Shedding light on human gonadal development : perspectives from in vitro models for fertility preservation

Impaired fertility is a common, often under-discussed lifelong side effect associated with treatments for malignant and non-malignant diseases. With effective interventions, cancer survivorship has steadily increased over the past decades, bringing to light the consequences that impact reproductive health. Adult males can conserve their fertility by cryopreservation of mature gametes before undergoing gonadotoxic treatments. To safeguard the future fertility for pre- and early pubertal patients, the only option is to cryopreserve immature testicular tissue. This experimental procedure aims to preserve the constituent spermatogonial stem cells (SSCs) in the hope that an experimental method can differentiate these cells later in life. The long-term objective is to restore fertility through either in vitro or in vivo clinical procedures. However, up until today, protocols to restore fertility using human immature testicular tissue remain under development, and ways to study the effects of gonadotoxic treatment before the onset of puberty remain flawed.

This thesis aimed to improve in vitro systems that mimic the human testicular microenvironment, allowing us to better understand human gonadal development from early embryonic development until prepubertal maturation. Moreover, knowledge was gained about the adverse effects of gonadotoxic medical treatment on the prepubertal testis, concerning both the stem cells and their supportive somatic environment. The studies within this thesis include prepubertal testicular samples collected as part of a fertility preservation program and first-trimester gonadal tissue.

The aim of study I was to model the human first-trimester gonadal environment (testes and ovaries) that recapitulates in vivo tissue organization. An established Matrigel-based gradient system, the three-layer gradient system (3-LGS), was utilized. When cell suspensions from human embryonic testicular tissue (5-9.5 weeks post conception) were used, the cells self-organized into structures resembling testis-like organoids within seven days. The organoids displayed well- defined features similar to those in vivo. More specifically, a two-compartment architecture separated by a basement membrane was formed with seminiferous- like cords containing germ cells and an interstitial space with supporting somatic cells. However, when mesonephric cells were added to the tissue digest, this organization did not persist. The somatic cells within the testis-like organoids also displayed indications of differentiation. The SRY-Box transcription factor 9 (SOX9)-positive Sertoli cells within the seminiferous-like cords expressed anti- Müllerian hormone (AMH), whereas the Leydig cells in the interstitial space presented indications of steroidogenic enzyme activity. While compartmentalized testis-like organoids that supported somatic functions could be generated, germ cells did not survive. In contrast, ovarian-like organoids exhibited less organization but could maintain germ cells in the 3-LGS. This underlines the challenge of preserving germ cells in male organoid cultures. We conclude that the 3-LGS is a promising method for studying gonadal development, which could be a stepping stone for more complex in vitro models in development and regenerative medicine.

Subsequently, our focus shifted from early gonadal development to male prepubertal maturation. A greater understanding of how testicular somatic cells develop and nurture SSCs could help advance tissue culture methodologies. Study II explored the ability of testicular cells originating from pre- and peripubertal patients (N=49, age: 0.8-13.4 years), with malignant diseases who have undergone chemotherapy, to culture testicular organoids. Eleven freshly obtained biopsy samples (age: 7.7 + 4.1 years [mean + SD]) were dissociated to acquire cell suspensions, which were then applied to the 3-LGS. Here, four out of eleven testicular cell suspensions self-organized into testicular organoids within one week. The patient-derived organoids presented distinct cord-like structures housing Sertoli cell marker-positive cells. Furthermore, levels of AMH and testosterone in the culture medium displayed an increasing trend within seven days. Additionally, SOX9 expression was positively correlated with testicular organoid formation, emphasizing its potential function as an indicator for successful organoid formation. In turn, prior exposure to alkylating agents before the biopsy was negatively associated with SOX9 expression, which draws attention to the adverse effects of chemotherapy. This study highlights the importance of supporting somatic cell populations, especially Sertoli cells, for the promotion of testicular organoid formation.

