Integrity rate of pronuclei after cryopreservation of pronuclear-zygotes as a criteria for subsequent embryo development and pregnancy.

Abstract

BACKGROUND: The aim of this study was to examine whether the integrity rate of pronuclei, after cryopreservation of pronuclear zygotes, could be a predictor of future embryo development and implantation. METHODS: Two-pronuclei stage zygotes (n = 862) were cryopreserved by aseptic rapid freezing in 15% ethylene glycol + 15% DMSO + 0.2 M sucrose with a 4-step exposure in 12% (v:v), 25, 50 and 100% rapid freezing solution for 2, 1, 1 min and 30-50 s, respectively, at room temperature, and then plunged into liquid nitrogen. Zygotes were rapidly warmed at a speed of 30,000 degrees C/min and subsequently expelled into a graded series of sucrose solutions (1.0, 0.5, 0.25, 0.12 and 0.06 M) at 2.5 min intervals and in vitro cultured for 5 days. If embryos had developed to blastocysts on the 5th day, they were transferred to the recipients. The rest of the embryos were fixed for evaluation of chromatin. RESULTS: Zygote development, up to the expanded blastocyst stage, after in vitro culture, was 40%, if the integrity rate of pronuclei was high, and 4% if the integrity rate was low (P < 0.05). The pregnancy rate after transfer of the 5-day blastocysts depended on the pronuclear integrity rate: 43% (125 pregnancies aftertransfer of 291 blastocysts) for those with a high integrity, and only 1 in 5 (20%) for the few blastocysts which had shown a pronuclear low integrity rate (P < 0.05). CONCLUSIONS: Integrity rate of pronuclei after cryopreservation of pronuclear zygotes was a predictor of future embryo development and implantation: high integrity rate resulted in high pregnancy rate, while zygotes with low integrity rate of pronuclei after cryopreservation had low developmental potential.