Preservation of male fertility

Summary:Chemotherapy and radiation treatments for cancer or other conditions can cause infertility. Adult men can preserve their future fertility by cryopreserving a semen sample prior to initiating gonadotoxic therapy. Unfortunately, this is not an option for prepubertal boys who are not producing sperm. These prepubertal patients can cryopreserve testicular tissue prior to therapy in anticipation that spermatogonial stem cells (SSCs) in that tissue can be used in the future to restore fertility. The application of SSC transplantation to restore fertility in this patient population will be challenged by the small amount of tissue, and therefore SSCs, that can be obtained via testicular biopsy and by the risk of reintroducing malignant cells back into a cancer survivor.

In the present study we showed that testicular biopsies from prepubertal cancer patient boys before or even after chemotherapy express biologically active spermatogonial cells. Our results indicate that using cryopreserved testicular biopsies for future isolation and use are better than using cryopreserved testicular cell suspension. We also characterized the molecular phenotype of human spermatogonia, and have identified cell surface markers that can be used to enrich human spermatogonia by FACS and remove malignant contamination. These results have implications for the safety and feasibility of using SSC transplantation to restore the fertility of cancer survivors. In addition, xenografting the experimental portion of testicular tissues from our prepubertal patients into castrated immune deficient nude mice led to proliferation of spermatogonial cells and their differentiation up to the stage of spermatocytes. On the other hand, in some samples we could induce proliferation and differentiation of isolated cells that been cultured in vitro, in methylcellulose and different growth factors, to meiotic and postmeiotic stages. In addition, using decellularize human testes that produced a human testis extracellular matrix (htECM) hydrogel (to provide a substrate for serum-free), we could induce proliferation of the isolated spermatogonial cells in vitro.

The ability of isolated spermatogonial cells to proliferate and differentiate in vitro was dependent on the amount of the biopsy used and as a sequence to the number of isolated cells cultured in vitro.

Our results may suggest the methylcellulose culture condition as a possible in vitro system to induce proliferation and differentiation of spermatogonial cells from prepubertal cancer patient boys. However, it still needs optimization to show constant repetition and also to induce complete spermatogenesis including sperm.

In conclusion, our results may be used as a base for future development of in vitro culture system to develop spermatogenesis in vitro, which may assist in development future therapeutic strategies for fertility preservation of prepubertal cancer patient boys.