DEVELOPMENT OF A BIOMIMETIC TESTICULAR SCAFFOLD MODEL TO INVESTIGATE ADIPOCYTE DERIVED PARACRINE EFFECTS ON OBESITY - ASSOCIATED HYPOGONADISM.

Resumo

Introduction: Male hypogonadism is a condition character ized by insufficient testicular function, particularly reduced testosterone production, leading to hormonal, metabolic, and reproductive alterations. Obesity has emerged as a contributing factor to this condition, since excessive adipose tissue accumulation promotes the release of inflammatory mediators, adipokines, and metabolic signals capable of impairing testicular function, mainly through reduced steroidogenesis and inflammatory dysregulation of the testicular microenvironment. However, the mechanisms by which adipose tissue negatively affects testicular cells remain incompletely understood, partly due to the limitations of conventional two -dimensional culture systems, which do not adequately reproduce the three -dimensional architecture, extracellular m atrix composition, and cell -matrix interactions present in the native testicular niche. Hypothesis: A biomimetic three - dimensional scaffold derived from decellularized canine testicular extracellular matrix can support testicular somatic cells and partial ly reproduce key aspects of the native testicular microenvironment. Moreover, soluble factors released by differentiated adipocytes will induce a partial hypogonadal- like phenotype in this model, characterized by impaired steroidogenic activity, inflammato ry activation, and altered cell-matrix interactions. Objective: This study aims to develop a biomimetic three- dimensional in vitro model of obesity -associated male hypogonadism using decellularized canine testicular extracellular matrix scaffolds recellul arized with testicular somatic cells, and to evaluate the indirect paracrine effects of differentiated adipocytes on this biomimetic testicular microenvironment. Materials and Methods: Canine testicular extracellular matrix will be obtained under UNIMAR Et hics Committee protocol 15/2026 and decellularized to produce biomimetic three-dimensional scaffolds that preserve important structural and biochemical components of native testicular tissue. These scaffolds will be recellularized with testicular somatic c ells, particularly Sertoli and Leydig cells, to partially reconstruct the functional somatic compartment of the testicular niche. To model obesity -associated hypogonadal dysfunction, differentiated adipocytes will be cultured in Transwell® inserts. This sy stem will allow soluble factors released by adipocytes to act indirectly on the recellularized testicular scaffolds, without direct cell -to-cell contact. Therefore, the model will specifically evaluate the paracrine effects of adipocytes on the biomimetic testicular microenvironment. Functional validation will include testosterone quantification, gene expression analysis of steroidogenic, inflammatory, and testicular functional markers, as well as morphological and cell viability assessments. This model is not intended to reproduce complete spermatogenesis, but to mimic selected features of hypogonadal dysfunction, mainly impaired steroidogenesis, inflammatory activation, and altered cell -matrix interactions. Quantitative data will be analyzed according to the experimental design. Comparisons between two groups will be performed using Student’s t -test, while comparisons among three or more groups will be analyzed by one -way analysis of variance, followed by Tukey’s post hoc test when applicable. Statistical significance will be set at p < 0.05. Expected outcomes: The decellularized testicular matrix will provide a biomimetic scaffold capable of supporting the adhesion, viability, and organization of Sertoli and Leydig cells. Exposure to adipocyte -derived solub le factors is expected to induce a partial hypogonadal -like phenotype, characterized by reduced testosterone production, altered expression of steroidogenic and testicular functional markers, increased inflammatory signaling, and changes in cell viability or morphology. Therefore, this approach may provide a controlled 3D platform to investigate the indirect effects of adipocytes on testicular function under obesity-related conditions.