title: Preserved seminiferous tubule integrity with spermatogonial survival and induction of Sertoli and Leydig cell maturation after long-term organotypic culture of prepubertal human testicular tissue.
abstract: Is an organotypic culture system able to provide the appropriate testicular microenvironment for in-vitro maturation of human immature testicular tissue (ITT)?
  
  
  
  Our organotypic culture system provided a microenvironment capable of preserving seminiferous tubule (ST) integrity and Leydig cell (LC) functionality and inducing Sertoli cell (SC) maturation.
  
  
  
  Cryopreservation of human ITT is a well-established strategy to preserve fertility in prepubertal boys affected by cancer, with a view for obtaining sperm. While spermatogenesis in mice has been replicated in organotypic culture, yielding reproductively efficient spermatozoa, this process has not yet been achieved in humans.
  
  
  
  The aim of this study was to in vitro mature frozen-thawed ITT. To this end, 1 mm3tissue fragments from three prepubertal patients aged 2 (P1), 11 (P2) and 12 (P3) years were placed in organotypic culture for 139 days. Culture media, supplemented with either testosterone or hCG, were compared.
  
  
  
  ST integrity and tissue viability were assessed by histological score and lactate dehydrogenase (LDH) levels in supernatants. Spermatogonia (SG), proliferating cells and proliferating SG were identified by the use of MAGE-A4 and Ki67 immunohistochemical markers. Glial cell line-derived neurotrophic factor (GDNF) was used as a marker of SC functionality, while SC maturation was evaluated by androgen receptor (AR), anti-Müllerian hormone (AMH) immunohistochemistry (IHC) and AMH immunoenzymatic assay. LC functionality was determined by testosterone levels in supernatants and by 3β-hydroxysteroid dehydrogenase (3β-HSD) IHC. Apoptosis was studied by IHC with active caspases 3 and 8 and by TUNEL (terminal deoxynubocleotidyl transferase-mediated dUTP nick end labeling) analysis.
  
  
  
  Tissue viability was preserved, as demonstrated by the decrease in and stabilization of LDH release, and evolution of ST scoring, with the percentage of well-preserved STs showing no statistical differences during culture in either medium. GDNF was expressed until Day 139, demonstrating SC functionality. Moreover, a significant reduction in AMH expression and release indicated SC maturation. Testosterone concentrations in supernatants increased in both culture media, demonstrating LC functionality with paracrine interactions. SG were present up to Day 139, although the ratio between MAGE-A4-positive cells and well-preserved tubules was significantly reduced over the course of culture (P ≤ 0.001). SCs exhibited a decreased proliferation rate over time (P ≤ 0.05). The proliferation rate of SG remained stable until Day 64, but over the total culture period (139 days), it was found to have decreased (P ≤ 0.05). The number of apoptotic cells did not vary during culture, nor was any statistical difference observed between the two culture media for any of the studied parameters.
  
  
  
  N/A LIMITATIONS, REASONS FOR CAUTION: Loss of SG constitutes a limitation for evaluating full functionality of spermatogonial stem cells and warrants further investigation. The scarcity of human immature material is the reason for the limited amount of tissue available for experiments, precluding more comprehensive analysis.
  
  
  
  Our culture system, mimicking the peripubertal testicular microenvironment with SC maturation, LC functionality and preserved paracrine interactions, and the first to use human ITT, opens the door to a deeper understanding of niche and culture conditions to obtain sperm from cryostored ITT, with the ultimate goal of restoring fertility after gonadotoxic treatments.
  
  
  
  This project was supported by a grant from the Fond National de la Recherche Scientifique de Belgique (grant Télevie N° 7.4554.14F and N° 7.4512.15F) and the Fondation Salus Sanguinis. No conflict of interest is declared.
authors.affiliation: Institut de Recherche Expérimentale et Clinique (IREC), Université Catholique de Louvain, Avenue Mounier 52, 1200 Brussels, Belgium.|Department of Gynecology-Andrology, Cliniques Universitaires Saint-Luc, Avenue Hippocrate 10, 1200 Brussels, Belgium.
authors.forename: F
authors.initials: F
authors.lastname: de Michele
authors.affiliation: Institut de Recherche Expérimentale et Clinique (IREC), Université Catholique de Louvain, Avenue Mounier 52, 1200 Brussels, Belgium.
authors.forename: J
authors.initials: J
authors.lastname: Poels
authors.affiliation: Institut de Recherche Expérimentale et Clinique (IREC), Université Catholique de Louvain, Avenue Mounier 52, 1200 Brussels, Belgium.
authors.forename: L
authors.initials: L
authors.lastname: Weerens
authors.affiliation: Department of Gynecology-Andrology, Cliniques Universitaires Saint-Luc, Avenue Hippocrate 10, 1200 Brussels, Belgium.
authors.forename: C
authors.initials: C
authors.lastname: Petit
authors.affiliation: Department of Gynecology-Andrology, Cliniques Universitaires Saint-Luc, Avenue Hippocrate 10, 1200 Brussels, Belgium.
authors.forename: Z
authors.initials: Z
authors.lastname: Evrard
authors.affiliation: Institut de Recherche Expérimentale et Clinique (IREC), Centre de Technologies Moléculaires Appliquées (CTMA), Clos Chapelle-aux-Champs 30, 1200 Brussels, Belgium.
authors.forename: J
authors.initials: J
authors.lastname: Ambroise
authors.affiliation: Department of Clinical Biochemistry, Cliniques Universitaires Saint-Luc, Avenue Hippocrate 10, 1200 Brussels, Belgium.
authors.forename: D
authors.initials: D
authors.lastname: Gruson
authors.affiliation: Institut de Recherche Expérimentale et Clinique (IREC), Université Catholique de Louvain, Avenue Mounier 52, 1200 Brussels, Belgium christine.wyns@uclouvain.be.|Department of Gynecology-Andrology, Cliniques Universitaires Saint-Luc, Avenue Hippocrate 10, 1200 Brussels, Belgium.
authors.forename: C
authors.initials: C
authors.lastname: Wyns
mesh_terms: D054304:Anti-Mullerian Hormone / Q000378:metabolism; D017209:Apoptosis / Q000502:physiology; D049109:Cell Proliferation / Q000502:physiology; D002470:Cell Survival / Q000502:physiology; D006063:Chorionic Gonadotropin / Q000378:metabolism; D003470:Culture Media; D051100:Glial Cell Line-Derived Neurotrophic Factor / Q000378:metabolism; D006801:Humans; D007985:Leydig Cells / Q000166:cytology* / Q000378:metabolism; D008297:Male; D009924:Organ Culture Techniques / Q000379:methods*; D011944:Receptors, Androgen / Q000378:metabolism; D012671:Seminiferous Tubules / Q000254:growth & development* / Q000378:metabolism; D012708:Sertoli Cells / Q000166:cytology* / Q000378:metabolism; D013093:Spermatogonia / Q000166:cytology* / Q000378:metabolism; D013737:Testis / Q000254:growth & development* / Q000378:metabolism; D013739:Testosterone / Q000378:metabolism
chemical_list: D006063:Chorionic Gonadotropin; D003470:Culture Media; D051100:Glial Cell Line-Derived Neurotrophic Factor; D011944:Receptors, Androgen; D013739:Testosterone; D054304:Anti-Mullerian Hormone
keywords: fertility preservation; in-vitro maturation; prepubertal boys; spermatogonial stem cells; testicular tissue