Effects of chemotherapy on the immature testis using human testicular models

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

Maintenance of Sertoli Cell Number and Function in Immature Human Testicular Tissues Exposed to Platinum-Based Chemotherapy-Implications for Fertility Restoration

Background: Retrospective studies in adult survivors of childhood cancer show long-term impacts of exposure to alkylating chemotherapy on future fertility. We recently demonstrated germ cell loss in immature human testicular tissues following exposure to platinum-based chemotherapeutic drugs. This study investigated the effects of platinum-based chemotherapy exposure on the somatic Sertoli cell population in human fetal and pre-pubertal testicular tissues. Methods: Human fetal (n = 23; 14–22 gestational weeks) testicular tissue pieces were exposed to cisplatin (0.5 or 1.0 μg/ml) or vehicle for 24 h in vitro and analysed 24–240 h post-exposure or 12 weeks after xenografting. Human pre-pubertal (n = 10; 1–12 years) testicular tissue pieces were exposed to cisplatin (0.5 μg/ml), carboplatin (5 μg/ml) or vehicle for 24 h in vitro and analysed 24–240 h post-exposure; exposure to carboplatin at 10-times the concentration of cisplatin reflects the relative clinical doses given to patients. Immunohistochemistry was performed for SOX9 and anti-Müllerian hormone (AMH) expression and quantification was carried out to assess effects on Sertoli cell number and function respectively. AMH and inhibin B was measured in culture medium collected post-exposure to assess effects on Sertoli cell function. Results: Sertoli cell (SOX9(+ve)) number was maintained in cisplatin-exposed human fetal testicular tissues (7,647 ± 459 vs. 7,767 ± 498 cells/mm(2); p > 0.05) at 240 h post-exposure. No effect on inhibin B (indicator of Sertoli cell function) production was observed at 96 h after cisplatin (0.5 and 1.0 μg/ml) exposure compared to control (21 ± 5 (0.5 μg/ml cisplatin) vs. 23 ± 7 (1.0 μg/ml cisplatin) vs. 25 ± 7 (control) ng/ml, p > 0.05). Xenografting of cisplatin-exposed (0.5 μg/ml) human fetal testicular tissues had no long-term effect on Sertoli cell number or function (percentage seminiferous area stained for SOX9 and AMH, respectively), compared with non-exposed tissues. Sertoli cell number was maintained in human pre-pubertal testicular tissues following exposure to either 0.5 μg/ml cisplatin (6,723 ± 1,647 cells/mm(2)) or 5 μg/ml carboplatin (7,502 ± 627 cells/mm(2)) compared to control (6,592 ± 1,545 cells/mm(2)). Conclusions: This study demonstrates maintenance of Sertoli cell number and function in immature human testicular tissues exposed to platinum-based chemotherapeutic agents. The maintenance of a functional Sertoli cell environment following chemotherapy exposure suggests that fertility restoration by spermatogonial stem cell (SSC) transplant may be possible in boys facing platinum-based cancer treatment

Cisplatin and carboplatin result in similar gonadotoxicity in immature human testis with implications for fertility preservation in childhood cancer

Background Clinical studies indicate chemotherapy agents used in childhood cancer treatment regimens may impact future fertility. However, effects of individual agents on prepubertal human testis, necessary to identify later risk, have not been determined. The study aimed to investigate the impact of cisplatin, commonly used in childhood cancer, on immature (foetal and prepubertal) human testicular tissues. Comparison was made with carboplatin, which is used as an alternative to cisplatin in order to reduce toxicity in healthy tissues. Methods We developed an organotypic culture system combined with xenografting to determine the effect of clinically-relevant exposure to platinum-based chemotherapeutics on human testis. Human foetal and prepubertal testicular tissues were cultured and exposed to cisplatin, carboplatin or vehicle for 24 h, followed by 24-240 h in culture or long-term xenografting. Survival, proliferation and apoptosis of prepubertal germ stem cell populations (gonocytes and spermatogonia), critical for sperm production in adulthood, were quantified. Results Cisplatin exposure resulted in a significant reduction in the total number of germ cells (- 44%, p <0.0001) in human foetal testis, which involved an initial loss of gonocytes followed by a significant reduction in spermatogonia. This coincided with a reduction (- 70%, p <0.05) in germ cell proliferation. Cisplatin exposure resulted in similar effects on total germ cell number (including spermatogonial stem cells) in prepubertal human testicular tissues, demonstrating direct relevance to childhood cancer patients. Xenografting of cisplatin-exposed human foetal testicular tissue demonstrated that germ cell loss (- 42%, p <0.01) persisted at 12 weeks. Comparison between exposures to human-relevant concentrations of cisplatin and carboplatin revealed a very similar degree of germ cell loss at 240 h post-exposure. Conclusions This is the first demonstration of direct effects of chemotherapy exposure on germ cell populations in human foetal and prepubertal testis, demonstrating platinum-induced loss of all germ cell populations, and similar effects of cisplatin or carboplatin. Furthermore, these experimental approaches can be used to determine the effects of established and novel cancer therapies on the developing testis that will inform fertility counselling and development of strategies to preserve fertility in children with cancer.Peer reviewe

The Dynamic Transcriptional Cell Atlas of Testis Development during Human Puberty

