Reproductive biomedicine online · 2025
Towards a universal rapid warming protocol for cryopreserved human ovarian tissue.
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Färber Cara Maria, Einenkel Rebekka, Emrich Norah LA, Kestermann Emily Zia, Parmegiani Lodovico, Schallmoser Andreas, Sänger Nicole
Аннотация
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.
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.