Apoptosis and ultrastructural assessment after cryopreservation of whole human ovaries with their vascular pedicle

ObjectiveTo investigate possible damage caused by freeze-thawing whole human ovaries.DesignProspective experimental study.SettingAcademic gynecology research unit in a university hospital.Patient(s)Ovaries were obtained from three women (aged 29–36 years).Intervention(s)Ovaries were perfused and bathed in cryoprotective solution, and slow freezing was performed. Rapid thawing was achieved by perfusion and bathing with a decreased sucrose gradient.Main Outcome Measure(s)Apoptosis was assessed by the terminal deoxynucleotidyl transferase-mediated biotinylated deoxyuridine triphosphates nick end-labeling (TUNEL) method and by immunohistochemistry for active caspase-3 in fresh ovaries, after cryoprotectant exposure, and after thawing. Morphometric analysis of TUNEL-positive surface area was performed. Ultrastructure was assessed by transmission electron microscopy (TEM) in the thawed tissue.Result(s)No primordial or primary follicles were found to be positive for either TUNEL or active caspase-3. No statistically significant difference in mean TUNEL-positive surface area values was found between the three groups: fresh, 0.05% ± 0.03%, with 134 high-power fields (HPFs); cryoperfused, 0.02% ± 0.01%, with 130 HPFs; and thawed, 0.09% ± 0.03%, with 622 HPFs. By means of TEM, follicles and vessels showed a well-preserved ultrastructure, with 96.7% (29/30) healthy-looking primordial and primary follicles, and 96.3% (180/187) healthy-looking endothelial cells.Conclusion(s)Cryopreservation of intact human ovary with its vascular pedicle, according to the freeze-thawing protocol described here, is not associated with any signs of apoptosis or ultrastructural alterations in any cell types. Whole-organ vascular transplantation may thus be a viable option in the future.