of the cryodevice strip in liquid nitrogen during vitrification. To prevent damage, bending of the strip should be avoided and the embryos should be placed near the tip of the strip.