BackgroundSpermatogonial stem cell transplantation (SSCT) could become a fertility restoration tool for childhood cancer survivors. However, since in mice, the colonization efficiency of transplanted spermatogonial stem cells (SSCs) is only 12%, the efficiency of the procedure needs to be improved before clinical implementation is possible. Co-transplantation of mesenchymal stem cells (MSCs) might increase colonization efficiency of SSCs by restoring the SSC niche after gonadotoxic treatment.MethodsA mouse model for long-term infertility was developed and used to transplant SSCs (SSCT, n = 10), MSCs (MSCT, n = 10), a combination of SSCs and MSCs (MS-SSCT, n = 10), or a combination of SSCs and TGFß1-treated MSCs (MSi-SSCT, n = 10).ResultsThe best model for transplantation was obtained after intraperitoneal injection of busulfan (40 mg/kg body weight) at 4 weeks followed by CdCl2 (2 mg/kg body weight) at 8 weeks of age and transplantation at 11 weeks of age. Three months after transplantation, spermatogenesis resumed with a significantly better tubular fertility index (TFI) in all transplanted groups compared to non-transplanted controls (P < 0.001). TFI after MSi-SSCT (83.3 ± 19.5%) was significantly higher compared to MS-SSCT (71.5 ± 21.7%, P = 0.036) but did not differ statistically compared to SSCT (78.2 ± 12.5%). In contrast, TFI after MSCT (50.2 ± 22.5%) was significantly lower compared to SSCT (P < 0.001). Interestingly, donor-derived TFI was found to be significantly improved after MSi-SSCT (18.8 ± 8.0%) compared to SSCT (1.9 ± 1.1%; P < 0.001), MSCT (0.0 ± 0.0%; P < 0.001), and MS-SSCT (3.4 ± 1.9%; P < 0.001). While analyses showed that both native and TGFß1-treated MSCs maintained characteristics of MSCs, the latter showed less migratory characteristics and was not detected in other organs.ConclusionCo-transplanting SSCs and TGFß1-treated MSCs significantly improves the recovery of endogenous SSCs and increases the homing efficiency of transplanted SSCs. This procedure could become an efficient method to treat infertility in a clinical setup, once the safety of the technique has been proven.Electronic supplementary materialThe online version of this article (10.1186/s13287-018-1065-0) contains supplementary material, which is available to authorized users.

STUDY QUESTION Can full spermatogenesis be achieved after xenotransplantation of prepubertal primate testis tissue to the mouse, in testis or subcutaneously? SUMMARY ANSWER Intratesticular xenotransplantation supported the differentiation of immature germ cells from marmoset ( Callithrix jacchus ) into spermatids and spermatozoa at 4 and 9 months post-transplantation, while in subcutaneous transplants, spermatogenic arrest was observed at 4 months and none of the transplants survived at 9 months. WHAT IS KNOWN ALREADY Auto-transplantation of cryopreserved immature testis tissue (ITT) could be a potential fertility restoration strategy for patients with complete loss of germ cells due to chemo- and/or radiotherapy at a young age. Before ITT transplantation can be used for clinical application, it is a prerequisite to demonstrate the feasibility of the technique and identify the conditions required for establishing spermatogenesis in primate ITT transplants. Although xenotransplantation of ITT from several species has resulted in complete spermatogenesis, in human and marmoset, ITT has not been successful. STUDY DESIGN, SIZE, DURATION In this study, we used marmoset as a pre-clinical animal model. ITT was obtained from two 6-month-old co-twin marmosets. A total of 147 testis tissue pieces (~0.8–1.0 mm 3 each) were transplanted into the testicular parenchyma (intratesticular; n = 40) or under the dorsal skin (ectopic; n = 107) of 4-week-old immunodeficient Swiss Nu/Nu mice ( n = 20). Each mouse received one single marmoset testis tissue piece in each testis and 4–6 pieces subcutaneously. Xenotransplants were retrieved at 4 and 9 months post-transplantation and evaluations were performed with regards to transplant survival, spermatogonial quantity and germ cell differentiation. PARTICIPANTS/MATERIALS, SETTING, METHODS Transplant survival was histologically evaluated by haematoxylin-periodic acid Schiff (H/PAS) staining. Spermatogonia were identified by MAGE-A4 via immunohistochemistry. Germ cell differentiation was assessed by morphological identification of different germ cell types on H/PAS stained sections. Meiotically active germ cells were identified by BOLL expression. CREM immunohistochemistry was performed to confirm the presence of post-meiotic germ cells and ACROSIN was used to determine the presence of round, elongating and elongated spermatids. MAIN RESULTS AND THE ROLE OF CHANCE Four months post-transplantation, 50% of the intratesticular transplants and 21% of the ectopic transplants were recovered ( P = 0.019). The number of spermatogonia per tubule did not show any variation. In 33% of the recovered intratesticular transplants, complete spermatogenesis was establis...

