Background: Survival rates among childhood cancer patients have progressively increased over the past four decades particularly due to the development of more effective anti-cancer treatments. Today, 85% of these children will be cured of their disease and approx. 12,000 in Sweden, are adult childhood cancer survivors (CCSs). An increasing number of cured patients will face late effects of the disease itself or due to the anti-cancer treatments. Treatments, such as high cumulative doses of cytotoxic agents, exposure to gonadal and pituitary gland irradiation, as well as conditioning therapies for haematopoietic stem cell transplantations (HSCTs), have been identified as risk factors for germ cell loss and infertility in adulthood. At the tissue level, male fertility relies on the spermatogenic process leading to the production of fertile male gametes (sperm). This process includes the proliferation of spermatogonial stem cells (SSCs), meiotic division, and the differentiation and maturation of spermatids, ultimately resulting in functional sperm. Advanced research techniques, including single cell RNA-sequencing (scRNA-Seq), have revealed various SSC sub-populations. However, our understanding of the highly complex process of human SSC self-renewal and differentiation, especially in patients treated for childhood cancer, remains inadequate. Proposal and aims of the project: The two main aims of this project are to define gonadotoxic risk factors of anti-cancer treatments used in paediatric oncology/haematology and to establish an in vitro model for human spermatogenesis with the ultimate goal to rescue fertility in prepubertal boys subjected to gonadotoxic treatment regimens. Impact of our research: The outcomes of this project will increase the basic information on effects of anti-cancer treatments on human gametogenesis in boys and its physiological regulation, which has previously been difficult to study. Thereby, we will be able to provide more information on treatment related fertility problems, which will be beneficial for physicians when informing patients and families about potential gonadotoxic late effects connected to the planned treatments. Experiments focusing on the quality status of fresh and cryopreserved testicular samples will clarify not only the morphological and molecular profile of the cells, but also the functional status of the clinically collected testicular tissue samples for fertility preservation. These quality controls will ensure that the tissue is cryopreserved in the best way for future use.