- Cryopreservation of the two-cell stage embryos was performed in accordance to the method of Eto et. al. .
2. The CPS used a pre-treatment solution (P10; 10% propylene glycol in PB1) and vitrification solution (PEPeS; 10% propylene glycol, 30% ethylene glycol, 0.3 mol sucrose, and 20% Percoll in PB1).
3. For warming of vitrified embryos, SPB1 (0.3 mol sucrose in PB1) was used.
4. Percoll was from GE Healthcare (Upsalla, Sweden), and other reagents for preparing the solutions were purchased from Sigma-Aldrich Chemical Co.
5. Two-cell stage embryos of the C57BL/6J strain were used in the experiments.
6. The methods for the vitrification and warming of two-cell stage embryos were as follows.
7. The two-cell stage embryos were exposed to the P10 at 25°C±0.5°C for 5min.
8. The embryos and 5 μl of P10 was then placed into the cryotubes (MS-4501W; Sumitomo Bakelite Co. Ltd., Tokyo, Japan) and cooled to 0°C for 1min.
9. We then added 95 μl of precooled (0°C) vitrification solution to the cryotubes, and 1 min later the cryotubes were placed in liquid nitrogen for vitrification.
10. The vitrified two-cell stage embryos were stored in liquid nitrogen for at least 1 week.
- The vitrified embryos were warmed by shifting the Cryotube from liquid nitrogen to a room temperature of approximately 25oC, 30 seconds later 900μl of SPB1 at 37oC was added to the cryotube and rapidly stirred 5 to 6 times.
12. The warmed embryos were placed in PB1 2 min after the addition of SPB1, left at rest for 2 min, washed with PB1 three times, then washed with mW medium three times, and used for IVC.
13. In the experiment, after the two-cell stage embryos were exposed to CPS, SPB1 was infused into the cryotubes and a group in which the two-cell stage embryos were collected by the same method as used in the warming procedures was prepared.
14. The survival rate of the vitrified embryos and the fresh embryos exposed to CPS was evaluated under an inverted microscope after the embryos were placed into mW medium.
15. The surviving two-cell stage embryos underwent IVC for 72 h and development to blastocysts was examined.
As a fresh control group, untreated embryos were also subjected to IVC.