Jan-Bernd Stukenborg

This study aimed to investigate the ex vivo properties of prepubertal testicular biopsy samples collected for fertility preservation, with particular focus on how patient-specific factors and tissue handling may affect the hormone-secreting capacity of testicular somatic cells. Testicular biopsy samples from 59 boys aged 0.7 to 14.3 years, obtained before or during treatment for malignant or haematological diseases within a fertility preservation research program, were cultured short-term using an organotypic culture system. Culture medium was collected periodically and analysed for secretion of anti-Müllerian hormone, inhibin B, and testosterone. Hormone secretion was evaluated in relation to age at biopsy, spermatogonial density, previous chemotherapy exposure, exposure to alkylating agents, and biopsy transportation time. The tissues retained the ability to secrete hormones outside the body after transportation and, in some cases, previous chemotherapy exposure. Hormone secretion was not significantly associated with spermatogonial density, suggesting that somatic cell function may be preserved even when germ cells are depleted. Age at biopsy and exposure to alkylating agent chemotherapy were significant predictors of hormone secretion in culture. These findings indicate that short-term hormone secretion assays can be used to assess the functional capacity of somatic cell populations in prepubertal testicular tissue collected for fertility preservation, and that both developmental stage and previous treatment exposure should be considered when interpreting tissue function. Lay Summary: Some boys with cancer or blood diseases may have a small piece of testicular tissue frozen before they start treatment, with the hope of preserving future fertility. In this study, testicular tissue from 59 boys aged 0.7 to 14.3 years was kept in a laboratory dish for a short time after biopsy. We measured whether the tissue could still release testosterone and other hormones that support germ cells that will make sperm in adulthood. The tissue of was able to release hormones after being transported from the operating room to the laboratory premises and, in some cases, even after previous chemotherapy. Hormone release was linked to the child’s age and to previous treatment with a type of chemotherapy known to damage fertility. It was not clearly linked to the number of germ cells in the tissue. This suggests that hormone producing testicular cells may remain active even when germ cells are reduced. These tests may help assess tissue quality before future fertility treatments.

Male reproductive health is in a state of decline that is characterised, in part, by an apparent global decrease in sperm concentrations. A functional preclinical model of the testis would provide an increased understanding of male reproductive health and facilitate the development of effective treatments for the rising incidence of male infertility. However, current preclinical models of the human testis have yet to successfully replicate the process of spermatogenesis, one of the primary functions of the testis. Understanding the micromechanical environment of the human testis could provide insights into overcoming the challenge of mimicking human spermatogenesis in vitro and aid in the development of representative material substrates for use in testicular models. This study characterised the mechanical properties of human testis tissue obtained from three cohorts, including adults undergoing microsurgical testicular sperm extraction (n = 6) and gender affirmation surgery (n = 24), and developmental tissues (foetal and adolescence) (n = 7). A nanoindentation technique, combined with histological investigation, was used to spatially characterise the elastic (Young's modulus) and viscoelastic properties (storage and loss moduli and Tan(δ)) of testis tissue across numerous morphological states. The tissue exhibited Young's modulus values between 0.1 and 1 kPa, while seminiferous tubules exhibited significantly higher Young's and storage moduli compared to interstitial space. Tan(δ) exhibited a significant negative correlation with spermatogenesis status in adult cohorts. This study informs the development of improved diagnostics for male infertility and facilitates the fabrication of mechanically representative material substrates for use in systems that aim to achieve in vitro spermatogenesis using human testicular cells.

Figure S3. TH-MYCN tumor microenvironment is composed of Schwannian and mesenchymal stroma, and a diverse immune-cell repertoire. Related to Figures 2 and 3.

Table S1 shows characteristics of TH-MYCN mouse samples used for tumoroid cultures, scRNA-seq and immunostainings.

Figure S9. Immunofluorescence staining of hemizygous TH-MYCN tumors and matched ex vivo tumoroids. Related to Figure 4.

Figure S5. Identification of malignant cells by inferCNV. Related to Figure 3.

Figure S4. Tumor hallmark gene expression patterns in human and mouse tumors.

Figure S15. Cross-species integration analysis of common cell clusters. Related to Figure 5.

Figure S13. Survival probability for neuroblastoma patients non-MYCN amplified tumors with tumoroid enriched gene expression programs. Related to Figure 5.

Figure S14. Survival probability for neuroblastoma patients MYCN amplified tumors with tumoroid enriched gene expression programs. Related to Figure 5.