Ultra-rapid freezing of human multipronuclear zygotes using electron microscope grids

Developmental capacity of human multipronuclear (PN) zygotes cryopreserved using an ultra-rapid freezing method and electron microscope (EM) grids was studied. Multipronuclear zygotes obtained from a human IVF programme were used as an alternative to normal 2PN zygotes; they were divided into 3PN or greater than or equal to 4PN zygotes and their in-vitro development and cryo-injury were compared according to PN number. EFS30, which consisted of 30% ethylene glycol, 18% Picoll, 0.5 mol/l sucrose and 10% fetal bovine serum with added modified Dulbecco's phosphate buffered saline was used as the freezing solution, After ultra-rapid freezing and thawing 85.5% of multipronuclear zygotes survived. A comparison of cleavage rates between the control and cryopreserved groups showed no significant difference (3PN; 81.3 and 85.4% and greater than or equal to 4PN; 90.0 and 95.7% respectively). Comparing the in-vitro development after thawing up to blastocyst formation on day 5 after IVF, the outcome of the frozen 3PN group (22.0%) was not different from that of control 3PN group (38.5%), while the outcome of the frozen greater than or equal to 4PN group (4.5%) was significantly lower than that of control greater than or equal to 4PN group (44.4%) (P < 0.05).