Cryopreservation of human and rabbit oocytes and one-cell embryos: a comparison of DMSO and propanediol

Abstract

The aim of this study was to improve the cryopreservation of human oocytes and pronuclear embryos. One-step and multiple-step addition of dimethyl sulphoxide (DMSO) and 1,2-propanediol (PROH) and three different freezing protocols with intermediate temperatures of -35, -70 and -110 degrees C were investigated. This work was performed using rabbit oocytes as well as human oocytes and one-cell embryos from the routine IVF programme. Also, human polyploid pronucleate oocytes were used in controlled prospective studies of morphological intactness and development in vitro. Rabbit oocytes survived best (113/126) when PROH was added in one step and controlled freezing stopped at -110 degrees C. But the development was better (141/187) if DMSO was added in multiple steps and the oocytes were cooled to -70 degrees C before being plunged into liquid nitrogen. The mode of addition of the cryoprotectant influenced development only if slow freezing was stopped at -35 degrees C (51 versus 34%). Using PROH, the development after thawing was also better if cooling was stopped at -35 degrees C (51 versus 37%) and DMSO was superior to PROH when the oocytes were cooled slowly to -110 degrees C (66 versus 37%). In the human, significantly more pronucleated than unfertilized oocytes developed after freezing (92 versus 50%). The best results were achieved with pronuclear embryos using 1.5 M PROH and cooling to -110 degrees C, when 91.7% of the surviving oocytes developed further. This is a marked improvement of the development rate and comparable to embryo freezing.