Ovarian injury during cryopreservation and transplantation in mice: a comparative study between cryoinjury and ischemic injury

• Published in: Human reproduction (Oxford) 2016-08, Vol.31 (8), p.1827-1837
• Main Authors: Lee, Jaewang, Kong, Hyun Sun, Kim, Eun Jung, Youm, Hye Won, Lee, Jung Ryeol, Suh, Chang Suk, Kim, Seok Hyun
• Format: Article
• Language: English
• Subjects: Animals; Cryopreservation - methods; Cryoprotective Agents - adverse effects; Female; Ischemia - etiology; Ischemia - pathology; Mice; Ovary - blood supply; Ovary - pathology; Ovary - transplantation; Vitrification
• ISSN: 0268-1161; 1460-2350
• DOI: 10.1093/humrep/dew144

Abstract STUDY QUESTION What is the main cause of ovarian injury during cryopreservation and transplantation in mice: cryoinjury or ischemic injury? SUMMARY ANSWER Post-transplantation ischemia is the main cause of ovarian injury during cryopreservation and transplantation for restoring ovarian function. WHAT IS KNOWN ALREADY During cryopreservation and the transplantation of ovaries, cryoinjury and ischemic injury inevitably occur, which has a detrimental effect on ovarian quality and reserve. STUDY DESIGN, SIZE, DURATION 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). PARTICIPANTS/MATERIALS, SETTING, METHODS 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. MAIN RESULTS AND THE ROLE OF CHANCE 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-p