Novel direct cover vitrification for cryopreservation of ovarian tissues increases follicle viability and pregnancy capability in mice.
BACKGROUND
Cryopreservation of ovarian tissue is valuable for fertility preservation. We develop an innovative vitrification method using less concentrated cryoprotectants and direct application of liquid nitrogen to the ovarian tissue (direct cover vitrification, DCV) to improve its efficiency.


METHODS
Ovaries of 5- to 6-week-old C57BL/6J mice were randomly allocated to four groups: DCV, conventional vitrification, slow-freezing and non-frozen controls. Experiment 1: observing the follicle morphology. Experiment 2: assessing viability. Experiment 3: investigating the ultrastructure. Experiment 4: examining the follicle number after grafting. Experiment 5: ascertaining pregnancy potential by allogeneic orthotopic transplantation.


RESULTS
The percentages of morphologically normal or viable follicles from DCV were significantly greater than those achieved from conventional vitrification and slow freezing (P < 0.01). The ultrastructure of primordial follicles from DCV appeared better than that achieved from conventional vitrification and slow freezing. After grafting, the follicle number from DCV was greater than conventional vitrification (P = 0.001) and slow freezing (P = 0.021). The pregnancy rate of DCV was higher than conventional vitrification (P < 0.01). The litter size from DCV was comparable with that from non-frozen graft and was significantly greater than that achieved from conventional vitrification and slow freezing (P < 0.01).


CONCLUSIONS
DCV is highly efficient for cryopreservation of ovarian tissue. Using less concentrated cryoprotectants appears to reduce toxicity. Direct cover by liquid nitrogen maximizes cooling that could facilitate vitrification and prevent ice crystal injury.
Chen
Shee-Uan
SU
Department of Obstetrics and Gynecology, National Taiwan University Hospital and College of Medicine, Taipei.
Chien
Chung-Liang
CL
Wu
Ming-Yih
MY
Chen
Tzu-Hsin
TH
Lai
Shu-Mei
SM
Lin
Chung-Wu
CW
Yang
Yu-Shih
YS
D000818:Animals; D002470:Cell Survival; D015925:Cryopreservation / Q000379:methods*; D005260:Female; D051379:Mice; D008810:Mice, Inbred C57BL; D006080:Ovarian Follicle / Q000166:cytology* / Q000648:ultrastructure; D010053:Ovary / Q000166:cytology* / Q000648:ultrastructure; D011247:Pregnancy; D011270:Pregnancy, Animal / Q000502:physiology*