A new safe, simple and successful vitrification method for bovine and human blastocysts

Survival, re-expansion and cell survival of human blastocysts following vitrification and warming using two vitrification systems

“…Furthermore, the distinct developmental stages of the embryos in different groups did not affect overall cell survival. Overall, these results confirmed that when combined with ethylene glycol, both DMSO, with its fast penetrating characteristics and low molecular weight, and glycerol, with higher molecular weight but moving across the plasma membrane predominantly by facilitated diffusion through aquaporins 3 [26], are efficient cryoprotectants for blastocyst vitrification, as earlier demonstrated by Stachecki et al [12,13]. Thus, blastocyst vitrification could be effectively achieved with larger carriers, which were sealed to avoid cross contamination during long term storage, using longer loading and exposure times and without the need of blastocoel collapse.…”

“…The results obtained with the Fast Freeze direct plunge were in accordance with those observed in a preliminary study where a small sample of surplus human blastocysts donated for research were vitrified with the S3 method (basis of the Global Fast Freeze media, using a protocol similar to that used in the Fast Freeze-direct plunge group) and subsequently stained, leading to a survival rate following warming of 84 % and a cell survival rate of 87 % [12]. Thus, we could document and confirm that the simplified version of the Fast Freeze protocol, including the direct plunge of the freezing straw into liquid nitrogen (Fast Freezedirect plunge), leads to equivalent results to those obtained with the original protocol version (S3 vitrification system; [13]).…”

“…Thus, blastocyst vitrification could be effectively achieved with larger carriers, which were sealed to avoid cross contamination during long term storage, using longer loading and exposure times and without the need of blastocoel collapse. Slower cooling rates were achieved with the Fast Freeze-direct plunge and with the Fast Freeze −100°C step when compared with the Vit Kit-direct plunge group, but high and reproducible pregnancy rates have been reported using this method of vitrification (mean pregnancy rate of 65 %, according to Stachecki et al [12]). …”

“…Blastocysts were stained with propidium iodide (PI) for staining dead cells (Life-dead cell staining kit; Biovision, CA, US) and Hoechst stain 33342 (Thermo scientific, IL, USA) for staining the nucleus of the fixed cells, as described by Stachecki and collegues [12], with slight modifications. Briefly, blastocysts were incubated in 400 μl of staining buffer medium with 1 μl of PI (2.5 mg/ml), in the dark, at 37°C for 15 min, washed in buffer medium, fixed individually in 70 % ethanol at 5°C for 5 min, and incubated in 400 μl of 70 % ethanol containing 1 μl of Hoechst stain 33342 (12.3 mg/ml) at room temperature for 10 min.…”

“…Rapid-I™ kit, Vitro Life, Sweden, [11]), while others are volume independent, use larger straws which are easier to handle and allow for longer embryo exposure to vitrification solutions (e.g. Global Fast Freeze kit, LifeGlobal, LLC, Canada, [12,13]). …”