title: Optimal vitrification protocol for mouse ovarian tissue cryopreservation: effect of cryoprotective agents and in vitro culture on vitrified-warmed ovarian tissue survival.
abstract: OBJECTIVE
  What is the optimal vitrification protocol according to the cryoprotective agent (CPA) for ovarian tissue (OT) cryopreservation?
  
  
  CONCLUSIONS
  The two-step protocol with 7.5% ethylene glycol (EG) and 7.5% dimethyl sulfoxide (DMSO) for 10 min then 20% EG, 20% DMSO and 0.5 M sucrose for 5 min showed the best results in mouse OT vitrification.
  
  
  BACKGROUND
  Establishing the optimal cryopreservation protocol is one of the most important steps to improve OT survival. However, only a few studies have compared vitrification protocols with different CPAs and investigated the effect of in vitro culture (IVC) on vitrified-warmed OT survival. Some recent papers proposed that a combination of CPAs has less toxicity than one type of CPA. However, the efficacy of different types and concentrations of CPA are not yet well documented.
  
  
  METHODS
  A total of 644 ovaries were collected from 4-week-old BDF1 mice, of which 571 ovaries were randomly assigned to 8 groups and vitrified using different protocols according to CPA composition and the remaining 73 ovaries were used as controls. After warming, each of the eight groups of ovaries was further randomly divided into four subgroups and in vitro cultured for 0, 0.5, 2 and 4 h, respectively. Ovaries of the best two groups among the eight groups were autotransplanted after IVC.
  
  
  METHODS
  The CPA solutions for the eight groups were composed of EDS, ES, ED, EPS, EF, EFS, E and EP, respectively (E, EG; D, DMSO; P, propanediol; S, sucrose; F, Ficoll). The IVC medium was composed of α-minimal essential medium, 10% fetal bovine serum and 10 mIU/ml follicle-stimulating hormone (FSH). Autotransplantation of vitrified-warmed OTs after IVC (0 to 4 h) using the EDS or ES protocol was performed, and the grafts were recovered after 3 weeks. Ovarian follicles were assessed for morphology, apoptosis, proliferation and FSH level.
  
  
  RESULTS
  The percentages of the morphologically intact (G1) and apoptotic follicles in each group at 0, 0.5, 2 and 4 h of IVC were compared. For G1 follicles at 0 and 4 h of IVC, the EDS group showed the best results at 63.8 and 46.6%, respectively, whereas the EP group showed the worst results at 42.2 and 12.8%, respectively. The apoptotic follicle ratio was lowest in the EDS group at 0 h (8.1%) and 0.5 h (12.7%) of IVC. All of the eight groups showed significant decreases in G1 follicles and increases in apoptotic follicles as IVC duration progressed. After autotransplantation, the EDS 0 h group showed a significantly higher G1 percentage (84.9%) than did the other groups (42.4-58.8%), while only the ES 4 h group showed a significant decrease in the number of proliferative cells (80.6%, 87.6-92.9%). However, no significant differences in apoptotic rates and FSH levels were observed between the groups after autotransplantation.
  
  
  CONCLUSIONS
  The limitation of this study was the absence of in vitro fertilization using oocytes obtained from OT grafts, which should be performed to confirm the outcomes of ovarian cryopreservation and transplantation.
  
  
  CONCLUSIONS
  We compared eight vitrification protocols according to CPA composition and found the EDS protocol to be the optimal method among them. The data presented herein will help improve OT cryopreservation protocols for humans or other animals.
authors.affiliation: Department of Obstetrics and Gynecology, Seoul National University Bundang Hospital, 300 Gumi-dong, Bundang-gu, Seongnam 463-707, Korea.
authors.forename: Hye Won
authors.initials: HW
authors.lastname: Youm
authors.forename: Jung Ryeol
authors.initials: JR
authors.lastname: Lee
authors.forename: Jaewang
authors.initials: J
authors.lastname: Lee
authors.forename: Byung Chul
authors.initials: BC
authors.lastname: Jee
authors.forename: Chang Suk
authors.initials: CS
authors.lastname: Suh
authors.forename: Seok Hyun
authors.initials: SH
authors.lastname: Kim
mesh_terms: D000818:Animals; D017209:Apoptosis; D049109:Cell Proliferation; D015925:Cryopreservation / Q000379:methods*; D003451:Cryoprotective Agents / Q000494:pharmacology*; D005260:Female; D005640:Follicle Stimulating Hormone / Q000378:metabolism; D051379:Mice; D008815:Mice, Inbred Strains; D010053:Ovary / Q000166:cytology / Q000187:drug effects* / Q000502:physiology; D058989:Vitrification
chemical_list: D003451:Cryoprotective Agents; D005640:Follicle Stimulating Hormone
keywords: fertility preservation; in vitro culture; ovarian tissue; transplantation; vitrification