The human testis undergoes dramatic developmental and structural changes during puberty, including proliferation and maturation of somatic niche cells, and the onset of spermatogenesis. To characterize this understudied process, we profiled and analyzed single-cell transcriptomes of similar to 10,000 testicular cells from four boys spanning puberty and compared them to those of infants and adults. During puberty, undifferentiated spermatogonia sequentially expand and differentiate prior to the initiation of gametogenesis. Notably, we identify a common pre-pubertal progenitor for Leydig and myoid cells and delineate candidate factors controlling pubertal differentiation. Furthermore, pre-pubertal Sertoli cells exhibit two distinct transcriptional states differing in metabolic profiles before converging to an alternative single mature population during puberty. Roles for testosterone in Sertoli cell maturation, antimicrobial peptide secretion, and spermatogonial differentiation are further highlighted through single-cell analysis of testosterone-suppressed transfemale testes. Taken together, our transcriptional atlas of the developing human testis provides multiple insights into developmental changes and key factors accompanying male puberty

The bii4africa dataset of faunal and floral population intactness estimates across Africa’s major land uses

Sub-Saharan Africa is under-represented in global biodiversity datasets, particularly regarding the impact of land use on species’ population abundances. Drawing on recent advances in expert elicitation to ensure data consistency, 200 experts were convened using a modified-Delphi process to estimate ‘intactness scores’: the remaining proportion of an ‘intact’ reference population of a species group in a particular land use, on a scale from 0 (no remaining individuals) to 1 (same abundance as the reference) and, in rare cases, to 2 (populations that thrive in human-modified landscapes). The resulting bii4africa dataset contains intactness scores representing terrestrial vertebrates (tetrapods: ±5,400 amphibians, reptiles, birds, mammals) and vascular plants (±45,000 forbs, graminoids, trees, shrubs) in sub-Saharan Africa across the region’s major land uses (urban, cropland, rangeland, plantation, protected, etc.) and intensities (e.g., large-scale vs smallholder cropland). This dataset was co-produced as part of the Biodiversity Intactness Index for Africa Project. Additional uses include assessing ecosystem condition; rectifying geographic/ taxonomic biases in global biodiversity indicators and maps; and informing the Red List of Ecosystems

The bii4africa dataset of faunal and floral population intactness estimates across Africa’s major land uses

Sub-Saharan Africa is under-represented in global biodiversity datasets, particularly regarding the impact of land use on species’ population abundances. Drawing on recent advances in expert elicitation to ensure data consistency, 200 experts were convened using a modified-Delphi process to estimate ‘intactness scores’: the remaining proportion of an ‘intact’ reference population of a species group in a particular land use, on a scale from 0 (no remaining individuals) to 1 (same abundance as the reference) and, in rare cases, to 2 (populations that thrive in human-modified landscapes). The resulting bii4africa dataset contains intactness scores representing terrestrial vertebrates (tetrapods: ±5,400 amphibians, reptiles, birds, mammals) and vascular plants (±45,000 forbs, graminoids, trees, shrubs) in sub-Saharan Africa across the region’s major land uses (urban, cropland, rangeland, plantation, protected, etc.) and intensities (e.g., large-scale vs smallholder cropland). This dataset was co-produced as part of the Biodiversity Intactness Index for Africa Project. Additional uses include assessing ecosystem condition; rectifying geographic/taxonomic biases in global biodiversity indicators and maps; and informing the Red List of Ecosystems

The bii4africa dataset of faunal and floral population intactness estimates across Africa’s major land uses

International audienceSub-Saharan Africa is under-represented in global biodiversity datasets, particularly regarding the impact of land use on species' population abundances. Drawing on recent advances in expert elicitation to ensure data consistency, 200 experts were convened using a modified-Delphi process to estimate 'intactness scores': the remaining proportion of an 'intact' reference population of a species group in a particular land use, on a scale from 0 (no remaining individuals) to 1 (same abundance as the reference) and, in rare cases, to 2 (populations that thrive in human-modified landscapes). The resulting bii4africa dataset contains intactness scores representing terrestrial vertebrates (tetrapods: ±5,400 amphibians, reptiles, birds, mammals) and vascular plants (±45,000 forbs, graminoids, trees, shrubs) in sub-Saharan Africa across the region's major land uses (urban, cropland, rangeland, plantation, protected, etc.) and intensities (e.g., large-scale vs smallholder cropland). This dataset was co-produced as part of the Biodiversity Intactness Index for Africa Project. Additional uses include assessing ecosystem condition; rectifying geographic/ taxonomic biases in global biodiversity indicators and maps; and informing the Red List of Ecosystems

bi4africa dataset - open source

The bii4africa dataset is presented in a multi-spreadsheet .ods file. The raw data spreadsheet (‘Scores_Raw’) includes 31,313 individual expert estimates of the impact of a sub-Saharan African land use on a species response group of terrestrial vertebrates or vascular plants. Estimates are reported as intactness scores - the remaining proportion of an ‘intact’ reference (pre-industrial or contemporary wilderness area) population of a species response group in a land use, on a scale from 0 (no individuals remain) through 0.5 (half the individuals remain), to 1 (same as the reference population) and, in limited cases, to 2 (two or more times the reference population). For species that thrive in human-modified landscapes, scores could be greater than 1 but not exceeding 2 to avoid extremely large scores biasing aggregation exercises. Expert comments are included alongside respective estimates