Towards a universal rapid warming protocol for cryopreserved human ovarian tissue.

RESEARCH QUESTION: Is it feasible to apply a universal warming protocol to slow-frozen human ovarian tissue, using the rapid-warming protocols already established for vitrified tissue, so that a single protocol could be used for both methods?

DESIGN

Slow-frozen ovarian tissue samples from 25 patients were thawed by conventional protocol (slow freezing-thawing) or by a rapid-warming protocol produced 'in-house' or by commercially available media. Anti-Müllerian hormone values and follicular viability were determined before cryopreservation. Twenty-four hours after thawing, follicular viability was determined. Hypoxic tissue culture was conducted for 48 h to determine VEGF-A levels to estimate the angiogenic potential. Tissue sections were analysed with TUNEL staining for apoptosis and with haematoxylin and eosin staining for primordial follicles.

RESULTS

Follicular survival did not statistically differ: based on fresh tissue (67.5), viable follicle numbers after conventional thawing (44.5), rapid warming with 'in-house' produced (54.5) and commercially available media (58.5) revealed no statistically significant differences between the groups, but were the highest using the rapid warming protocols. After 48 h in culture, preservation of primordial follicles was highest in the rapid warming groups (52% and 57%) compared with the regular thawing group (33%). No significant differences in vascular endothelial growth factor-A were observed comparing conventional thawing protocol with the rapid warming protocol using 'in-house'-produced media. Although VEGF-A secretion was significantly lower in tissues after using commercially available rapid-warming media (P ≤ 0.0001) compared with conventional thawing, apoptosis level was significantly lower (P = 0.014) indicating improved tissue preservation.

CONCLUSIONS

Rapid-warming protocols provide similar to superior results compared with the conventional thawing approach. A universal protocol would increase the efficiency of ovarian tissue thawing-warming, potentially optimizing costs and improving outcome.