As a functional somatic compartment is essential for potential fertility restoration and supporting future spermatogenesis, insights into the impact of chemotherapy on the testicular somatic compartment in samples collected for fertility preservation are needed. Study III examined how patient-specific factors (age, spermatogonial density, prior chemotherapy exposure) and tissue handling process affect the capacity of the somatic compartment to sustain hormone secretions ex vivo using fresh prepubertal testicular tissues from 59 patients (0.7-14.3 years). Testicular fragments were maintained in organotypic cultures at air-liquid interface conditions, and three hormones were quantified after a seven- day short-term culture. Levels of secreted AMH and inhibin B served as a measure of Sertoli cell function, whereas testosterone production was considered an indicator of Leydig cell activity. At large, the prepubertal testicular tissues sustained the secretion of hormones ex vivo, despite prior exposure to alkylating chemotherapy and transportation. The findings identified age at the time of biopsy and exposure to alkylating agent chemotherapy as significant predictors of in vitro hormone secretion. Interestingly, somatic cell functionality was maintained despite germ cell depletion. These observations underscore the importance of accounting for patient age as a significant variable in interpreting in vitro hormone secretion assays of prepubertal testicular tissue.

Altogether, the findings presented in this thesis provide evidence of the feasibility of a Matrigel-based gradient system to promote the self-organization of human testicular cells into organoid structures. The human first-trimester embryonic organoid-like structures, as well as the patient-derived prepubertal testicular organoids, displayed testis-like features in terms of in vivo spatial architecture, including a compartmentalized organization and indicators of function. This improves on multi-layered in vitro approaches to investigate gonadal development. It is noteworthy that the success of these novel approaches was linked to the functional capacity of the supporting somatic compartment of the SSC, which was, in turn, impacted by prior exposure to chemotherapy. This body of evidence, combined with the findings that the somatic compartment is capable of sustaining hormone secretion in organotypic cultures, highlights the need to understand how somatic cells mediate the impact of chemotherapy on supporting germ cells in the prepubertal testis.

List of scientific papers

I. Elizabeth Oliver*, João Pedro Alves-Lopes*, Femke Harteveld, Rod T Mitchell, Elisabet Åkesson, Olle Söder, Jan-Bernd Stukenborg. Self-organising human gonads generated by a Matrigel-based gradient system. BMC Biology. 2021 Dec;19:1-1. * denotes that these authors contributed equally to this work. https://doi.org/10.1186/s12915-021-01149-3

II. Yanhua Cui, Femke Harteveld, Hajar Ali Mohammed Ba Omar, Yifan Yang, Ragnar Bjarnason, Patrik Romerius, Mikael Sundin, Ulrika Norén Nyström, Cecilia Langenskiöld, Hartmut Vogt, Lars Henningsohn, Per Frisk, Kaisa Vepsäläinen, Cecilia Petersen, Rod T Mitchell, Jingtao Guo, João Pedro Alves-Lopes, Kirsi Jahnukainen, Jan-Bernd Stukenborg. Prior exposure to alkylating agents negatively impacts testicular organoid formation in childhood cancer patients. Human Reproduction Open. 2024 Aug 13;2024(3):hoae049. https://doi.org/10.1093/hropen/hoae049

III. Femke Harteveld, João Pedro Alves-Lopes, Anu Haavisto, Yanhua Cui, Magdalena Kurek, Halima Albalushi, Olle Söder, Ragnar Bjarnason, Patrik Romerius, Mikael Sundin, Ulrika Noren Nyström, Cecilia Langenskiöld, Hartmut Vogt, Lars Henningsohn, Per Frisk, Kaisa Vepsäläinen, Rod T. Mitchell, Cecilia Petersen, Konstantin Svechnikov, Kirsi Jahnukainen, Jan-Bernd Stukenborg Short-term organotypic culture and in vitro hormone secretion analysis of prepubertal testicular tissue collected for fertility preservation. [Manuscript]