Manipulation of the spermatogonial stem cell niche in immature human testis – could it act as a fertility preservation strategy for boys with cancer?

Abstract

In the United Kingdom, childhood cancers account for around 1% of all new cancer cases reported, with boys being affected more often than girls. Improvement in cancer treatments has resulted in more than 80% of children with cancer surviving for more than 5 years. It is estimated that 1 in 500 young adults is a childhood cancer survivor. Although lifesaving, radiotherapy and chemotherapy administered to childhood cancer patients usually have side effects, one of them being damage to reproductive function. Retrospective follow-up studies show that children who were exposed to chemotherapy agents during childhood have a significantly lower chance of siring a healthy pregnancy when they are adults compared to their healthy siblings. This is especially of importance to male patients as currently there are no established fertility preservation options for pre-pubertal boys who undergo cancer treatment. Pre-pubertal boys do not produce sperm, therefore, the established fertility preservation option of freezing sperm that is available for adult men is not applicable to boys. Currently, several experimental fertility preservation strategies are being investigated as a way to preserve fertility in pre-pubertal boys. All of the strategies include obtaining a testicular biopsy prior to cancer treatment and most depend on finding efficient ways to successfully isolate the spermatogonial stem cells (SSCs) from the tissue and mature these cells or the tissue itself in vitro. Another emerging experimental strategy of protecting the testes in situ by administering another drug either before, at the same time or after the chemotherapy treatment is becoming of interest. This may be possible by manipulating the SSC niche using agents that are shown to prevent the chemotherapy-induced damage. However, to establish the manipulation of the SSC niche using exogenous agents as a fertility preservation strategy, it is important to, understand more about the SSC niche in the immature human testis, investigate how individual chemotherapeutic drugs affect the SSC niche, and find a potential chemoprotective agent that could prevent the damage to the SSC niche. This study aimed to address all of these aspects. The aim of the first part of this study was to take into account recent single-cell sequencing findings and characterise the germ cell populations in immature human testicular tissues. Triple immunostaining was performed to determine the expression of a putative SSC marker, namely Undifferentiated Embryonic Cell Transcription Factor 1 (UTF1), alongside gonocyte and (pre)spermatogonial markers. Expression of UTF1 was detected in a sub-population of spermatogonia in the pre- and peri-pubertal testicular tissues. The same population was also present in the second trimester fetal testicular tissues which are often used as a model for pre-pubertal testicular tissues. This finding further supports the use of second trimester human fetal testicular tissues as a model to study effects of exposure to pharmaceutical compounds on pre-pubertal testicular tissues. Interestingly, sub-populations of gonocytes and (pre)spermatogonia were identified in the human fetal testicular tissues which had not been previously reported. This suggests differentiation of germ cells occurring during fetal development to be more complex than previously thought. In the second part of this study, a well-established in vitro hanging drop system was utilised to culture pieces of the second trimester human fetal testicular tissues. This system was employed to study the effects of two commonly used chemotherapeutic drugs in paediatric oncology – cisplatin and carboplatin. Cisplatin is classed as an alkylating-like platinum-based drug which is known to be gonadotoxic. Carboplatin is a second generation platinum drug, deemed to have less severe side effects, although fertility-related effects are unknown. Results in this part of the study showed that exposure to a clinically-relevant concentration of cisplatin induced an acute reduction in germ cell number in human fetal testicular tissues. No effect was observed on Sertoli cell number. An increase in apoptotic cells within cords was observed in cisplatin-exposed tissues, with most of the expression of apoptotic marker present in the germ cells. Taken together, this would suggest direct damage to the germ cells. Comparison between exposure to cisplatin and carboplatin revealed the same extent of germ cell reduction between the two drugs. Effect of exposure to cisplatin and carboplatin was not observed on human pre- and peri-pubertal testicular tissues, however, this was a preliminary experiment including a small sample size due to limited tissue availability. In the final part of this study, second trimester human fetal testicular tissue pieces were exposed to combined treatment which included exposure to cisplatin in combination with various regimens and concentrations of granulocyte-colony stimulating factor (G-CSF). G-CSF was identified as a potential chemoprotective agent which was shown to partially prevent the loss of spermatogenesis in chemotherapy-exposed adult male mice. Regimens used included exposure to G-CSF before, during and after exposing tissue pieces to chemotherapy. None of the regimens of G-CSF exposure prevented cisplatin-induced germ cell loss. Human fetal and pre-pubertal testicular tissues were also exposed to G-CSF without cytotoxic insult and this had no effect on the number of germ cells present. As these results were in contrast to findings in published studies using animal models, mouse pre-pubertal testicular tissues were used for in vitro exposure to G-CSF with and without cisplatin exposure. Germ cell quantification revealed no differences between vehicle- and G-CSF-exposed tissues. Similar to the results obtained for human tissues, combined G-CSF + cisplatin did not protect from cisplatin-induced germ cell loss in mouse pre-pubertal testicular tissues. Taken together, this study showed that exposure to commonly used chemotherapeutic drugs induced germ cell reduction in immature human testicular tissues. Combined exposure with G-CSF did not prevent the chemotherapy-induced damage on germ cell number in either immature human or mouse testicular tissues