Cryopreservation of human ovarian tissue by direct plunging into liquid nitrogen

Aim: To establish a prospective direction for further development of the protocol for cryopreservation of ovarian tissue by direct plunging into liquid nitrogen. Materials and methods: Human ovarian biopsies from 20 patients (cut in similar to0.5 mm(3) pieces) were exposed to: 40% ethylene glycol + 0.35 M sucrose + 5% egg yolk; 40% ethylene glycol + 18% Ficoll-70 + 0.35 M sucrose; 20% ethylene glycol + 20% dimethyl sulphoxide. Cryopreservation of pieces was accomplished by plunging 0.25 ml straws or copper grids into liquid nitrogen or 0.25 ml straws into precooled (-196degreesC) metallic powder. Thawed pieces were transferred to sucrose solution for incremental dilution of cryoprotectants. Histological observation of the tissue was performed after cryopreservation and in vitro culture was done to study hormone production ability after cryopreservation. Results: Only ultrarapid cooling in ethylene glycol-sucrose-egg yolk solution protected both follicles and stroma from damage. Conclusion: The following parameters were established as required for a protocol of human ovarian tissue cryopreservation by direct plunging into liquid nitrogen: the vitrification medium should include ethylene glycol, disaccharide and egg yolk; ultrarapid cooling/thawing should take place using standard 0.25 straws or copper grids. (C) 2003 Published by Elsevier Science Ireland Ltd.