title: Quality and functionality of human ovarian tissue after cryopreservation using an original slow freezing procedure.
abstract: OBJECTIVE
  To evaluate the efficiency of an original slow freezing protocol on the quality and function of human ovarian cortex.
  
  
  METHODS
  Human ovarian tissues were cryopreserved using a freezing medium supplemented with propanediol and raffinose as cryoprotectants and antioxidants (L-glutamine, taurine). Samples were then frozen using a faster cooling rate than the usual one. Viability and morphology of follicles, DNA fragmentation in follicles and stroma as well as histology of the vascular endothelium were analyzed before and after freezing/thawing. Moreover, a functional analysis was performed based on the evaluation of follicular growth and development in thawed ovarian tissues that were cultured in vitro.
  
  
  RESULTS
  Our freezing/thawing protocol allows preservation of a high proportion of viable follicles and the preservation of the different follicle developmental stages (p>0.05 versus fresh control). 70.5 ± 5.2 % of follicles retained an intact morphology after cryopreservation (p=0.04). Stroma cells but not follicles exhibited a slight increase of DNA fragmentation after thawing (p<0.05). Microvessel endothelium within thawed tissues appeared to be preserved. Granulosa cells showed signs of proliferation in follicles cultured for 12 days. Secretion of 17β-oestradiol significantly increased during in vitro culture.
  
  
  CONCLUSIONS
  This protocol leads to good preservation of ovarian integrity and functionality post-thawing and thus appears as a suitable technique of ovarian tissue cryopreservation in clinical settings. Further research could be extended to optimize conditions of in vitro culture.
authors.affiliation: Laboratoire de Biologie de la Reproduction, Université Clermont 1, UFR Médecine, E.A. 975, 28 Place Henri Dunant, 63001, Clermont-Ferrand Cedex 1, France. sanfilipposandra@gmail.com
authors.forename: Sandra
authors.initials: S
authors.lastname: Sanfilippo
authors.forename: Michel
authors.initials: M
authors.lastname: Canis
authors.forename: Sergio
authors.initials: S
authors.lastname: Romero
authors.forename: Benoît
authors.initials: B
authors.lastname: Sion
authors.forename: Pierre
authors.initials: P
authors.lastname: Déchelotte
authors.forename: Jean-Luc
authors.initials: JL
authors.lastname: Pouly
authors.forename: Laurent
authors.initials: L
authors.lastname: Janny
authors.forename: Johan
authors.initials: J
authors.lastname: Smitz
authors.forename: Florence
authors.initials: F
authors.lastname: Brugnon
mesh_terms: D000328:Adult; D049109:Cell Proliferation; D048430:Cell Shape; D002470:Cell Survival; D002478:Cells, Cultured; D015925:Cryopreservation / Q000379:methods*; D003451:Cryoprotective Agents / Q000494:pharmacology*; D053938:DNA Fragmentation; D042783:Endothelial Cells / Q000166:cytology / Q000187:drug effects; D004958:Estradiol / Q000378:metabolism; D005260:Female; D005615:Freezing; D006107:Granulosa Cells / Q000166:cytology / Q000187:drug effects; D006801:Humans; D007150:Immunohistochemistry; D008315:Malondialdehyde / Q000494:pharmacology; D009924:Organ Culture Techniques; D006080:Ovarian Follicle / Q000166:cytology / Q000187:drug effects*; D011887:Raffinose / Q000494:pharmacology; D017154:Stromal Cells / Q000166:cytology / Q000187:drug effects; D013696:Temperature; D013997:Time Factors
chemical_list: D003451:Cryoprotective Agents; D004958:Estradiol; D008315:Malondialdehyde; D011887:Raffinose