| In the recent decades, assisted reproductive techniques (ART) have beenone of the important treatment methods for the infertility and sterility, and ithas been considerably developed. Embryo cryopreservation technique hasbecome increasingly important in ART since it offers many potentialadvantages, such as increasing delivery rate per retrieval cycle, reducing therisk of multiple pregnancies, reducing the severity of ovarian hyperstimulationby selecting proper time for embryo transfer, avoiding the supernumeraryembryos from destruction and reducing the treatment costs. How to improvethe survival rate and the developmental potential of cryopreserved embryoshas been the highlight of the researches for a long time.In this study, the techniques of cell culture, embryo cryopreservation,laser-assisted hatching, micromanipulation and laser confocal microscopewere used toⅰ) evaluate the effects of different cryoprotectants oncryopreservation of mouse embryos,ⅱ) evaluate the effects of differentcryopreservation protocols on cryopreservation of mouse embryos,ⅲ) study the influences on the developmental potential of embryo by removal ofnecrotic blastomeres with laser-assisted hatching and micromanipulation frompartially damaged cryopreserved embryos, andⅳ) evaluate the effects ofcryopreservation on the cytoskeleton of mouse embryo. The results were asfollows.When 2～8-cell mouse embryos were cryopreserved by theslowing-freezing protocol, the survival rate, the blastocyst formation rate andthe blastocyst hatching rate with propandiol (PROH) as cryoprotectant weresignificantly higher than those with demethyl-sulphoxide (DMSO) andglycerol (G) (P＜0.05, respectively), but there were no significant differencecompared with ethylene glycerol (EG) (P＞0.05). Furthermore, there were nosignificant difference among the survival rate, the blastocyst formation rateand the blastocyst hatching rate compared with DMSO, G and EG ascryoprotectant (P＞0.05, respectively).When 2～8-cell mouse embryos were cryopreserved by Vit-Mastervitrification protocol, the survival rate, the blastocyst formation rate and theblastocyst hatching rate with EG as cryoprotectant were significantly higherthan those with PROH, DMSO and G as cryoprotectant (P＜0.05,respectively). However, there were no significant difference among thesurvival rate, the blastocyst formation rate and the blastocyst hatching ratecompared with PROH, DMSO and G as cryoprotectant (P＞0.05,respectively).The survival rate, the blastocyst formation rate and the blastocysthatching rate of cryopreserved 2～8-cell mouse embryos by Vit-Mastervitrification protocol with EG as cryoprotectant were not significantly different compared with those by the slowing-freezing protocol with PROH (P＞0.05).After 8-cell mouse embryos were thawed, the blastocyst formation rateand the blastocyst hatching rate of intact thawed embryos were significantlyhigher than those of partially damaged thawed embryos (P＞0.05).Laser-assisted hatching of mouse cryopreserved fully intact embryossignificantly increased, blastocyst hatching (63.4％vs. 48.3％, respectively, P＜0.05), but had little effect on blastocyst formation (72.0％vs. 70.1％,respectively, P＞0.05). The removal of necrotic blastomeres from partiallydamaged mouse cryopreserved embryos with micromanipulation significantlyincreased blastocyst formation (52.9％vs. 32.0％, respectively, P＜0.05)andblastocyst hatching (41.2％vs. 22.0％, respectively, P＜0.05) compared withthe control group.The retrospective analysis of human cryopreserved embryos transfershowed that the clinical pregnancy rate and implantation rate of the embryostransferred with both intact state and partial damage were significantly higherthan those transferred with only partial damage (P＜0.05). Laser assistedhatching has little effect on the clinical pregnancy rate and implantation rate ofthe transferred human cryopreserved embryos (P＞0.05). Removal of necroticblastomeres from partially damaged cryopreserved human embryos beforetransfer significantly increased the clinical pregnancy rate (43.90％vs. 24.00％,respectively, P＜0.05) and implantation rate (19.44％vs. 10.30％, respectively,P＜0.05) compared with the control group.The slowing-freezing and fast-thawing protocol with PROH ascryoprotectant had little effect on the distributing characteristic of cytoskeleton of 2-cell, 4-cell mouse embryos, and had little effect on the fluorescentintensity by fluorescence quantitive analysis.In conclusion, PROH was the most effective cryoprotectant for thecryopreservation of 2～8-cell mouse embryos by slowing-freezing andfast-thawing protocol. EG was the most effective cryoprotectant for thecryopresevation of 2～8-cell mouse embryos by Vit-Master vitrificationprotocol, and which may be a substituted method for slowing-freezing andfast-thawing protocol. Laser assisted hatching could improve the blastocysthatching of mouse embryos. The developmental potential of partially damagedcryopreserved embryos could be improved by removal of necrotic blastomereswith micromanipulation. The slowing-freezing and fast-thawing protocol withPROH as cryoprotectant had little effect on the cytoskeleton of 2～4-cell mouseembryos. The results of our study will afford some valuable data for theclinical application of the techniques of embryos cryopreservation.
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