Toxic Effect of Cryoprotectants on Embryo Development in a Murine Model / 대한불임학회지

OBJECTIVES: The aim of this study was to assess toxicities of cryoprotectants. METHODS: Toxicities of two cryoprotectants, dimethyl sulfoxide (DMSO) and 1,2-propanediol (PROH), were investigated using a murine embryo model. Female F-1 mice were stimulated with gonadotropin, induced ovulation with hCG and mated. Two cell embryos were collected and cultured after exposure to either DMSO or PROH. Embryo development was evaluated up to the blastocyst stage. Blastocysts were stained with bis-benzimide to evaluate the cell count and with terminal deoxynucleotidyl transferase mediated dUTP nick labeling (TUNEL) to assess apoptosis. RESULTS: The total cell count of blastocysts that were treated with DMSO at the 2-cell stage was significantly lower than that were treated with PROH (75.9+/-27.0) or the control (99.0+/-18.3) (p<0.001). On comparison of two cryoprotectant treated groups, the DMSO treated group showed a decreased cell count compared with the PROH treated group (p<0.05). Both DMSO (14.2+/-1.5) and PROH (11.2+/-1.4) treated groups showed higher apoptosis rates of cells in the blastocyst compared with the control (6.2+/-0.9, p<0.0001). In addition, the DMSO treated group showed more apoptotic cells than the PROH treated group (p<0.001). CONCLUSIONS: The potential toxicity of cryoprotectants was uncovered by prolonged exposure of murine embryos to either DMSO or PROH at room temperature. When comparing two cryoprotective agents, PROH appeared to be less toxic than DMSO at least in a murine embryo model.

The Experimental Study on Cryopreservation of Mouse Embryo / 대한불임학회지

OBJECTIVES: This study was carried out to evaluate the effects of embryonic stage, cryoprotectant, and freezing-thawing method on the rates of survival and development of the cryopreserved mouse early embryo and finally to establish the cryopreservation method of surplus embryos obtained during assisted reproductive technology (ART). MATERIALS AND METHODS: Two to eight cell embryos were obtained from oviducts of mated F1 hybrid female mice superovulated by pregnant mare's serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG). Two-step 1,2-propanediol (PROH), dimethylsulfoxide (DMSO) and 4-step PROH, DMSO were used as cryoprotectant and dehydration and rehydration method of embryos, and slow- cooling or rapid-cooling method was used as frozen program. The survival rates of embryos were measured after thawing and rehydration, and the developmental rates of embryos were compared and observed during culturing embryos for 24, 48, 72, 96 hrs. RESULTS: As for the survival and development rates of embryos according to embryonic stage, the survival rate of 2 cell stage in PROH and DMSO was significantly higher than 4-8 cell (64.5% versus 62.1%, 79.7% versus 73.2%) (p<0.01, p<0.01), but the development rates of 4-8 cell embryos in PROH and DMSO were significantly higher than 2 cell embryos for whole culture period (p<0.01) and the development rates of 4-8 cell embryos in PROH were significantly higher than 2 cell embryos in DMSO (p<0.01). As for the survival and development rates of embryos according to cryoprotectant, the survival rate of 2 cell embryo in DMSO was significantly higher than that in PROH (74.4% versus 64.5%) (p<0.01), whereas the development rate of embryos was not differ till 24 hrs. The development rate from morular to hatching blastocyst, however, was significantly higher in PROH than in DMSO during 48 hr (p<0.01). The survival rate of 4-8 cell embryo was 62.1% in PROH and 73.2% in DMSO. The development rates of embryo in PROH were significantly higher for whole culture periods (p<0.01, 0.05). In respect to the effect of freezing and thawing program on the survival and development rates of embryos, method of slow cooling and rapid thawing was more effective than that of rapid cooling and rapid thawing. CONCLUSIONS: The survival rate of embryo in 2 cell stage was higher than in 4-8 cell stage, and PROH appears more effective cryoprotectant than DMSO because PROH showed better development rates of embryos in 2 and 4-8 cell stage. Moreover, slow cooling and rapid thawing method was considered as the best cryopreservation program.

Development of Mice Embryo after Cryopreservation and Thawing / 대한산부인과학회잡지

Cryopreservation has proved to be a highly successful method for long term storage of viable embryos and it is necessary for human embryo cryopreservation in assisted reproductive technology to reduce the risk of multiple gestation and severe ovarian hyperstimulation syndrome as well as contributing to an overall increase in pregnancy rates. Mouse embryo cryopreservation was carried out in order to establish the most ideal developmental stage of embryo for cryopreservation. F1 hybrid mouse embryo were collected according to its developmetnal stage; 1-cell, 2- cell, 4-cell, morula and blastocyst. 1-cell stage embryo were cryopreserved by slow coolingrapid thawing method using PROH(1,2 propanediol) and 2- cell stage embryo and 4-cell stage embryo were cryopreserved by the same method using PROH or DMSO(dimethyl sulfoxide). And morula and blastocyst stage embryo were cryopreserved by the same cooling method using glycerol. The frozen-thawed embryo showed significantly lower(p 0.05). The proportion of frozen embryos developing to hatched blastocysts was significantly decreased in 1-cell stage embryos and blastocysts compared to the 2-cell, 4-cell and morula stage embryo(p < 0.05). According to in vitro development of mouse embryo, the highest hatching rate was obtained from 4-cell stage embryo(43.5%), so the ideal cell stage for embryo cryopreservation is 4-cell stage embryo using DMSO as a cryoprotectant.