Effects of chemotherapy on the immature testis using human testicular models
Creators: name: Tharmalingam, Melissa
name: Tharmalingam, Melissa Durgahshree
Publisher: name: The University of Edinburgh
Year: 2022

description: Chemotherapy is widely used to eliminate proliferative malignant cells in patients with cancer. Therapy not only targets malignant cells but other healthy mitotic cells in the body. Gonadotoxicity, a side effect that reduces the fecundity of cancer survivors, has been highlighted in recent years due to reduced mortality following cancer treatment. This acquired infertility, whether transient or permanent is dependent on the chemotherapeutics and regimens administered and age of the patient. Contrary to previous belief that the prepubertal testis is not susceptible to chemotherapy, retrospective clinical analysis has shown that boys who have had chemotherapy had a lower chance of siring a pregnancy compared to their healthy sibling. A reduction in tubular fertility index in testis biopsies of prepubertal boys post chemotherapy demonstrates germ cell loss. Elevated FSH levels reported in adulthood is indicative of dysfunctional Sertoli cells, which are an important component of the germ cell niche. This raises the question of whether chemotherapy impacts the germ cells directly or through the somatic niche. In addition, reported clinical effects on the prepubertal testis often involve a treatment regimen consisting of several drugs, rather than a single agent, discounting the contribution of individual chemotherapeutics to gonadotoxicity. Therefore, it is important to study the gonadotoxicty of individual agents on the testis, which may facilitate the development of cancer treatment protocols with a reduced risk of gonadotoxicity. Thus far, studies investigating the effects of chemotherapeutics on the testis have been largely restricted to animal models. This thesis aimed to determine the effects of chemotherapy on the various cell populations within the testis and to assess whether these treatments impair testicular function using models that involve the use of human immature testis tissue. Firstly, the project investigated the suitability of the second trimester human fetal testis as a model for the prepubertal testis by demonstrating the similarities and differences between the human second trimester fetal and prepubertal testis. Presence of gonocytes, (pre)spermatogonia (precursor spermatogonial stem cells) and an immature somatic environment very closely mimic the prepubertal testis. In vitro culture of the second trimester fetal testis was used to investigate the acute, short-term impact of chemotherapeutics. The xenograft model was utilised to investigate the long-term impact on the human fetal testis tissue following in vitro exposure to chemotherapy, thereby avoiding the need to administer chemotherapy to the host animal. The project went on to interrogate the effects of chemotherapy on the immature testis using agents from two different drugs classes. This included the cell cycle independent platinum agent, cisplatin and a cell cycle dependent anti-tubulin agent, paclitaxel. The short-term effects were initially investigated using in vitro exposure of the human fetal testis tissues. Tissue fragments were exposed to either drug at clinically relevant doses and analysis was performed at 2 time points. Some of the testis tissue fragments were subcutaneously xenografted 24 hours post exposure for a period of 12 weeks to investigate the long-term effects. These studies demonstrated differential effects on the (i) germ cell subpopulations (ii) Sertoli cell number and function and (iii) proliferation and apoptosis of the testicular cell populations. Post drug exposure, a decrease in gonocytes and (pre)spermatogonia was observed after the in vitro culture period. After 12 weeks, only the (pre)spermatogonia population was significantly reduced in the paclitaxel exposed xenografts, and both gonocytes and (pre)spermatogonia remained significantly reduced in the cisplatin exposed xenografts. Proliferative germ cells were not reduced in the paclitaxel exposed tissue. However, during the in vitro culture a significant reduction in proliferative gonocytes and (pre)spermatogonia were observed. The Sertoli cell number and consequently AMH was reduced after the in vitro culture period at 96 hours post paclitaxel exposure but not with cisplatin exposure. No change in proliferative Sertoli cells was observed with both drugs’ exposure. In the presence of hCG (human chorionic gonadotrophin), Leydig cells in xenografts exposed to both drugs were steroidogenic. Although there were differential effects, the (pre)spermatogonia population, the precursor germ cells to SSCs (spermatogonial stem cells), remained reduced in the xenografts 12 weeks after exposure. Subsequently, in vitro cisplatin exposure experiments similar to the human fetal testis tissue in vitro experiments were performed with scarcely available human prepubertal testis tissue obtained from children with cancer prior to initiation of chemotherapy. Before carrying out the experiments, an optimal culture condition was selected that could maintain the prepubertal testis tissue in its prepubertal state. A reduction in spermatogonia including SSCs which express Undifferentiated Embryonic Cell Transcription Factor 1 (UTF1)) was observed mirroring the gonadotoxicity observed in the human fetal testis model. Although, there was no change in Sertoli cell number, a decrease in AMH expression was observed, suggesting a change in Sertoli cell function. In conclusion, this work provides evidence that exposure to different classes of chemotherapy agents can have differential effects on cell subpopulations in the immature human testis, both acutely and over the long term after exposure. This warrants further work to investigate the mechanisms involved in chemotherapy-induced damage and to investigate the impacts of other classes of chemotherapy agents using the human immature testis as a model. Together, such information could facilitate selection of chemotherapy regimens that are less gonadotoxic and to develop interventions with agents that can protect the testis from chemotherapy-induced damage. Clinicians would be able to better provide patients with information on fertility risks and suggest relevant fertility preservation options, such as testicular tissue cryopreservation, prior to chemotherapy.