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 subjected to vitrification using cooling by direct submerging of open-pulled straws in liquid nitrogen versus vitrification by cool-ing of open-pulled straws located inside a closed 0.5 ml straw (aseptic system). METHODS: Two- and three-pronu-clei stage oocytes (n 5 114) were cryopreserved in super-open-pulled straws by vitrification in 20 % ethylene glycol 120 % dimethylsulphoxide (DMSO) 1 osmotic active and neutral non-permeable cryoprotectants with a four-step exposure in 20, 33, 50 and 100 % vitrification solution for 2, 1 and 1 min, and 30–50 s, respectively at room temperature, and plunging into liquid nitrogen. Oocytes of group 1 (n 5 42) were rapidly cooled at a speed of 20 0008C/min by direct plunging of open-pulled straws into liquid nitrogen. Oocytes of group 2 (n 5 44) were first located in 0.5 ml straws, which were closed at both sides by metal balls, and then plunged into liquid nitrogen. This method resulted in a cooling speed of 2008C/min. For both groups, oocytes were thawed rapidly at a speed of 20 0008C/min using an identical protocol. Oocytes subsequently were expelled into a graded series of sucrose solutions (1.0, 0.75, 0.5, 0.25 and 0.12 mol/l) at 2.5 min intervals. RESULTS: Oocyte development up to expanded 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 potentia