Cryopreservation of immature testicular tissue (ITT) prior to chemo/radiotherapy is now ethically accepted and is currently the only way to preserve fertility of prepubertal boys about to undergo cancer therapies. So far, three-dimensional culture of testicular cells isolated from prepubertal human testicular tissue was neither efficient nor reproducible to obtain mature spermatozoa, and ITT transplantation is not a safe option when there is a risk of cancer cell contamination of the testis. Hence, generation of testicular organoids (TOs) after cell selection is a novel strategy aimed at restoring fertility in these patients. Here, we created TOs using hydrogels developed from decellularized porcine ITT and compared cell numbers, organization and function to TOs generated in collagen only hydrogel. Organotypic culture of porcine ITT was used as a control. Rheological and mass spectrometry analyses of both hydrogels highlighted differences in terms of extracellular matrix stiffness and composition, respectively. Sertoli cells (SCs) and germ cells (GCs) assembled into seminiferous tubule-like structures delimited by a basement membrane while Leydig cells (LCs) and peritubular cells localized outside. TOs were maintained for 45 days in culture and secreted stem cell factor and testosterone demonstrating functionality of SCs and LCs, respectively. In both TOs GC numbers decreased and SC numbers increased. However, LC numbers decreased significantly in the collagen hydrogel TOs (p < 0.05) suggesting a better preservation of growth factors within TOs developed from decellularized ITT and thus a better potential to restore the reproductive capacity.
Cryopreservation of immature testicular tissue before chemo/radiotherapy is the only option to preserve fertility of cancer-affected prepubertal boys. To avoid reintroduction of malignant cells, development of a transplantable scaffold by decellularization of pig immature testicular tissue (ITT) able to support decontaminated testicular cells could be an option for fertility restoration in these patients. We, therefore, compared decellularization protocols to produce a cytocompatible scaffold. Fragments of ITT from 15 piglets were decellularized using three protocols: sodium dodecyl sulfate (SDS)-Triton (ST), Triton-SDS-Triton (TST) and trypsin 0.05%/ethylenediaminetetraacetic acid (EDTA) 0.02%-Triton (TET) with varying detergent concentrations. All protocols were able to lower DNA levels. Collagen retention was demonstrated in all groups except ST 1%, and a significant decrease in glycosaminoglycans was observed in the TST 1% and TET 1% groups. When Sertoli cells (SCs) were cultured with decellularized tissue, no signs of cytotoxicity were detected. A higher SC proliferation rate and greater stem cell factor secretion were observed than with SCs cultured without scaffold. ST 0.01% and TET 3% conditions offered the best compromise in terms of DNA elimination and extracellular matrix (ECM) preservation, while ensuring good attachment, proliferation and functionality of human SCs. This study demonstrates the potential of using decellularized pig ITT for human testicular tissue engineering purposes.
While improvements made in the field of cancer therapy allow high survival rates, gonadotoxicity of chemo- and radiotherapy can lead to infertility in male and female pre- and postpubertal patients. Clinical options to preserve fertility before starting gonadotoxic therapies by cryopreserving sperm or oocytes for future use with assisted reproductive technology (ART) are now applied worldwide. Cryopreservation of pre- and postpubertal ovarian tissue containing primordial follicles, though still considered experimental, has already led to the birth of healthy babies after autotransplantation and is performed in an increasing number of centers. For prepubertal boys who do not produce gametes ready for fertilization, cryopreservation of immature testicular tissue (ITT) containing spermatogonial stem cells may be proposed as an experimental strategy with the aim of restoring fertility. Based on achievements in nonhuman primates, autotransplantation of ITT or testicular cell suspensions appears promising to restore fertility of young cancer survivors. So far, whether in two- or three-dimensional culture systems, in vitro maturation of immature male and female gonadal cells or tissue has not demonstrated a capacity to produce safe gametes for ART. Recently, primordial germ cells have been generated from embryonic and induced pluripotent stem cells, but further investigations regarding efficiency and safety are needed. Transplantation of mesenchymal stem cells to improve the vascularization of gonadal tissue grafts, increase the colonization of transplanted cells, and restore the damaged somatic compartment could overcome the current limitations encountered with transplantation.
Despite their important contribution to the cure of both oncological and benign diseases, gonadotoxic therapies present the risk of a severe impairment of fertility. Sperm cryopreservation is not an option to preserve prepubertal boys’ reproductive potential, as their seminiferous tubules only contain spermatogonial stem cells (as diploid precursors of spermatozoa). Cryobanking of human immature testicular tissue (ITT) prior to gonadotoxic therapies is an accepted practice. Evaluation of cryopreserved ITT using xenotransplantation in nude mice showed the survival of a limited proportion of spermatogonia and their ability to proliferate and initiate differentiation. However, complete spermatogenesis could not be achieved in the mouse model. Loss of germ cells after ITT grafting points to the need to optimize the transplantation technique. Tissue engineering, a new branch of science that aims at improving cellular environment using scaffolds and molecules administration, might be an approach for further progress. In this review, after summarizing the lessons learned from human prepubertal testicular germ cells or tissue xenotransplantation experiments, we will focus on the benefits that might be gathered using bioengineering techniques to enhance transplantation outcomes by optimizing early tissue graft revascularization, protecting cells from toxic insults linked to ischemic injury and exploring strategies to promote cellular differentiation.
Fertility preservation for prepubertal boys relies exclusively on cryopreservation of immature testicular tissue (ITT) containing spermatogonia as the only cells with reproductive potential. Preclinical studies that used a nude mice model to evaluate the development of human transplanted ITT were characterized by important spermatogonial loss. We hypothesized that the encapsulation of testicular tissue in an alginate matrix supplemented with nanoparticles containing a necrosis inhibitor (NECINH-NPS) would improve tissue integrity and germ cells’ survival in grafts. We performed orthotopic autotransplantation of 1 mm³ testicular tissue fragments recovered form mice (aged 4–5 weeks). Fragments were either non-encapsulated, encapsulated in an alginate matrix, or encapsulated in an alginate matrix containing NECINH-NPs. Grafts were recovered 5- and 21-days post-transplantation. We evaluated tissue integrity (hematoxylin-eosin staining), germ cells survival (immunohistochemistry for promyelocytic leukemia zinc-finger, VASA, and protein-boule-like), apoptosis (immunohistochemistry for active-caspase 3), and lipid peroxidation (immunohistochemistry for malondialdehyde). NECINH-NPs significantly improved testicular tissue integrity and germ cells’ survival after 21 days. Oxidative stress was reduced after 5 days, regardless of nanoparticle incorporation. No effect on caspase-dependent apoptosis was observed. In conclusion, NECINH-NPs in an alginate matrix significantly improved tissue integrity and germ cells’ survival in grafts with the perspective of higher reproductive outcomes.
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