O-22: Bioenergy/Oxidative Status of Human Ovarian Tissue Cryopreserved by Slow Freezin or Vitrification
Ovarian tissue cryopreservation represents a promising strategy to preserve the ovarian function in cancer patients. It is usually performed by slow freezing/rapid thawing (SF/RT). Recent studies emphasize an ultrarapid cryopreservation procedure, vitrification/warming (V/W), since it might prevent damages due to ice crystal formation. Comparative studies between the cryopreservation procedures are primarily based on morphological evaluation of ovarian tissue. This study aims to investigate the bioenergy/oxidative status using Confocal Laser Scanning Microscopy (CLSM) of ovarian tissue cryopreserved by SF/RT and V/W, in association with a morpho-ultrastructural analysis by Light (LM) and Transmission Electron Microscopy (TEM).
Materials and methods
Six ovarian biopsies of consent cancer patients were cryopreserved by SF/RT and V/W. Fresh and cryopreserved tissues were processed for CLSM using MitoTracker Orange CMTM Ros (mitochondria activity) and DCHFDA (intracellular reactive oxygen species levels-ROS) fluorescent probes and for ematossilin/eosin staining (LM) and TEM. Mitochondria activity and ROS levels of fresh, SF/ RT and V/W samples were compared by ANOVA.
Bioenergy/oxidative status resulted significantly different in fresh and cryopreserved ovarian tissues. Fresh samples presented higher mitochondria activity and ROS levels than SF/RT (P=0.002 and P<0.001, respectively) and V/W (P<0.001) samples. The cryopreservation protocols also differed for mitochondria activity and ROS levels (P<0.001). Morpho-ultrastructural analysis showed a well-preserved fresh tissue and stromal and follicle damages in cryopreserved tissues. The most alterations were interstitial oedema and oocytes cytoplasm vacuolization in SF/RT samples, and slight stromal vacuolization and swelling of oocyte mitochondria in V/W samples
CLSM analysis of bioenergy/oxidative status revealed cryopreservation-induced functional ovarian damages, as demonstrated by the reduction of mitochondrial oxidative phosphorylation activity and by the decrease of ROS levels. The application of CLSM, in association with LM and TEM, can be used as an efficient test to evaluate the effectiveness of cryopreservation procedures.