No abstract

While the incidence of cancer in children and adolescents has significantly increased over the last decades, improvements made in the field of cancer therapy have led to an increased life expectancy for childhood cancer survivors. However, the gonadotoxic effect of the treatments may lead to infertility. Although semen cryopreservation represents the most efficient and safe fertility preservation method for males producing sperm, it is not feasible for prepubertal boys. The development of an effective strategy based on the pharmacological protection of the germ cells and testicular function during gonadotoxic exposure is a non-invasive preventive approach that prepubertal boys could benefit from. However, the progress in this field is slow. Currently, cryopreservation of immature testicular tissue (ITT) containing spermatogonial stem cells is offered to prepubertal boys as an experimental fertility preservation strategy by a number of medical centers. Several in vitro and in vivo fertility restoration approaches based on the use of ITT have been developed so far with autotransplantation of ITT appearing more promising. In this review, we discuss the pharmacological approaches for fertility protection in prepubertal and adolescent boys and the fertility restoration approaches developed on the utilization of ITT.

BackgroundSpermatogonial stem cell transplantation (SSCT) is a promising therapy in restoring the fertility of childhood cancer survivors. However, the low efficiency of SSCT is a significant concern. SSCT could be improved by co-transplanting transforming growth factor beta 1 (TGFβ1)-induced mesenchymal stem cells (MSCs). In this study, we investigated the reproductive efficiency and safety of co-transplanting spermatogonial stem cells (SSCs) and TGFβ1-induced MSCs.MethodsA mouse model for long-term infertility was used to transplant SSCs (SSCT, n = 10) and a combination of SSCs and TGFβ1-treated MSCs (MSi-SSCT, n = 10). Both transplanted groups and a fertile control group (n = 7) were allowed to mate naturally to check the reproductive efficiency after transplantation. Furthermore, the testes from transplanted males and donor-derived male offspring were analyzed for the epigenetic markers DNA methyltransferase 3A (DNMT3A) and histone 4 lysine 5 acetylation (H4K5ac).ResultsThe overall tubular fertility index (TFI) after SSCT (76 ± 12) was similar to that after MSi-SSCT (73 ± 14). However, the donor-derived TFI after MSi-SSCT (26 ± 14) was higher compared to the one after SSCT (9 ± 5; P = 0.002), even after injecting half of the number of SSCs in MSi-SSCT. The litter sizes after SSCT (3.7 ± 3.7) and MSi-SSCT (3.7 ± 3.6) were similar but differed significantly with the control group (7.6 ± 1.0; P < 0.001). The number of GFP+ offspring per litter obtained after SSCT (1.6 ± 0.5) and MSi-SSCT (2.0 ± 1.0) was also similar. The expression of DNMT3A and H4K5ac in germ cells of transplanted males was found to be significantly reduced compared to the control group. However, in donor-derived offspring, DNMT3A and H4K5ac followed the normal pattern.ConclusionCo-transplanting SSCs and TGFβ1-treated MSCs results in reproductive efficiency as good as SSCT, even after transplanting half the number of SSCs. Although transplanted males showed lower expression of DNMT3A and H4K5ac in donor-derived germ cells, the expression was restored to normal levels in germ cells of donor-derived offspring. This procedure could become an efficient method to restore fertility in a clinical setup, but more studies are needed to ensure safety in the long term.