title: Investigation of in vitro parameters and fertility of mouse ovary after storage at an optimal temperature and duration for transportation.
abstract: OBJECTIVE
  How do the temperature and duration of storage affect ovaries during transportation?
  
  
  CONCLUSIONS
  Fertility is reduced with the extension of the storage duration.
  
  
  BACKGROUND
  Live birth has been reported after ovarian transport overnight on ice before freezing ovarian tissue, but there have been no basic investigations of ovarian storage conditions focused on fertility. There are no guidelines on optimal ovarian storage conditions and the maximum storage time during transportation.
  
  
  METHODS
  Experiments were performed using C57BL/6J mice. Ovaries of 4-week-old mice were harvested, stored at 4, 14, 37 °C or room temperature (RT) for 24 h, and subjected to histological examination. Next, ovaries were stored at 4 °C for 4, 8 or 24 h and subjected to histological examination. Then orthotopic transplantation of ovaries, stored at 4 °C for 4, 8 or 24 h, was performed in 6-week-old C57BL/6J mice, and fertility was assessed by in vitro fertilization and embryo transfer. Freshly harvested ovaries were used as controls for comparison with ovaries stored under the above-mentioned conditions and experiments were repeated at least three times.
  
  
  METHODS
  In experiments on the ovarian storage temperature, haematoxylin-eosin (HE) staining was performed for histological examination. In experiments on the storage duration, HE staining, the terminal deoxynucleotidyl transferase dUTP nick end labelling assay, Ki-67 staining and electron microscopy were performed, and the numbers of follicles were counted. Fertility was assessed from the number of oocytes, and the rates of fertilization, embryo development, implantation and live birth.
  
  
  RESULTS
  Histological changes were minimal after storage of ovaries at 4 °C for up to 24 h. At 4 °C, there were no significant changes in the number of MII oocytes, fertilization rate or blastocyst development rate with storage up to 24 h. The implantation rate was 82.7 ± 17.3% in the control group, while it was 82.2 ± 7.7, 14.6 ± 14.6 and 4.4 ± 4.4% after storage for 4, 8 or 24 h, respectively. After 8 or 24 h of storage, the implantation rate was significantly lower in than in the control group (P< 0.05). The rate of live pups was 24.8 ± 13.2% in the control group, while it was 23.9 ± 6.6, 4.2 ± 4.2 and 4.4 ± 4.4% after storage for 4, 8 or 24 h, respectively. After 8 or 24 h of storage, the rate of live pups was significantly lower than in the control group (P< 0.05).
  
  
  CONCLUSIONS
  Further investigations are needed in mammals with ovaries of a similar size to human ovaries, and should include the assessment of fertility following transplantation of frozen and thawed ovaries.
  
  
  CONCLUSIONS
  The present results suggest that prolonging the ovarian storage time reduces fertility in mice. Thus, ovaries should be frozen immediately after harvesting or transported as rapidly as possible to minimize damage. To allow young cancer patients to preserve fertility, regional medical centres need adequate ovarian tissue cryopreservation techniques.
  
  
  BACKGROUND
  This study supported by Department of Obstetrics and Gynecology, St. Marianna University School of Medicine. The authors have no competing interests to declare.
authors.affiliation: Department of Obstetrics and Gynecology, St. Marianna University School of Medicine, Kanagawa 216-8512, Japan Department of Obstetrics and Gynecology, The Jikei University School of Medicine, Tokyo 105-8461, Japan.
authors.forename: K
authors.initials: K
authors.lastname: Kamoshita
authors.affiliation: Department of Obstetrics and Gynecology, St. Marianna University School of Medicine, Kanagawa 216-8512, Japan.
authors.forename: N
authors.initials: N
authors.lastname: Okamoto
authors.affiliation: Department of Obstetrics and Gynecology, St. Marianna University School of Medicine, Kanagawa 216-8512, Japan.
authors.forename: M
authors.initials: M
authors.lastname: Nakajima
authors.affiliation: Department of Obstetrics and Gynecology, The Jikei University School of Medicine, Tokyo 105-8461, Japan.
authors.forename: T
authors.initials: T
authors.lastname: Haino
authors.affiliation: Department of Obstetrics and Gynecology, The Jikei University School of Medicine, Tokyo 105-8461, Japan.
authors.forename: K
authors.initials: K
authors.lastname: Sugimoto
authors.affiliation: Department of Obstetrics and Gynecology, The Jikei University School of Medicine, Tokyo 105-8461, Japan.
authors.forename: A
authors.initials: A
authors.lastname: Okamoto
authors.affiliation: Department of Obstetrics and Gynecology, St. Marianna University School of Medicine, Kanagawa 216-8512, Japan.
authors.forename: Y
authors.initials: Y
authors.lastname: Sugishita
authors.affiliation: Department of Obstetrics and Gynecology, St. Marianna University School of Medicine, Kanagawa 216-8512, Japan nao@marianna-u.ac.jp.
authors.forename: N
authors.initials: N
authors.lastname: Suzuki
mesh_terms: D000818:Animals; D002585:Cesarean Section / Q000662:veterinary; D003080:Cold Temperature / Q000009:adverse effects; D015925:Cryopreservation / Q000379:methods / Q000662:veterinary*; D004624:Embryo Transfer / Q000662:veterinary; D005260:Female; D005307:Fertilization in Vitro / Q000662:veterinary; D007564:Japan; D050498:Live Birth / Q000662:veterinary; D008810:Mice, Inbred C57BL; D008813:Mice, Inbred ICR; D046529:Microscopy, Electron, Transmission / Q000662:veterinary; D010053:Ovary / Q000166:cytology / Q000378:metabolism / Q000637:transplantation* / Q000648:ultrastructure; D013997:Time Factors; D014186:Transportation
keywords: cryopreservation; fertility; in vitro fertilization; mouse; ovarian storage