Morphological appearance of the cryopreserved mouse blastocyst as a tool to identify the type of cryoinjury

BACKGROUND: If it were possible to deduce the mechanism of injury in cryopreserved embryos by their appearance, it would help to optimize cryopreservation protocols. METHODS: Mouse blastocysts were treated so that they were damaged by the six types of cryoinjuries listed below, and their appearance was observed at recovery in sucrose solution and a modified phosphate-buffered saline (PB1), and after culture for 1 and 24 h. RESULTS: (i) Intracellular ice: the embryos shrank normally in sucrose solution, but swelled in PB1 and collapsed after culture. (ii) Chemical toxicity of the cryoprotectant: the embryos looked normal in sucrose solution and PB1. After 1 h of culture, however, the blastomeres showed decompaction and degenerated thereafter. If the toxicity was extremely high, embryos looked nearly normal in PB1, but the surface of the cytoplasm was wrinkled as if they were `fixed'. (iii) Osmotic swelling: the embryos looked normal in PB1, but after culture they shrank. (iv) Osmotic shrinkage: the embryos swelled in PB1, and then collapsed. (v) Fracture damage: the zona pellucida of the embryos was dissected. (vi) Extracellular ice: the zona of the embryos was elongated. CONCLUSIONS: It was often possible to deduce the type of injury that had occurred in cryopreserved embryos from their appearance at recovery and during subsequent culture. This may help to improve cryopreservation protocols for embryos of many species, including man.