Cisplatin effects on the human fetal testis - establishing the sensitive period for (pre)spermatogonial loss and relevance for fertility preservation in pre-pubertal boys

BACKGROUND: Exposure to chemotherapy during childhood can impair future fertility. Studies using in vitro culture have shown exposure to platinum-based alkylating-like chemotherapy reduces the germ cell number in the human fetal testicular tissues. We aimed to determine whether effects of exposure to cisplatin on the germ cell sub-populations are dependent on the gestational age of the fetus and what impact this might have on the utility of using human fetal testis cultures to model chemotherapy exposure in childhood testis. METHODS: We utilised an in vitro culture system to culture pieces of human fetal testicular tissues (total n=23 fetuses) from three different gestational age groups (14-16 (early), 17-19 (mid) and 20-22 (late) gestational weeks; GW) of the second trimester. Tissues were exposed to cisplatin or vehicle control for 24 hours, analysing the tissues 72 and 240 hours post-exposure. Number of germ cells and their sub-populations, including gonocytes and (pre)spermatogonia, were quantified. RESULTS: Total germ cell number and number of both germ cell sub-populations were unchanged at 72 hours post-exposure to cisplatin in the testicular tissues from fetuses of the early (14-16 GW) and late (20-22 GW) second trimester. In the testicular tissues from fetuses of mid (17-19 GW) second trimester, total germ cell and gonocyte number were significantly reduced, whilst (pre)spermatogonial number was unchanged. At 240 hours post-exposure, the total number of germ cells and that of both sub-populations was significantly reduced in the testicular tissues from fetuses of mid- and late-second trimester, whilst germ cells in early-second trimester tissues were unchanged at this time-point. CONCLUSIONS: In vitro culture of human fetal testicular tissues can be a useful model system to investigate the effects of chemotherapy-exposure on germ cell sub-populations during pre-puberty. Interpretation of the results of such studies in terms of relevance to later (infant and pre-pubertal) developmental stages should take into account the changes in germ cell composition and periods of germ cell sensitivity in the human fetal testis

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

Identification of a window of androgen sensitivity for somatic cell function in human fetal testis cultured ex vivo

BACKGROUND: Reduced androgen action during early fetal development has been suggested as the origin of reproductive disorders comprised within the testicular dysgenesis syndrome (TDS). This hypothesis has been supported by studies in rats demonstrating that normal male development and adult reproductive function depend on sufficient androgen exposure during a sensitive fetal period, called the masculinization programming window (MPW). The main aim of this study was therefore to examine the effects of manipulating androgen production during different timepoints during early human fetal testis development to identify the existence and timing of a possible window of androgen sensitivity resembling the MPW in rats. METHODS: The effects of experimentally reduced androgen exposure during different periods of human fetal testis development and function were examined using an established and validated human ex vivo tissue culture model. The androgen production was reduced by treatment with ketoconazole and validated by treatment with flutamide which blocks the androgen receptor. Testicular hormone production ex vivo was measured by liquid chromatography-tandem mass spectrometry or ELISA assays, and selected protein markers were assessed by immunohistochemistry. RESULTS: Ketoconazole reduced androgen production in testes from gestational weeks (GW) 7–21, which were subsequently divided into four age groups: GW 7–10, 10–12, 12–16 and 16–21. Additionally, reduced secretion of testicular hormones INSL3, AMH and Inhibin B was observed, but only in the age groups GW 7–10 and 10–12, while a decrease in the total density of germ cells and OCT4(+) gonocytes was found in the GW 7–10 age group. Flutamide treatment in specimens aged GW 7–12 did not alter androgen production, but the secretion of INSL3, AMH and Inhibin B was reduced, and a reduced number of pre-spermatogonia was observed. CONCLUSIONS: This study showed that reduced androgen action during early development affects the function and density of several cell types in the human fetal testis, with similar effects observed after ketoconazole and flutamide treatment. The effects were only observed within the GW 7–14 period—thereby indicating the presence of a window of androgen sensitivity in the human fetal testis. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12916-022-02602-y

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

Effects of environmental Bisphenol A exposures on germ cell development and Leydig cell function in the human fetal testis

