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

Abstract

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/mm2; 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/mm2) or 5 μg/ml carboplatin (7,502 ± 627 cells/mm2) compared to control (6,592 ± 1,545 cells/mm2).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.

Alkylating agents‐induced gonadotoxicity in prepubertal males: Insights on the clinical and preclinical front

“…Still, it remains plausible that apoptosis plays a crucial role, and its detection might be overlooked due to a lack of synchronization between the evaluation times and its occurrence. Indeed, a recent study conducted by the same authors unveiled an increase in apoptosis in human germ cells at an intermediate timepoint which had not been assessed in the previous study 91 …”

“…Using cultured human testicular fragments, neither cisplatin at 0.5 μg/mL nor carboplatin at 5.0 μg/mL impacted Sertoli cell density or apparent function, as evidenced by the largely unchanged AMH and inhibin B levels 91 …”

“…Still, both drugs were not compared at equivalent doses, which are in the range of 4:1–6:1 (carboplatin: cisplatin) in clinical practice 146 . Three preclinical studies showed a similar toxicity profile, 89,91,120 but carboplatin was administered at 10× the concentration of cisplatin. This dosage equivalency difference could, at least in part, contribute to the discrepancy in the findings.…”

“…Another developing aspect of preclinical research is the use of human‐derived tissue. So far, only cisplatin and carboplatin have been tested in such models 89–92 . Possibly due to easier accessibility, research was carried out on fetal tissue after it was found to exhibit a similar pattern and timing of damage to child‐derived tissue following treatment 89 .…”

“…Interestingly, apoptosis seemed to either not be apparent or very rare in vitro when human tissues were used: It was not found to be increased at either 24 h or 96 h after a 24 h culture with 0.5 μg/mL 89 . Likewise, 72 h and 240 h after culturing testis fragments for 24 h with 0.5 μg/mL there was no significant increase in the number of apoptotic germ or Sertoli cells 91 . As such, germ cell loss was hypothesized to occur mainly due to the loss of their proliferative capacity 89 …”