Cryopreservation of human ovarian tissue: comparison of novel direct cover vitrification and conventional vitrification.
OBJECTIVE
To compare the effect of novel direct cover vitrification (DCV) and conventional vitrification (CV) for human ovarian tissue.


METHODS
Ovarian biopsy specimens obtained from 12 patients were randomly allocated into five groups: Fresh, DCV1, DCV2, DCV3 and CV. Three concentrations of cryoprotectants were used in DCV group. The equilibration solution of DCV1, DCV2, DCV3 was 5% EG+5% DMSO+DPBS, 7.5% EG+7.5%DMSO+DPBS, 10% EG+10% DMSO+DPBS, respectively. And the vitrification solution of DCV1, DCV2, DCV3 was 10% EG+10% DMSO+DPBS, 15%EG+15% DMSO+DPBS, 20% EG+20% DMSO+DPBS, respectively. The equilibration solution and the vitrification solution of CV group was same as DCV3 group. The effects of cryopreserved procedure on human ovarian tissue were studied by histology, TUNEL assay, transmission electron microscopy (TEM) and heterotopic allograft.


RESULTS
The percentages of morphologically normal and viable follicles of DCV2 were significantly higher than DCV1, DCV3 and CV groups (P<0.05). TUNEL assay demonstrated that the incidence of apoptotic cell in vitrification ovarian tissue was significantly higher than fresh tissue (P<0.05), but there were no difference in various groups with cryopreservation. TEM showed that less damage was detected in DCV2 group. After grafting, the follicle density of DCV2 was greater than DCV1, DCV3 and CV groups (P<0.05).


CONCLUSIONS
The novel cover vitrification with optimal concentration of cryoprotectants is superior to conventional vitrification. It is suitable for human ovarian tissue fragments with high efficiency and facility.
Department of Gynecology, First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, China.
Xin-Hui
XH
Zhou
Yi-Jun
YJ
Wu
Jin
J
Shi
Ya-xian
YX
Xia
Shu-Sen
SS
Zheng
D000328:Adult; D000818:Animals; D017209:Apoptosis; D015925:Cryopreservation / Q000379:methods*; D003451:Cryoprotective Agents; D005260:Female; D006801:Humans; D020287:In Situ Nick-End Labeling; D051379:Mice; D008819:Mice, Nude; D046529:Microscopy, Electron, Transmission; D006080:Ovarian Follicle / Q000166:cytology / Q000648:ultrastructure; D010053:Ovary / Q000033:anatomy & histology / Q000637:transplantation / Q000648:ultrastructure; D014183:Transplantation, Heterologous; D055815:Young Adult
D003451:Cryoprotective Agents