Fertility preservation in pre-pubertal boys with cancer: a three-dimensional (3D) prepubertal testicular organoid culture system for in vitro spermatogonial stem cell (SSCs) propagation and spermatogenesis
Keywords: vitrification
immature testicular tissues
three-dimensional
testicular organoids
transportation time

The overall survival rate of childhood cancer has increased over the last decades due to improvements in cancer therapies. However, aggressive cancer treatments, such as chemo- or radiotherapy, can leave young prepubertal boys infertile. Cryogenically ‘banking’ sperm from prepubertal boys is impossible because spermatogonial stem cells (SSCs) only start to produce sperm after puberty. Thus, to help these patients preserve their fertility, it is recommended to cryopreserve their immature testicular tissues (ITTs) before receiving cancer therapies. To date, there is no standardised procedure for ITT transportation and cryopreservation in clinical practice. Approaches to fertility restoration using frozen/thawed ITTs are in the experimental stages. This thesis aimed to investigate the effects of different cryopreservation methods and potential transportation times on ITTs, and developed a three-dimensional (3D) testicular organoids (TOs) system to support the proliferation and development of SSCs. In Chapter 2, I compared the effects of uncontrolled slow freezing (USF), controlled slow freezing (CSF), and vitrification on the cryopreservation of neonatal gonocyte-containing ITTs using a bovine model. All three methods had similar effects in preserving germ cells, Sertoli cells and proliferating cells in seminiferous cords (p>0.05). Vitrified ITTs were found to have lower cell apoptosis but higher cords-basement membrane detachment (p<0.05). In chapter 3, I investigated the effects of transportation times of 1 hour, 6 hours, 24 hours, and 48 hours on ITTs. Transportation times up to 48 hours did not affect the viability, percentage of Sertoli cells and proliferating cells, and expressions of selected key genes (p>0.05). However, ITTs in the 48-hour group had higher levels of deterioration in terms of the structure of the seminiferous cords (16.43%±2.14%) and decreased percentage of seminiferous cords with germ cells (43.19%±6.45%; p<0.05). Next in Chapter 4 and chapter 5, for the first time, a 3D TO model was developed for in vitro culturing neonatal bovine testicular cells. Firstly, I optimized the dissociation and enrichment of germ cells in neonatal bovine ITTs. Next, by comparing different extracellular matrix (ECM), I found that Matrigel was optimal in the formation of germ cell aggregations. The TOs emerged from single cell suspensions and developed into 3D structures where germ cells were in the centre with Sertoli cells at the outer layers in neonatal bovine. Furthermore, growth factors glial cell line-derived neurotrophic factor (GDNF), fibroblast growth factor 2 (FGF2), and leukemia inhibitory factor (LIF) were found to promote the transformation of gonocytes into SSCs, while follicle-stimulating hormone (FSH) and testosterone maintained the viability and proliferation of cells in TOs. In summary, this thesis provided evidence that vitrification could be an alternative method for cryopreservation of ITTs, and tissue transportation times of up to 24 hours does not affect tissue quality and could be used in clinical practice. In addition, a novel 3D TO system was developed in the bovine model, thus offering an in vitro platform for the propagation and development of SSCs.