51 optimization of vitrification parameters for rhesus macaque blastocysts

The cryobiological characteristics of rhesus macaque embryos are not well defined, and only a few studies report limited success rates after recovery from slow-freezing cryopreservation. Vitrification is the method currently in use by many infertility clinics with 85 to 96% embryos surviving post-thaw. However, success of thawing rhesus macaque embryos after vitrification is not well documented. Therefore, multiple vitrification parameters were evaluated on rhesus expanded blastocysts by comparing culture medium, vitrification method, and embryo integrity. Embryos (n=204) were produced by standard IVF and cultured in either Global (single-step; LifeGlobal Group, Guilford, CT, USA) or Quinns Advantage (sequential; SAGE, Trumbull, CT, USA) in a humidified tri-gas mixture (6% CO2, 5% O2, 89% N2) at 37°C until blastocyst expansion was observed after 7 to 9 days of culture. Embryos were then vitrified in straws using the Global Fast Freeze Vitrification protocol or in cryotops using the Kitazato method (VT 601-Top-Kitazato, Japan). Additional embryos were biopsied with an objective-mounted laser to remove ~10 to 20 trophectoderm cells before vitrification with Global Fast Freeze in straws. Embryos were thawed at 30°C and cultured at 37°C for an additional 48h or until arrested. Morphology was graded as good (re-expanded), poor (shrunken cytoplasm), or lysed, following thaw, and success of good or hatched embryos was analysed by Fisher’s exact test with P<0.05. Because our goal was to provide good quality embryos for embryo transfer, the ability to hatch was our standard for determining success. There was no significant difference in embryo quality between culture medium, vitrification method, or embryo integrity. Overall, 90% of the embryos were scored as morphologically good regardless of culture medium based on re-expansion post-thaw <24h (55/61) but had a relatively low hatching rate of 25% (15/61), demonstrating that not all re-expanded embryos will go on to achieve the hatched state. Initial trials vitrifying with the Global protocol were associated with decreased recovery rates as many embryos were lost. Cryotops were able to increase our recovery rate to 100% (n=22) but with only 5% surviving post-thaw (1/22). Decreasing the amount of media used to recover embryos from straws enabled a re-evaluation of the cryotop technique with an improved recovery of 92% (60/65). Biopsied embryos tended to have a higher hatch rate (48%; 10/21) compared with intact embryos frozen at the same stage (26%; 9/35). The trophectoderm biopsy likely allows for better permeation of the cryoprotectant before vitrification in embryos due to a collapsed blastocoel. Taken together, these data suggest that vitrification is a viable method of preserving embryos of genetic value and those that undergo manipulation, including trophectoderm.