title: Use of trypan blue staining to assess the quality of ovarian cryopreservation.
abstract: OBJECTIVE
  To check the efficiency of the cryopreservation procedure by using trypan blue staining of ovarian test fragments to assess the quality of frozen-thawed ovarian tissue.
  
  
  METHODS
  Prospective study.
  
  
  METHODS
  University hospital.
  
  
  METHODS
  Patients with polycystic ovary syndrome undergoing laparoscopic ovarian drilling.
  
  
  METHODS
  Ovarian cortical biopsies obtained from polycystic ovary syndrome patients were frozen using a slow freezing-rapid thawing protocol.
  
  
  METHODS
  Primordial and primary follicle viability was assessed with trypan blue staining. Unstained isolated follicles were studied by transmission electron microscopy. Histologic and immunohistochemical analysis of apoptosis was performed on tissue sections.
  
  
  RESULTS
  The percentage of unstained follicles considered live was lower (P=.015) after freezing/thawing (71.9%) than before cryopreservation (87.3%). Transmission electron microscopy on follicles considered live confirmed the lack of ultrastructural damage. After freezing/thawing, tissue morphology was preserved, but immunohistochemical analysis shows a significant increase in the apoptosis process.
  
  
  CONCLUSIONS
  Ovarian cortical test fragments combined with trypan blue staining on enzymatically isolated follicles is a useful and quick method of assessing the initial quality and viability of follicles in cryopreserved ovarian tissue. This type of test should be used routinely as quality control in ovarian cryopreservation procedures.
authors.affiliation: Service de Génétique, Histologie, Biologie du Développement et de la Reproduction, Centre d'Etude et de Conservation des Oeufs, Hôpital Saint Jacques, University of Franche-Comté, Besançon, France.
authors.forename: Patricia
authors.initials: P
authors.lastname: Fauque
authors.forename: Anis
authors.initials: A
authors.lastname: Ben Amor
authors.forename: Christiane
authors.initials: C
authors.lastname: Joanne
authors.forename: Germain
authors.initials: G
authors.lastname: Agnani
authors.forename: Jean Luc
authors.initials: JL
authors.lastname: Bresson
authors.forename: Christophe
authors.initials: C
authors.lastname: Roux
mesh_terms: D000328:Adult; D015925:Cryopreservation / Q000379:methods* / Q000592:standards; D003451:Cryoprotective Agents / Q000032:analysis; D005260:Female; D006801:Humans; D010053:Ovary / Q000737:chemistry / Q000648:ultrastructure*; D011085:Polycystic Ovary Syndrome / Q000473:pathology; D011446:Prospective Studies; D013194:Staining and Labeling / Q000379:methods* / Q000592:standards; D014343:Trypan Blue / Q000032:analysis*
chemical_list: D003451:Cryoprotective Agents; D014343:Trypan Blue