Vitrification of human pronuclear oocytes by direct plunging into cooling agent: Non sterile liquid nitrogen vs. sterile liquid air

In fact, a full sterilization of commercially-produced liquid nitrogen contaminated with different pathogens is not possible. The aim of this study was to compare the viability of human pronuclear oocytes subjected to cooling by direct submerging of open carrier in liquid nitrogen versus submerging in clean liquid air (aseptic system). One- and three-pronuclei stage embryos (n = 444) were cryopreserved by direct plunging into liquid nitrogen (vitrified) in ethylene glycol (15%), dimethylsulphoxide (15%) and 0.2M sucrose. Oocytes were exposed in 20, 33, 50 and 100% vitrification solution for 2, 1 and 1 min, and 30-50 s, respectively at room temperature. Then first part of oocytes (n = 225) were directly plunged into liquid nitrogen, and second part of oocytes (n = 219) into liquid air. Oocytes were thawed rapidly at a speed of 20,000 °C/min and then subsequently were placed into a graded series of sucrose solutions (0.5, 0.25, 0.12 and 0.06M) at 2.5 min intervals and cultured in vitro for 3 days. In both groups, the rate of high-quality embryos (Grade 6A: 6 blastomeres, no fragmentation; Grade 8A: 8 blastomeres, no fragmentation; Grade 8A compacting: 8 blastomeres, beginning of compacting) was noted. The rates of high-quality embryos developed from one-pronuclear oocytes vitrified by cooling in liquid nitrogen and liquid air were: 39.4% ± 0.6 and 38.7% ± 0.8, respectively (P > 0.1). These rates for three-pronuclear oocytes were: 45.8 ± 0.8% and 52.0 ± 0.7%, respectively (P < 0.05). In conclusion, vitrification by direct submerging of oocytes in clean liquid air (aseptic system) is a good alternative for using of not sterile liquid nitrogen.