title: Viability and function of the cryopreserved whole rat ovary: comparison between slow-freezing and vitrification.
abstract: OBJECTIVE
  To investigate four different protocols for cryopreservation of the whole rat ovary with intact vasculature to evaluate whether differences exist in post-thawing viability of the ovary after either vitrification or slow freezing.
  
  
  METHODS
  Experimental study.
  
  
  METHODS
  Obstetrics and gynecology department.
  
  
  METHODS
  Immature Sprague-Dawley female rats.
  
  
  METHODS
  Ovaries were isolated with the vascular tree intact up to the bifurcation of the abdominal aorta and were subsequently cannulated. The ovaries were flushed with increasing concentrations of the cryoprotectant dimethyl sulfoxide (DMSO) to either 1.5 or 7 M. The ovaries underwent cryopreservation by vitrification or passive slow freezing.
  
  
  METHODS
  After thawing, the ovaries were subjected to neutral red viability staining to assess the density of viable small follicles and for long-term (48 hours) incubation evaluation of steroid secretion, histology, and apoptosis assay.
  
  
  RESULTS
  The follicular viability was decreased in both vitrification groups and in the slow-freezing group with the high concentration of DMSO, as compared with fresh controls. Estradiol levels in the incubation medium followed the same pattern. Light microscopy revealed well-preserved morphology in all groups after 48 hours' incubation. Apoptosis was increased in both vitrified and cryopreserved ovaries.
  
  
  CONCLUSIONS
  We have developed a new method that can be used in basic studies to improve cryopreservation protocols. Our initial findings suggest that a moderate concentration of the cryoprotectant DMSO is superior to a high DMSO concentration for both vitrification and slow freezing.
authors.affiliation: Department of Obstetrics and Gynecology, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden.
authors.forename: Milan
authors.initials: M
authors.lastname: Milenkovic
authors.forename: César
authors.initials: C
authors.lastname: Diaz-Garcia
authors.forename: Ann
authors.initials: A
authors.lastname: Wallin
authors.forename: Mats
authors.initials: M
authors.lastname: Brännström
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D053148:Caspase 3 / Q000378:metabolism; D015925:Cryopreservation / Q000379:methods*; D003451:Cryoprotective Agents / Q000494:pharmacology*; D004121:Dimethyl Sulfoxide / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D004958:Estradiol / Q000378:metabolism; D005260:Female; D059247:Fertility Preservation / Q000379:methods*; D005615:Freezing; D007150:Immunohistochemistry; D009926:Organ Preservation / Q000379:methods*; D006080:Ovarian Follicle / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D010053:Ovary / Q000098:blood supply / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology / Q000637:transplantation; D010477:Perfusion; D051381:Rats; D017207:Rats, Sprague-Dawley; D013997:Time Factors; D046509:Tissue Culture Techniques; D014022:Tissue Survival; D058989:Vitrification
chemical_list: D003451:Cryoprotective Agents; D004958:Estradiol; C505410:Casp3 protein, rat; D053148:Caspase 3; D004121:Dimethyl Sulfoxide