Validation of a new metabolic marker to assess the vascular viability of vitrified whole ewe ovaries

Abstract

OBJECTIVE: To explore the pedicle metabolism of VS4 (cryoprotector avoiding ice formation in isolated ovarian pedicles but not in cortex) vitrified whole ovaries, by developing a drip of Thiazodyl Blue Tetrazolium (MTT) as metabolic marker. DESIGN: In-vitro study. MATERIALS AND METHODS: Pairs of ewe ovaries were perfused with MTT (1g/l) for 2 hours at 39°C. Group A: tissue lesions were induced by immersing ovaries into 53° or 65°C heated mediums or into liquid nitrogen, prior to MTT drip. Group B: different metabolic substrates (D-glucose, L-glucose) and inhibitor (2-deoxy D-glucose) were added to the MTT drip. Group C: ovaries were exposed to VS4 ± vitrified-thawed prior to MTT perfusion. Pedicle's MTT staining was either qualitatively analyzed on frozen sections (histological assessment) or solubilized in alcohol and quantified by optical density at 564 nm. Statistics: ANOVA, p<0.05 considered significant. Each experiment compared the MTT staining of 10 treated ovaries with their paired controls. RESULTS: MTT stained strongly and reproductively vessel's smooth muscle. Liquid nitrogen, 53°C or 65°C tissue lesions significantly reduce MTT staining by 93.9%, 48.0% and 94.3%, respectively. MTT could thus be considered as a viability marker. MTT staining partially depended on D-glucose metabolism: its inhibition by 2-deoxy D-glucose, privation of D-glucose or its replacement by unmetabolised L-glucose significantly reduce MTT staining by 23.6%, 40.9% and 31.6% respectively. While thawing, ice seeding from ovarian cortex, constantly invaded the fully VS4 vitrified pedicles (recrystallisation): they significantly stained 29.9% less than control, with histological focal unstained areas, whereas unvitrified VS4 exposed pedicles stained like controls. CONCLUSIONS: Thawing is critical for incompletely vitrified whole ovary. Thus MTT appears to be a proper marker to evaluate whole organ cryoprotection protocols.