Overcoming To Blastocyst Collapse And Improvement Of Blastocyst Development In Sequential Culture Media

Introduction: This study was started to improve in vitro blastocyst development and avoiding of blastocyst collapsment (no visibie cavity, returning of hatching blastocyst into zona pellucida, and tendency to degeneration).
Materials and Methods: 24hrs one - cell mouse embryos were recovered from positive plug females and cultured in following canditions: Experiments 1,2: During first 48hrs NMRI 1-cell mouse embryos were cultured in R1 and then transferred to R2 (group 1) for 3 days. Some of one-cell mouse embryos were clutured in R1 plus commercially vero line or fibroblast cells derived from 12-14 days mouse embryos. After 48hrs, embryos were transferred to R2 plus one of the cell lines (groups 2, 3). Experiment 3, 4: One-cell mouse embryos were cultured in R1 for 48hrs and then transferred to R2 plus vero cells or R2 plus embryonic fibroblast cells, (group 5) for 3 days. Experiment 5: The 24hrs and 48hrs R2-conditioned mediums of vero and embryonic fibroblasts cells were prepared by filtration through 0.22 µ filters and freezed in-20oC. 1-cell mouse embryose were cultured in R1 for 48hrs and transfeered to 24hrs or 48hrs-R1 conditioned mediums for other 3 days.
Results: Comparsion of blastocyst development in 1st, 2nd and 3th group did not show any significant difference (59%, 54 and 52% respectively). Howerer, a high percent of 1-cell embryo developed as hatching and hatched blastocyst (71%) in both cocultures after 5days. The conditioned media (24hrs and 48hrs) on both type cell lines were not improved the blastocyst development and lot of blastocysts were collapsed in 5th day.
Conclusion: These results showed that blastocyst development was significantly improved and blastocyst collapsment was markedly reduced, by using only sequential culture media during 2 days after one-cell embryo retrieval and simultaneously with coculture during following days. 2) Conditioned media did not improve blastocyst development.