Design and analysis strategies for robust microbiome ageing research

The gut microbiome changes systematically with age and associates with age-related morbidity and mortality, establishing it as a candidate biomarker and intervention target for ageing. Realising this potential requires methodological rigour, as distinguishing genuine biological signals from methodological artefacts remains challenging given variable findings across cohorts. This review provides an integrated framework for human microbiome-ageing research, organised around five methodological challenges that will collectively strengthen causal inference. We examine how age-associated factors can correlate with chronological age and may confound the microbiome-age associations, while selection biases shape old-age cohorts towards healthier profiles. We address within-host temporal dynamics and between-individual heterogeneity that require appropriate sampling to distinguish age-related signatures from transient states, and validation strategies that separate ageing from batch effects in predictive models. Mendelian randomisation provides causal leverage when triangulated with longitudinal and interventional evidence. Throughout, we examine how design choices determine the limits of analytical inference. The review concludes with a practical checklist, equipping researchers to strengthen reproducibility, improve generalisability and advance microbiome-based metrics towards validated indicators of biological ageing.