Ovarian injury during cryopreservation and transplantation in mice: a comparative study between cryoinjury and ischemic injury.
What is the main cause of ovarian injury during cryopreservation and transplantation in mice: cryoinjury or ischemic injury?



Post-transplantation ischemia is the main cause of ovarian injury during cryopreservation and transplantation for restoring ovarian function.



During cryopreservation and the transplantation of ovaries, cryoinjury and ischemic injury inevitably occur, which has a detrimental effect on ovarian quality and reserve.



A total of 80 B6D2F1 female mice were randomly allocated to 2 control and 6 experimental groups according to the presence or the absence of transplantation (n = 10/group). The control groups consisted of fresh or vitrified-warmed controls that had the whole ovary fixed without transplantation (fresh and vitri-con, respectively). The experimental groups were further divided according to the presence of vitrification (fresh or vitrified-warmed) and the transplantation period (2 [D2], 7 [D7] or 21 [D21] days).



In the control groups, fresh and vitrified-warmed ovaries were immediately fixed after the collection (fresh) and the vitrification-warming process (vitrification control, vitri-con), respectively. Of those experimental groups, three were auto-transplanted with fresh whole ovary (FrOT; FrOT-D2, FrOT-D7 and FrOT-D21). For the other three groups, the ovaries were harvested and stored in liquid nitrogen for 1 week after vitrification and then warmed to auto-transplant the vitrified whole ovaries (vitrified ovary [VtOT]; VtOT-D2, VtOT-D7 and VtOT-D21). After 2, 7 or 21 days of grafting, the grafts and blood sera were collected for analysis by hematoxylin-eosin staining, terminal deoxynucleotidyl transferase dUTP nick end labeling assay, CD31 immunohistochemistry and follicle-stimulating hormone enzyme-linked immunosorbent assay.



The vitrification-warming procedure decreased the proportion of intact follicles (Grade 1, G1) (vitri-con 50.3% versus fresh 64.2%) but there was a larger decrease due to ischemic injury after transplantation (FrOT-D2: 42.5%). The percentage of apoptotic follicles was significantly increased in the vitrified-warmed ovary group compared with the fresh control, but it increased more after transplantation without vitrification (fresh: 0.9%, vitri-con: 6.0% and FrOT-D2: 26.8%). The mean number of follicles per section and percentage of CD31-positive area significantly decreased after vitrification but decreased to a larger extent after transplantation (number of follicles, fresh: 30.3 ± 3.6, vitri-con: 20.6 ± 2.9, FrOT-D2: 17.9 ± 2.1; CD31-positive area, fresh: 10.6 ± 1.3%, vitri-con: 5.7 ± 0.9% and FrOT-D2: 4.2 ± 0.4%). Regarding the G1 follicle ratio and CD31-positive area per graft, only the FrOT groups significantly recovered with time after transplantation (G1 follicle ratio, FrOT-D2: 42.5%, FrOT-D7: 56.1% and FrOT-D21: 70.7%; CD31-positive area, FrOT-D2: 4.2 ± 0.4%, FrOT-D7: 5.4 ± 0.6% and FrOT-D21: 7.5 ± 0.8%). Although there was no significant difference between the two transplantation groups at each evaluation, the serum follicle-stimulating hormone level of both groups significantly decreased over time.



It is unclear how far these results can be extrapolated from mice to the human ovary.



Minimizing ischemic injury should be the first priority rather than preventing cryoinjury alone, and decreasing the combination of cryoinjury and ischemic injury is necessary to improve ovarian quality after cryopreservation and transplantation.



This study was supported by a grant of the Korea Healthcare Technology R&D Project, Ministry of Health & Welfare, Republic of Korea (HI12C0055). The authors have no conflict of interest to declare.
Department of Obstetrics and Gynecology, Seoul National University Bundang Hospital, 300 Gumi-dong, Bundang-gu, Seongnam 463-707, Korea Department of Obstetrics and Gynecology, Seoul National University College of Medicine, Seoul 110-744, Korea.
Jaewang
J
Lee
Department of Obstetrics and Gynecology, Seoul National University Bundang Hospital, 300 Gumi-dong, Bundang-gu, Seongnam 463-707, Korea Department of Obstetrics and Gynecology, Seoul National University College of Medicine, Seoul 110-744, Korea.
Hyun Sun
HS
Kong
Department of Obstetrics and Gynecology, Seoul National University Bundang Hospital, 300 Gumi-dong, Bundang-gu, Seongnam 463-707, Korea Department of Obstetrics and Gynecology, Seoul National University College of Medicine, Seoul 110-744, Korea.
Eun Jung
EJ
Kim
Department of Obstetrics and Gynecology, Seoul National University Bundang Hospital, 300 Gumi-dong, Bundang-gu, Seongnam 463-707, Korea.
Hye Won
HW
Youm
Department of Obstetrics and Gynecology, Seoul National University Bundang Hospital, 300 Gumi-dong, Bundang-gu, Seongnam 463-707, Korea Department of Obstetrics and Gynecology, Seoul National University College of Medicine, Seoul 110-744, Korea leejrmd@snu.ac.kr.
Jung Ryeol
JR
Lee
Department of Obstetrics and Gynecology, Seoul National University Bundang Hospital, 300 Gumi-dong, Bundang-gu, Seongnam 463-707, Korea Department of Obstetrics and Gynecology, Seoul National University College of Medicine, Seoul 110-744, Korea.
Chang Suk
CS
Suh
Department of Obstetrics and Gynecology, Seoul National University College of Medicine, Seoul 110-744, Korea.
Seok Hyun
SH
Kim
D000818:Animals; D015925:Cryopreservation / Q000379:methods*; D003451:Cryoprotective Agents / Q000009:adverse effects; D005260:Female; D007511:Ischemia / Q000209:etiology* / Q000473:pathology; D051379:Mice; D010053:Ovary / Q000098:blood supply / Q000473:pathology / Q000637:transplantation*; D058989:Vitrification
D003451:Cryoprotective Agents
comparative study; cryoinjury; cryopreservation; fertility preservation; ischemic injury; ovarian tissue; vitrification