Testicular organoid models in male reproductive toxicology: advances, applications, and future perspectives

Male reproductive health has declined markedly over the past decades, with environmental chemicals, pharmaceuticals, and lifestyle factors implicated in this trend. Conventional toxicological approaches-rodent bioassays and two-dimensional cell cultures-suffer from species-specific biases, limited physiological relevance, and inadequate throughput for screening the expanding universe of suspect compounds. Testicular organoid technology has emerged as a promising alternative, reconstituting three-dimensional cellular architecture, paracrine crosstalk, and partial spermatogenic progression in vitro. This narrative review was assembled from English-language studies retrieved from PubMed, Web of Science, and Scopus (2010-2024) using combinations of the terms testicular organoid, organ-on-chip, spermatogenesis, and reproductive toxicology, prioritizing original research and methodological reports on three-dimensional testicular models and excluding abstract-only records. On this basis the review maps current construction strategies, spanning primary cell isolation, pluripotent stem cell differentiation, and spermatogonial stem cell expansion, alongside matrix choices and microfluidic culture platforms. It then examines how testicular organoids have been deployed to assess endocrine-disrupting chemicals, chemotherapeutics, and other pharmacological agents, where several studies report the detection of effects at concentrations appreciably below those required in monolayer systems, particularly under chronic low-dose exposure. Integration with single-cell multi-omics and machine learning has advanced mechanistic dissection and candidate biomarker discovery across oxidative, mitochondrial, and epigenetic axes, although these computational applications remain exploratory given the absence of standardized organoid protocols. Morphological, functional, molecular, and epigenetic indicators are consolidated into a unified evaluation framework, and persistent bottlenecks are addressed, including incomplete blood-testis barrier reconstitution, long-term culture instability, inter-donor variability, and inefficient elongated spermatozoa formation. Overall, testicular organoids offer clear mechanistic and human-relevance advantages, yet they are not yet sufficiently mature or validated to broadly replace in vivo models in regulatory assessment. Future priorities center on protocol standardization, automated high-throughput adaptation, multi-organ-on-chip integration, and formal regulatory validation to enable transformative applications in drug safety evaluation, environmental risk management, and male reproductive health protection.