title: Vitrification of human ovarian tissue: a practical and relevant alternative to slow freezing.
abstract: BACKGROUND
  Cryopreservation of ovarian tissue can be used to preserve the fertility of patients who are about to receive treatment(s) that could compromise their future ovarian function. Here we evaluate the effectiveness of a vitrification protocol by carrying out a systematic comparison with a conventional slow-freezing method on human ovarian tissue.
  
  
  METHODS
  Human ovarian samples (mean age 28.0 ± 1.1 years) were processed in parallel for each cryopreservation procedure: vitrification and slow-freezing. Following warming/thawing, histological observations and a TUNEL assay in ovarian follicles were performed and compared to unfrozen control.
  
  
  RESULTS
  Both cryopreservation protocols gave comparable histological outcomes. Percentage of intact follicles was 83.6 % following vitrification in a 1.5 M 1,2-propanediol (PrOH), 1.5 M ethylene glycol (EG) and 0.5 M raffinose solution, 80.7 % after slow-freezing in 1.5 M PrOH and 0.025 M raffinose, and 99.6 % in fresh tissue. Follicle density was unchanged by vitrification (0.6 follicles/mm2) or slow-freezing (0.5 follicles/mm2) compared to fresh tissue (0.7 follicles/mm2). Percentage of follicles with DNA fragmentation was not statistically different in vitrified (20.8 %) or slow-frozen (31.3 %) tissues compared to the unfrozen control (35.0 %). There was no difference in proportion of stroma cells with DNA fragmentation in vitrified (6.4 %) and slow-frozen (3.7 %) tissues compared to unfrozen tissue (4.2 %).
  
  
  CONCLUSIONS
  This vitrification protocol enables good preservation of ovarian quality post-warming. The evaluation of endocrine function after vitrification need to be perform in a higher cohort to evaluate if this protocol may offer a relevant alternative to conventional slow-freezing for the cryopreservation of human ovarian tissue.
authors.affiliation: Centre international de chirurgie endoscopique, Clermont-Ferrand, France. sanfilipposandra@gmail.com.|CHU Clermont-Ferrand, CHU Estaing, Pôle gynécologie obstétrique et reproduction humaine - Assistance médicale à la procréation - CECOS, Clermont-Ferrand, France. sanfilipposandra@gmail.com.
authors.forename: Sandra
authors.initials: S
authors.lastname: Sanfilippo
authors.affiliation: Centre international de chirurgie endoscopique, Clermont-Ferrand, France. mcanis@chu-clermontferrand.fr.|CHU Clermont-Ferrand, CHU Estaing, Pôle gynécologie obstétrique et reproduction humaine - Assistance médicale à la procréation - CECOS, Clermont-Ferrand, France. mcanis@chu-clermontferrand.fr.
authors.forename: Michel
authors.initials: M
authors.lastname: Canis
authors.affiliation: Follicle biology laboratory, Vrije Universiteit Brussel, B - 1090, Jette, Belgium. Johan.Smitz@uzbrussel.be.
authors.forename: Johan
authors.initials: J
authors.lastname: Smitz
authors.affiliation: Laboratoire de physiologie, Université Clermont 1, UFR Pharmacie, Inserm U1107 NEURO-DOL, Clermont-Ferrand, France. benoit.sion@bbox.fr.
authors.forename: Benoît
authors.initials: B
authors.lastname: Sion
authors.affiliation: CHU Clermont-Ferrand, CHU Gabriel Montpied, Anatomie et cytologie pathologiques, Centre de biologie, Clermont-Ferrand, France. cdarcha@chu-clermontferrand.fr.
authors.forename: Claude
authors.initials: C
authors.lastname: Darcha
authors.affiliation: CHU Clermont-Ferrand, CHU Estaing, Pôle gynécologie obstétrique et reproduction humaine - Assistance médicale à la procréation - CECOS, Clermont-Ferrand, France. ljanny@chu-clermontferrand.fr.|Faculté de médecine, CNRS-UMR 6293/INSERM U1103, Laboratoire de génétique, reproduction et développement, Université d'Auvergne, Clermont-Ferrand, France. ljanny@chu-clermontferrand.fr.
authors.forename: Laurent
authors.initials: L
authors.lastname: Janny
authors.affiliation: CHU Clermont-Ferrand, CHU Estaing, Pôle gynécologie obstétrique et reproduction humaine - Assistance médicale à la procréation - CECOS, Clermont-Ferrand, France. fbrugnon@chu-clermontferrand.fr.|Faculté de médecine, CNRS-UMR 6293/INSERM U1103, Laboratoire de génétique, reproduction et développement, Université d'Auvergne, Clermont-Ferrand, France. fbrugnon@chu-clermontferrand.fr.
authors.forename: Florence
authors.initials: F
authors.lastname: Brugnon
mesh_terms: D000328:Adult; D015925:Cryopreservation / Q000379:methods*; D003451:Cryoprotective Agents; D053938:DNA Fragmentation; D005260:Female; D005615:Freezing; D006801:Humans; D006080:Ovarian Follicle / Q000473:pathology*; D010053:Ovary / Q000473:pathology*; D019946:Propylene Glycol; D058989:Vitrification
chemical_list: D003451:Cryoprotective Agents; D019946:Propylene Glycol