title: Beneficial effect of directional freezing on in vitro viability of cryopreserved sheep whole ovaries and ovarian cortical slices.
abstract: OBJECTIVE
  Does directional freezing improve the structural and functional integrity of ovarian fragments compared with conventional slow freezing and to whole ovary cryopreservation?
  
  
  CONCLUSIONS
  Compared with slow freezing, the use of directional freezing significantly improves all structural and functional parameters of ovarian fragments assessed in vitro and, overall, whole ovaries were better preserved than ovarian fragments.
  
  
  BACKGROUND
  Directional freezing has been developed to provide an alternative way to cryopreserve large biological samples and it is known to improve the structural and functional integrity of whole ovaries. Conventional slow freezing of ovarian fragments is the procedure more widely used in clinical settings but it causes substantial structural damage that limits the functional period after transfer back into the patient.
  
  
  METHODS
  We performed a 2 × 2 factorial design experiment on a total of 40 sheep ovaries, divided into four groups (n = 10 ovaries per group): (i) directional freezing of whole ovary (DFwo); (ii) directional freezing of ovarian fragments (DFof); (iii) conventional freezing of whole ovary (CFwo); (iv) conventional freezing of ovarian fragments (CFof). An additional eight ovaries were used as fresh controls.
  
  
  METHODS
  Ewe ovaries were randomly assigned to one of the experimental groups and frozen accordingly. Upon thawing, ovarian tissue was examined morphologically and cultured in vitro for 7 days. Samples were analyzed for cell proliferation and apoptosis, for DNA damage and repair activity, and for the presence of a panel of heat shock proteins (HSPs) by immunohistochemistry.
  
  
  RESULTS
  Most studied parameters were significantly improved (P < 0.05) in all samples cryopreserved with directional compared with slow freezing. The proportion of primordial follicles, which developed to the primary stage in whole ovaries (53 ± 1.7%) and in ovarian fragments (44 ± 1.8%) cryopreserved with directional freezing, was greater than with slow frozen whole ovaries (6 ± 0.5%, P = 0.001) or fragments (32 ± 1.5%, P = 0.004). After 7 days of culture, cell proliferation in DFwo (28 ± 0.73%) was the highest of all groups (P < 0.05) followed by DFof (23 ± 0.81%), CFof (20 ± 0.79%) and CFwo (9 ± 0.85%). Directional freezing also resulted in a better preservation of the cell capacity to repair DNA damage compared with slow freezing both in whole ovaries and ovarian fragments. Apoptosis and HSP protein levels were significantly increased only in the CFwo group. Direct comparison demonstrated that, overall, DFwo had better parameters than DFof and was no different from the fresh controls.
  
  
  CONCLUSIONS
  The study is limited to an in vitro evaluation and uses sheep ovaries, which are smaller than human ovaries and therefore may withstand the procedures better.
  
  
  CONCLUSIONS
  Improved integrity of ovarian morphology may translate to improved outcomes after transplantation. Alternatively, the particularly good preservation of whole ovaries suggests they could provide a source of ovarian follicles for in vitro culture in those cases when the presence of malignant cells poses a substantial risk for the patient.
  
  
  BACKGROUND
  Supported by: Associazione Italiana per la Ricerca sul Cancro (AIRC) IG 10376, Carraresi Foundation and by Legge 7 Regione Autonoma Sardegna (R.A.S). There are no conflicts of interest.
authors.affiliation: Laboratory of Biomedical Embryology, Department of Health, Animal Science and Food Safety, Università Degli Studi di Milano, 20133 Milano, Italy.
authors.forename: S
authors.initials: S
authors.lastname: Maffei
authors.forename: G
authors.initials: G
authors.lastname: Pennarossa
authors.forename: T A L
authors.initials: TA
authors.lastname: Brevini
authors.forename: A
authors.initials: A
authors.lastname: Arav
authors.forename: F
authors.initials: F
authors.lastname: Gandolfi
mesh_terms: D000818:Animals; D049109:Cell Proliferation; D015925:Cryopreservation / Q000379:methods* / Q000662:veterinary; D004249:DNA Damage; D004260:DNA Repair; D005260:Female; D005615:Freezing; D006360:Heat-Shock Proteins / Q000096:biosynthesis; D006657:Histones / Q000096:biosynthesis; D009924:Organ Culture Techniques; D006080:Ovarian Follicle / Q000502:physiology; D010053:Ovary / Q000166:cytology* / Q000378:metabolism; D051135:Rad51 Recombinase / Q000096:biosynthesis; D012756:Sheep; D034561:Sheep, Domestic
chemical_list: D006360:Heat-Shock Proteins; D006657:Histones; D051135:Rad51 Recombinase
keywords: cryopreservation; directional freezing; follicles; ovarian fragments; whole ovaries