Male fertility preservation after gonadotoxic treatment

Recent advances in cancer therapy have resulted in an increased number of long-term cancer survivors. Unfortunately, aggressive chemotherapy, radiotherapy and preparative regimens for bone marrow transplantation can severely affect male germ cells, including spermatogonial stem cells (SSCs), and lead to permanent loss of fertility. Different options for fertility preservation are dependent on the pubertal state of the patient. While sperm cryopreservation prior to gonadotoxic treatment is a well established method after puberty, cryopreservation of immature tissue, either in the form of a cell suspension or whole pieces of tissue, is the only way of preserving fertility in prepubertal boys. To restore fertility in these boys, very limited and still experimental options exist, since their seminiferous tubules contain mainly diploid germ cells, i.e spermatogonia comprising stem cells, and Sertoli cells. These options include transplantation of isolated SSCs or testicular tissue pieces, and in vitro maturation of SSCs in order to obtain mature spermatozoa. Current data from animal models look promising, since healthy offspring have been obtained after transplantation of frozen testicular cell suspensions or tissue pieces, making application in humans likely in the near future. There are, however, many unresolved issues related to these technologies. Our objective is to offer young patients at risk of testicular failure after gonadotoxic therapies realistic and safe fertility preservation options. Our study therefore focused on the development of an appropriate cryopreservation protocol for immature testicular tissue, allowing survival and functioning of spermatogonia and Sertoli cells. In order to evaluate tissue and cell integrity after freezing and thawing, we developed an orthotopic xenografting model in immunodeficient nude mice. After cryopreservation and short-term xenografting of cryptorchid immature human testicular tissue, we observed good tissue survival, well preserved tissue integrity (88% intact seminiferous tubules), survival of spermatogonia (14.5% of the initial population) and Sertoli cells (100%), as well as proliferation in both cellular types. In order to investigate the capacity of cryopreserved tissue to complete spermatogenesis, we performed long-term grafting (6 months) of normal immature testicular tissue from young boys, who were to undergo fertility-threatening therapies. After freezing and 6 months' xenografting to immunodeficient mice, our results demonstrated the persistence of spermatogonia. While these cells maintained their mitotic capacity and the potential to enter the differentiation pathway, as spermatocytes in prophase of the first meiotic division were observed, no normal differentiation was encountered beyond this stage. Thus, our orthotopic xenografting model demonstrated survival of spermatogonia able to proliferate after cryopreservation. This model therefore looks promising for the further investigation of optimal transplantation conditions for immature testicular tissue, with a view to clinical implementation of autografting of stored tissue. Nevertheless, further studies are clearly required before this option can be offered to our young patients undergoing gonadotoxic treatment.