STUDY QUESTION: Is testicular transplantation of in vitro propagated spermatogonial stem cells associated with increased cancer incidence and decreased survival rates in recipient mice? SUMMARY ANSWER: Cancer incidence was not increased and long-term survival rate was not altered after transplantation of in vitro propagated murine spermatogonial stem cells (SSCs) in busulfan-treated recipients as compared to non-transplanted busulfan-treated controls. WHAT IS KNOWN ALREADY: Spermatogonial stem cell autotransplantation (SSCT) is a promising experimental reproductive technique currently under development to restore fertility in male childhood cancer survivors. Most preclinical studies have focused on the proofof- principle of the functionality and efficiency of this technique. The long-term health of recipients of SSCT has not been studied systematically. STUDY DESIGN, SIZE, DURATION: This study was designed as a murine equivalent of a clinical prospective study design. Long-term follow- up was performed for mice who received a busulfan treatment followed by either an intratesticular transplantation of in vitro propagated enhanced green fluorescent protein (eGFP) positive SSCs (cases, n = 34) or no transplantation (control, n = 37). Using a power calculation, we estimated that 36 animals per group would be sufficient to provide an 80% power and with a 5% level of significance to demonstrate a 25% increase in cancer incidence in the transplanted group. The survival rate and cancer incidence was investigated until the age of 18 months. PARTICIPANTS/MATERIALS, SETTING, METHODS: Neonatal male B6D2F1 actin-eGFP transgenic mouse testis were used to initiate eGFP positive germline stem (GS) cell culture, which harbor SSCs. Six-week old male C57BL/6 J mice received a single dose busulfan treatment to deplete the testis from endogenous spermatogenesis. Half of these mice received a testicular transplantation of cultured eGFP positive GS cells, while the remainder of mice served as a control group. Mice were followed up until the age of 18 months (497-517 days post-busulfan) or sacrificed earlier due to severe discomfort or illness. Survival data were collected. To evaluate cancer incidence a necropsy was performed and tissues were collected. eGFP signal in transplanted testis and in benign and malignant lesions was assessed by standard PCR. MAIN RESULTS AND THE ROLE OF CHANCE: We found 9% (95% CI: 2-25%) malignancies in the transplanted busulfan-treated animals compared to 26% (95% CI: 14-45%) in the busulfan-treated control group, indicating no statistically significant difference in incidence of malignant lesions in transplanted and control mice (OR: 0.3, 95% CI: 0.1-1.1). Furthermore, none of the malignancies that arose in the transplanted animals contained eGFP signal, suggesting that they are not derived from the in vitro propagated transplanted SSCs. Mean survivaltime after busulfan treatment was found to be equal, with a mean survival time for transplanted animals of 478 days and 437 days for control animals (P = 0.076). LARGE SCALE DATA: NA. LIMITATIONS, REASONS FOR CAUTION: Although we attempted to mimic the future clinical application of SSCT in humans as close as possible, the mouse model that we used might not reflect all aspects of the future clinical setting. WIDER IMPLICATIONS OF THE FINDINGS: The absence of an increase in cancer incidence and a decrease in survival of mice that received a testicular transplantation of in vitro propagated SSCs is reassuring in light of the future clinical application of SSCT in humans. STUDY FUNDING/COMPETING INTEREST(S): This study was funded by KiKa (Kika86) and ZonMw (TAS 116003002). The authors report no financial or other conflict of interest relevant to the subject of this article.