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

Abstract

Objective: To investigate possible damage caused by freeze-thawing whole human ovaries.

Design: Prospective experimental study.

Setting: Academic 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.