Experience with the cryopreservation of human embryos using the mouse as a model to establish successful techniques

Abstract

Mouse embryos at the one-, two-, and eight-cell stages have been used to optimize the conditions for cryopreservation of human oocytes and embryos. For storage in glass vials using 1.5 M dimethyl sulfoxide (DMSO) as a cryoprotectant and slow cooling (≈0.3°C/min), phosphate-buffered medium was superior to Hepes-buffered medium. Termination of slow cooling at −80°C before transfer to liquid nitrogen with subsequent slow thawing (−8°C/min) resulted in more embryos surviving than when cooling terminated at −40°C and rapid thawing (−500°C/min) was employed. Dilution of DMSO upon thawing with medium containing 0.5M sucrose gave higher embryo survival rates than a stepwise (0.25 M decrements) dilution. Using these techniques, three pregnancies were established upon the transfer of 11 frozenthawed embryos to seven patients. Rates of embryo survival using the simpler cryopreservation technique of ice-free vitrification in 0.25-ml straws have been disappointing.