title: Cryopreservation of the ovary by vitrification as an alternative to slow-cooling protocols.
abstract: OBJECTIVE
  To study the thermal properties of a cryoprotectant solution, called VS4, and of VS4-impregnated whole sheep ovaries with pedicle.
  
  
  METHODS
  Physical and experimental animal study.
  
  
  METHODS
  Academic research environment.
  
  
  METHODS
  Five- to 6-month-old ewes.
  
  
  METHODS
  Thermal properties on cooling of a cryoprotectant solution called VS4 were measured by differential scanning calorimetry. VS4 contains 2.75 mol/L dimethyl sulfoxide, 2.76 mol/L formamide, and 1.97 mol/L propylene glycol. Whole sheep ovaries were collected at the slaughterhouse and prepared for a vitrification procedure. Cortex and vessels underwent histologic examination before and after vitrification, and the thermal properties of VS4-impregnated ovarian cortex were then studied.
  
  
  METHODS
  Critical cooling rates (V(ccr)), vitreous transition temperature (Tg), end-of-melting temperature (Tm), follicle viability assessment by trypan blue test, and histologic examination of ovary and vessel structure.
  
  
  RESULTS
  The critical cooling rate (V(ccr)) of VS4 solution was estimated to be 14.3 +/- 1.1 degrees C/min. Its vitreous transition temperature (Tg) was -125.2 +/- 0.2 degrees C, and its end-of-melting temperature (Tm) -34.3 +/- 0.1 degrees C. Following our vitrification procedure, immediate follicle viability was 61.4% +/- 2.2%. The percentage of normal primordial follicles remaining after vitrification was 48% +/- 3.8%. The V(ccr) of VS4-impregnated cortex could not be determined because of the quantity of ice forming as of the top programmed cooling rate (-300 degrees C/min). The mean ovarian cortex cooling rate actually attained experimentally during vitrification was -342.9 +/- 49.6 degrees C/min.
  
  
  CONCLUSIONS
  Vitrification of entire organs, such as ovaries, is a great challenge in cryobiology and reproductive medicine. Physical studies seem indispensable for progress with this technique. Thus, our ovarian perfusion procedure needs improving to enhance ovarian cortex impregnation and bring down the V(ccr) rate in both tissue and VS4 solution.
authors.affiliation: Unité INSERM 418, Hôpital Debrousse, Lyon, France.
authors.forename: Blandine
authors.initials: B
authors.lastname: Courbiere
authors.forename: Valentina
authors.initials: V
authors.lastname: Odagescu
authors.forename: Anne
authors.initials: A
authors.lastname: Baudot
authors.forename: Jérôme
authors.initials: J
authors.lastname: Massardier
authors.forename: Claire
authors.initials: C
authors.lastname: Mazoyer
authors.forename: Bruno
authors.initials: B
authors.lastname: Salle
authors.forename: Jacqueline
authors.initials: J
authors.lastname: Lornage
mesh_terms: D000818:Animals; D001808:Blood Vessels / Q000187:drug effects; D002152:Calorimetry, Differential Scanning; D015925:Cryopreservation / Q000379:methods*; D003451:Cryoprotective Agents / Q000494:pharmacology; D005260:Female; D007552:Isotonic Solutions / Q000494:pharmacology; D006080:Ovarian Follicle / Q000187:drug effects / Q000473:pathology; D010053:Ovary / Q000098:blood supply; D012756:Sheep; D013997:Time Factors; D014022:Tissue Survival
chemical_list: D003451:Cryoprotective Agents; D007552:Isotonic Solutions