S 3 vitrification: a safe, simple, and successful method for blastocyst vitrification

Abstract

Vitrification is becoming an established way of storing embryos, but a safe, easy, and effective procedure has eluded researchers due to shortcomings of current techniques. We examined the effectiveness of a different vitrification method for human blastocysts. Current vitrification protocols, although successful, have shortcomings making them difficult to use effectively. Problems include use of dimethylsulfoxide, a relatively toxic cryoprotectant; micro-sized containers that are often open to contamination and difficult to manipulate; and short exposure times that are technically difficult leaving no room for error. We devised a different technique (S3 vitrification) that avoids these problems by using a large, sterile, sealable container, and longer exposure and handling times. Blastocysts from IVF patients that consented for freezing or research were vitrified. Also, bovine blastocysts were vitrified. Blastocysts were incubated in 2 solutions with ethylene glycol at 23°C for 5 min each, transferred to a vitrification solution of glycerol and ethylene glycol for 2 min while loading into a sterile 0.25 cc straw, heat-sealed, and vitrified. Straws were thawed for 5 sec in air then 10 sec in 30°C water. Cells were washed for 5 min each through 5 drops of medium with decreasing sucrose (0.85 M to 0 M). Blastocysts were graded and transferred to recipients 0–2 hours post-thaw. Implantation and pregnancy rate were examined. Bovine and human blastocysts (donated for research) were cultured 24 h after thaw and stained to analyze survival of all cells. Blastocyst survival was 92.7%, 57.1% implantation, and a clinical pregnancy of 54.2% (Table 1). Human and bovine blastocysts had a high rate of survival (83.3% n = 24 and 96.1% n = 102, respectively) and intact cells (86.4% n = 721 and 93.6% n = 6775, respectively). TableS3 Blastocyst Vitrification DataClinicThawedIntactReplacedImplantedFHBOngoing/DeliveredPreg/TransferA88 (100)87 (87.5)7 (87.5)74/4 (100)B3330 (90)2713 (48.1)12 (44.4)1211/17 (67.4)Total4138 (92.7)3520 (57.1)19 (54.2)1915/21 (71.4) Open table in a new tab The results show that blastocysts can be vitrified using a safe and simple method. We obtained high survival, pregnancy, and birth rates without dimethylsulfoxide; using a large, sealed container allowing easy manipulation with ample time for exposure and loading. The data suggest that S3 vitrification may be a promising, user-friendly replacement for current techniques.