Ex vivo testis explant culture of human testicular tissue

Introduction: Fertility preservation for boys subjected to gonadotoxic therapy, e.g. for cancer treatment, is increasingly in demand by patients and relatives, as long-term overall survival probability for pediatric cancer patients has risen to > 80% (German Childhood Cancer Registry, 2018). Consequently, most of the patients reach the reproductive age (nearly 50.000 in the GCCR long-term survivor cohort as of 2016). However, cryopreservation of sperm, applicable for later assisted reproduction techniques (ART) is no option for prepubertal boys since no spermatids are produced before puberty. Hence, the only possibility to restore fertility of young cancer survivors is cryopreservation of testicular tissue containing spermatogonial stem cells (SSCs). Material and Methods: In a first attempt to test the potential of juvenile human testicular tissue, we cultivated a rice grain sized testicular biopsy of a 13-year-old boy for 78 days. The tissue was fixed in modified Davidson’s fluid and tissue cells were visualized using immunostaining with cell-specific markers. The initial cellular status of the testis tissue prior to culture was determined on toluidine blue-stained semi-thin sections of glutaraldehyde-osmium-fixed and Epon-embedded tissue. Results: Both, germ cells, including SSCs, and somatic cells could be detected after the culture period. In one case, single PRM1+ structures resembling spermatids were focally detected. These cells are most likely remnants of individual spermatozoa already present in the boy’s testicular tissue, as these were also identified in the testicular tissue before organ culture. Conclusion: These findings demonstrate the potential to develop methods for in vitro spermatogenesis and the propagation of human SSCs within cultured human juvenile testis biopsies. If these techniques can be successfully established, this would pave the way for young male cancer survivors to fulfill their wish for biological paternity in later life.