Animal models of gut microbiota-male reproductive interactions: methodological assessment, translational bottlenecks, and optimization strategies.

Idiopathic infertility accounts for 30%-40% of male infertility cases, yet its etiology remains largely unexplained. The gut-testis axis hypothesis has recently emerged as a promising direction, with human cohort studies revealing statistical associations between gut dysbiosis and impaired semen quality. However, establishing mechanistic and causal evidence beyond observational associations requires complementary approaches, including animal models. Whether current models adequately capture the chronic, low-grade nature of gut-derived insults that may contribute to idiopathic infertility remains insufficiently examined. Here, we examine the methodological foundations of gut-testis axis animal models. We organize them according to the tier of causal question they address and evaluate their internal, external, and construct validity. Our analysis identifies a recurring pattern in which some existing models, particularly those testing the effects of individual molecular pathways, employ exposure magnitudes, routes, and time scales that may not fully reflect the prolonged, low-intensity disturbances hypothesized in gut-driven reproductive injury. The collective efforts of these model categories have identified several signaling axes that, under defined experimental conditions, can measurably affect the testis. However, the internal and external validity limitations of many of these models warrant caution when interpreting these demonstrations of conditional biological capacity as direct evidence that the same pathways carry etiological weight in human idiopathic infertility. To bridge this gap, we propose a logical framework for evaluating causal evidence that transitions from establishing a pathway's biological capacity to assessing its etiological contribution. This framework is anchored on three criteria: endogenous signal origin, pathomimetic exposure profile, and attributive exclusivity. We further propose a preliminary reporting framework, termed Minimal Information for Gut-Testis Axis Rodent Studies (MIGTARS), to support more transparent and methodologically informative reporting. These proposals together provide a methodological scaffold for moving the gut-testis field from pathway verification toward translational modeling.