Transplantation of testicular tissue in alginate hydrogel loaded with VEGF nanoparticles improves spermatogonial recovery.

Transplantation of cryopreserved immature testicular tissue (ITT) is a promising strategy to restore fertility in young boys facing gonadotoxic treatments. However, up to now, limited spermatogonial recovery has been achieved in xenografting models used to evaluate the potential of cryopreserved tissue transplantation. When comparing avascular xenografts of cryopreserved and fresh human ITT into a mouse model, the number of spermatogonia was significantly reduced, regardless of the cryopreservation procedure used. To improve tissue engraftment, revascularization and hence spermatogonial survival, ITT was embedded in two types of hydrogel loaded with VEGF nanoparticles. Small pieces (±1mm(3)) of testicular tissue were grafted in NMRI mice as follows: grafted without encapsulation, grafted after encapsulation in fibrin, in alginate, in fibrin-VEGF-nanoparticle (NP) and in alginate-VEGF-NP. Non-grafted tissue served as control. After 5 and 21days of implantation, seminiferous tubule integrity, revascularization and spermatogonial recovery were evaluated by histology and immunohistochemistry. Seminiferous tubule integrity ranged from 13.3% to 39.6% and 42.7% to 68.7% on day 5 and day 21, respectively. Vascular density on day 5 was found to be higher in VEGF supplemented groups, regardless of the hydrogel used. Staining for phosphorylated VEGF receptor 2 and endothelial proliferation on day 5 was higher in all groups compared to non-grafted avascular controls. Spermatogonial recovery ranged between 14.8% and 27.3% on day 21 and was significantly higher in the alginate and alginate-VEGF-NP groups. The present study demonstrates the potential of alginate hydrogel loaded with nanoencapsulated growth factors to improve cryopreserved tissue engraftment.

In Search of Better Spermatogonial Preservation by Supplementation of Cryopreserved Human Immature Testicular Tissue Xenografts with N-acetylcysteine and Testosterone.

Controlled slow-freezing is the procedure currently applied for immature testicular tissue (ITT) cryobanking in clinical practice. Vitrification has been proposed as a promising alternative, with a view to better preserve the spermatogonial stem cells for future fertility restoration by autografting in young boys suffering from cancer. It appears that besides the potential influence of the cryopreservation technique used, the transplantation procedure itself has a significant impact on spermatogonial loss observed in ITT xenografts. Eighteen ITT pieces issued from 6 patients aged 2-15 years were used. Fragments of fresh tissue (serving as ungrafted controls), frozen-thawed tissue, frozen-thawed tissue supplemented with N-acetylcysteine (NAC), and frozen-thawed tissue supplemented with testosterone xenografted to nude mice for 5 days were compared. Upon graft removal, histological and immunohistochemical analyses were performed to evaluate spermatogonia, intratubular proliferation, and intrinsic and extrinsic apoptosis. A significant decrease in the integrity of intact seminiferous tubules was found in all three grafted groups. Spermatogonia were observed by immunohistochemistry in all grafted groups, with recovery rates of 67, 63, and 53%, respectively, for slow-frozen tissue, slow-frozen tissue supplemented with NAC, and slow-frozen tissue supplemented with testosterone. Apoptosis evidenced by active caspase-3 and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling was similar in all grafts. The study is limited by the low availability of ITT samples of human origin, and no clear impact of graft supplementation was found. The mouse xenotransplantation model needs to be refined to investigate human spermatogenesis in human ITT grafts.