Aseptic technology of vitrification of human pronuclear oocytes using open-pulled straws

BACKGROUND: The aim of this study was to compare the viability of human pronuclear oocytes subject vitrification using cooling by direct submerging of open-pulled straws in liquid nitrogen versus vitrification by ing of open-pulled straws located inside a closed 0.5 ml straw (aseptic system). METHODS: Two- and three-pi clei stage oocytes (n = 114) were cryopreserved in super-open-pulled straws by vitrification in 20% ethylene g +20% dimethylsulphoxide (DMSO) + osmotic active and neutral non-permeable cryoprotectants with a four exposure in 20, 33, 50 and 100% vitrification solution for 2, 1 and 1min, and 30-50s, respectively at temperature, and plunging into liquid nitrogen. Oocytes of group 1 (n = 42) were rapidly cooled at a spec 20 000°C/min by direct plunging of open-pulled straws into liquid nitrogen. Oocytes of group 2 (n = 44) were located in 0.5 ml straws, which were closed at both sides by metal balls, and then plunged into liquid nitrogen. method resulted in a cooling speed of 200°C/min. For both groups, oocytes were thawed rapidly at a spec 20000°C/min using an identical protocol. Oocytes subsequently were expelled into a graded series of su solutions (1.0, 0.75, 0.5, 0.25 and 0.12 mol/l) at 2.5 min intervals. RESULTS: Oocyte development up to expa blastocyst stage after in vitro culture was 15% in group 1, 14% in group 2 and 29% in an untreated control group. CONCLUSION: The deposition of human pronuclear oocytes in open-pulled straws which are placed inside a hermetically closed container guarantees a complete isolation of oocytes from liquid nitrogen and avoids potential contamination by pathogenic microorganisms. The combination of direct plunging of this container into liquid nitrogen and rapid warning makes this process as efficient an conventional vitrification.