Preliminary study on cryopreservation of intact mouse ovaries by vitrification using ethylene glycol as cryoprotectant

Objective:Ultrarapid freezing method (vitrification) is an alterna tive method for cryopreserving ovarian tissue. The feasibility of int act murine ovaries cryopreservation was studied by vitrification.Methods: Intact mouse ovari es were cryopreserved using vitrification method and slow freezing method. The m orphological alterations after frozen-thawed procedure and the apoptosis rates o f primordial follicles were evaluated by histological examniation and TUNEL tech nique, and transmission electron microscopy (TEM) was used to observe the ultras tructural changes.Results:The percentages of normal primordial fol licles in fro zen-thawed groups were significantly lower than that in the control group (P<0.001 ), while no statistical difference was found between vitrification group and slo w freezing group (78.37%±4.34% vs 78.82%±3.13%). Moreover, the distribution of abnormalities (nucleus, cytoplasm, and nucleus and cytoplasm) was similar (P>0. 05) in these two groups. In the vitrification group, slow freezing group and the fresh control group, the percentages of the TUNEL-positive follicles were 16.3 7 %, 13.29% and 21.36%, respectively. The apoptosis rates in the two cryopreservat ion groups showed no significant difference (P>0.05). After the freezing-thawing procedure, subcellular structure was well preserved in both freezing groups wit h smililar ultrastructural changes.Conclusion:Vitrification is a convenient and efficient approach for small ovarian tissue cryopreservation.