title: Ficoll density gradient method for recovery of isolated human ovarian primordial follicles.
abstract: OBJECTIVE
  To develop a simple and efficient technique to allow rapid recovery of a maximum number of good quality isolated follicles.
  
  
  METHODS
  Prospective experimental study.
  
  
  METHODS
  Academic research unit of the department of gynecology in a university hospital.
  
  
  METHODS
  Biopsies were obtained from five women (between 26 and 31 years of age).
  
  
  METHODS
  Biopsies were cut with a tissue sectioner. Enzymatic digestion was performed in a collagenase solution for 90 min at 37 degrees C. The follicles were recovered using a discontinuous Ficoll density gradient method.
  
  
  METHODS
  The number of follicles present in the interface layers of Ficoll gradient was quantified. Follicular viability of these recovered follicles was assessed with live-dead stains, using calcein-AM and ethidium homodimer-I.
  
  
  RESULTS
  Out of a total of 6,811 recovered follicles, we found 63% (n = 4,201) at the medium-1.06 Ficoll interface and 36.9% (n = 2,590) at the 1.06-1.09 Ficoll interface, which represents 99.9% of total recovered follicles. Analysis by vital fluorescent staining showed that 95.8% of the follicles treated with Ficoll were totally viable.
  
  
  CONCLUSIONS
  The Ficoll density gradient method allows us to maximize the recovery of isolated human ovarian follicles and minimize the manipulation time while maintaining high follicular viability.
authors.affiliation: Department of Gynecology, Université Catholique de Louvain, Brussels, Belgium.
authors.forename: Belen
authors.initials: B
authors.lastname: Martinez-Madrid
authors.forename: Marie-Madeleine
authors.initials: MM
authors.lastname: Dolmans
authors.forename: Anne Van
authors.initials: AV
authors.lastname: Langendonckt
authors.forename: Sylvie
authors.initials: S
authors.lastname: Defrère
authors.forename: Anne-Sophie
authors.initials: AS
authors.lastname: Van Eyck
authors.forename: Jacques
authors.initials: J
authors.lastname: Donnez
mesh_terms: D000328:Adult; D001706:Biopsy; D002499:Centrifugation, Density Gradient; D017364:Collagenases / Q000494:pharmacology; D005260:Female; D005362:Ficoll; D006801:Humans; D008856:Microscopy, Fluorescence; D006080:Ovarian Follicle / Q000033:anatomy & histology / Q000502:physiology; D010053:Ovary / Q000187:drug effects / Q000473:pathology; D011446:Prospective Studies; D013048:Specimen Handling / Q000379:methods*; D014022:Tissue Survival
chemical_list: D005362:Ficoll; D017364:Collagenases