<div><p>Background</p><p>Using an organotypic culture system termed human Fetal Testis Assay (hFeTA) we previously showed that 0.01 μM BPA decreases basal, but not LH-stimulated, testosterone secreted by the first trimester human fetal testis. The present study was conducted to determine the potential for a long-term antiandrogenic effect of BPA using a xenograft model, and also to study the effect of BPA on germ cell development using both the hFETA and xenograft models.</p><p>Methods</p><p>Using the hFeTA system, first trimester testes were cultured for 3 days with 0.01 to 10 μM BPA. For xenografts, adult castrate male nude mice were injected with hCG and grafted with first trimester testes. Host mice received 10 μM BPA (~ 500 μg/kg/day) in their drinking water for 5 weeks. Plasma levels of total and unconjugated BPA were 0.10 μM and 0.038 μM respectively. Mice grafted with second trimester testes received 0.5 and 50 μg/kg/day BPA by oral gavage for 5 weeks.</p><p>Results</p><p>With first trimester human testes, using the hFeTA model, 10 μM BPA increased germ cell apoptosis. In xenografts, germ cell density was also reduced by BPA exposure. Importantly, BPA exposure significantly decreased the percentage of germ cells expressing the pluripotency marker AP-2γ, whilst the percentage of those expressing the pre-spermatogonial marker MAGE-A4 significantly increased. BPA exposure did not affect hCG-stimulated androgen production in first and second trimester xenografts as evaluated by both plasma testosterone level and seminal vesicle weight in host mice.</p><p>Conclusions</p><p>Exposure to BPA at environmentally relevant concentrations impairs germ cell development in first trimester human fetal testis, whilst gonadotrophin-stimulated testosterone production was unaffected in both first and second trimester testis. Studies using first trimester human fetal testis demonstrate the complementarity of the FeTA and xenograft models for determining the respective short-term and long term effects of environmental exposures.</p></div

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

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

Effect of BPA exposure on germ cell differentiation in first trimester human fetal testis xenografts.

<p>Human fetal testes (9.1–11.3 GW) were xenografted into castrate Nude (host) mice. Host mice received vehicle (Control) or 10μM BPA in the drinking water for five weeks. (A) Histological sections of testes after immunostaining for AP-2γ (gonocytes). Positive (red arrows) and negative (black arrows) germ cells can be identified. Scale bar: 60 μm. (B) Quantification of AP-2γ-positive cells displayed as mean ± SEM (n = 9) on the left panel and as individual values with a line drawn between the control and the corresponding BPA-treated testis from the same fetus on the right panel. (C) Histological sections of testes after immunostaining for MAGE-A4 (prespermatogonia). Positive (red arrows) and negative (black arrows) germ cells can be identified. Scale bar: 60 μm. (D) Quantification of MAGE-A4-positive cells displayed as mean ± SEM (n = 8) on the left part and as individual values with a line drawn between the control and the corresponding BPA-treated testis from the same fetus on the right part. Data analyzed using the Wilcoxon paired-test. *p<0.05, **p<0.01.</p

Effect of BPA exposure on germ cell apoptosis and proliferation in first trimester human fetal testes cultured using the FeTA system.

<p>Human fetal testes (6–12 GW, mean 8.7 ± 0.6 GW) were cultured using the ex vivo <u>h</u>uman <u>Fe</u>tal <u>T</u>estis <u>A</u>ssay system (hFeTA). After 24 hours in control medium, explants were cultured with 100 ng/mL of LH for the 3 subsequent days in the presence of ethanol vehicle (control explants) or BPA at concentrations ranging from 0.01 to 10 μM. Control and BPA-treated explants were paired samples from the same testis. (A) Histological sections after labeling with anti-cleaved caspase-3 antibody (brown) and anti-AMH antibody (green). Positive (red arrows) and negative (black arrows) germ cells can be identified. Scale bar: 10 μm. (B) Quantification of cleaved caspase-3 positive cells (mean ± SEM; n = 4–8). (C) Histological sections after labeling with anti-Ki-67 antibody (brown) and anti-AMH antibody (green). Positive (red arrows) and negative (black arrows) germ cells can be identified. Scale bar: 50 μm. (D) Quantification of Ki67 positive gonocytes (mean ± SEM; n = 4–8).</p