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

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 (approximately 0.3 degrees C/min), phosphate-buffered medium was superior to Hepes-buffered medium. Termination of slow cooling at -80 degrees C before transfer to liquid nitrogen with subsequent slow thawing (approximately 8 degrees C/min) resulted in more embryos surviving than when cooling terminated at -40 degrees C and rapid thawing (approximately 500 degrees C/min) was employed. Dilution of DMSO upon thawing with medium containing 0.5 M 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 frozen-thawed 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.