title: Factors related to unstained areas in whole ewe ovaries perfused with a metabolic marker.
abstract: OBJECTIVE
  What factors are associated with the presence of areas unexposed to the perfusate after whole ovary perfusion?
  
  
  CONCLUSIONS
  Over half the ovaries perfused with the metabolic marker methylthiazolyl blue tetrazolium (MTT) were incompletely stained. Incomplete staining was statistically significantly associated with a small ovarian slice surface area, inexperience of the experimenter, and the presence of a corpus luteum.
  
  
  BACKGROUND
  Whole ovary cryopreservation followed by vascular auto-transplantation has provided poor outcomes as an alternative way to safeguard fertility. Perfusion, commonly used to expose the ovaries to cryoprotectants, may miss areas excluded from the vascular network, explaining subsequent poor ovarian functionality.
  
  
  METHODS
  An observational study of 360 ewe ovaries stained by in vitro perfusion with MTT as a qualitative marker of tissue blood supply was performed. A logistic regression model was built to identify factors associated with incomplete ovary staining.
  
  
  METHODS
  Whole ewe ovaries with their vascular pedicles were perfused at 0.35 ml/min with 1 g/l MTT for 2 h at 39°C under 19 experimental conditions. The pedicles were removed and the ovaries cut in half sagittally and photographed. The unstained area of the slice surface was measured. Times from ovary collection to ovary rinsing and to MTT perfusion initiation, ovary weight and slice surface area, presence of a corpus luteum and operator experience (number of ovaries previously perfused) were recorded. Pedicle MTT staining was quantified at 564 nm after solubilization in alcohol.
  
  
  RESULTS
  Unstained areas were observed in 64.4% of the ovaries. Multivariate analysis found that incomplete ovary staining was independently associated with lower experimenter experience (P < 0.02), smaller ovary slice surface area (P < 0.0001) and presence of a corpus luteum (P < 0.01). The presence of unstained areas was independent from experimental conditions. The rate of incomplete ovary staining decreased from 83 to 60% beyond the 80th perfused ovary (P < 0.0001).
  
  
  CONCLUSIONS
  Descriptive study.
  
  
  CONCLUSIONS
  Blood-supply impairments that result in incomplete perfusion might adversely affect outcomes after whole ovary cryopreservation. Improved perfusion techniques should enhance success.
authors.affiliation: INSERM U846, Stem-cell and Brain Research Institute, 18 Avenue Doyen Lepine, Bron 69500, France. antoinetorre@voila.fr
authors.forename: A
authors.initials: A
authors.lastname: Torre
authors.forename: F
authors.initials: F
authors.lastname: Ben Brahim
authors.forename: T
authors.initials: T
authors.lastname: Popowski
authors.forename: R
authors.initials: R
authors.lastname: Boudjenah
authors.forename: B
authors.initials: B
authors.lastname: Salle
authors.forename: J
authors.initials: J
authors.lastname: Lornage
mesh_terms: D000818:Animals; D015415:Biomarkers / Q000032:analysis / Q000378:metabolism; D015925:Cryopreservation / Q000379:methods; D005260:Female; D059247:Fertility Preservation / Q000379:methods*; D016015:Logistic Models; D015999:Multivariate Analysis; D010053:Ovary / Q000098:blood supply / Q000378:metabolism*; D010477:Perfusion / Q000379:methods; D012756:Sheep; D013194:Staining and Labeling; D013778:Tetrazolium Salts / Q000032:analysis / Q000378:metabolism; D013844:Thiazoles / Q000032:analysis / Q000378:metabolism; D014021:Tissue Preservation / Q000379:methods*
chemical_list: D015415:Biomarkers; D013778:Tetrazolium Salts; D013844:Thiazoles; C022616:thiazolyl blue