Ultrasound localisation microscopy tracks testicular microvascular adaptations to endocrine function in male infertility

Background: Testicular disorders, including male infertility and hypogonadism, are increasingly prevalent and current diagnostic tools have important limitations. The testicular microcirculation underpins testicular function. Ultrasound localisation microscopy (ULM) enables super-resolution mapping of microvascular structure and flow at clinically relevant organ depth. Methods: Prospective case–control study of ULM-assessed testicular activity in men and rodents using clinical and research ultrasound systems. Study 1 compared healthy men (n = 10) with hypogonadotrophic hypogonadism (HH) (n = 9). Study 2 included men with HH receiving testosterone (n = 11), gonadotrophins (n = 9), or no treatment (n = 12). Study 3 assessed 12-month fertility treatment response in HH (n = 7). A rodent pubertal-blockade model was also studied (n = 5). Findings: ULM markers discriminated HH from controls (vessel density p < 0.01; diameter p = 0.01; tortuosity p < 0.01) and correlated with testosterone (r = 0.53–0.67, p < 0.05) and inhibin B (r = −0.61, p < 0.01). Vessel density, diameter, area and flow-related index were reduced in azoospermia (p < 0.01). ULM distinguished HH treatment groups (vessel density p < 0.001; diameter p < 0.05), with density and diameter correlating with testosterone (r = 0.69, 0.62; p < 0.001) and inhibin B (r = 0.64, 0.65; p < 0.001). Vessel density (p < 0.001) and diameter (p < 0.01) were reduced in azoospermia irrespective of treatment. During fertility therapy, ULM parameters increased (p < 0.05) and detected testicular activation earlier than volume or inhibin B. In rodents, pubertal development showed dynamic microvascular remodelling driven by testis growth. Interpretation: ULM provides a treatment-responsive, biologically grounded biomarker of testicular function enabling patient stratification, early detection of therapeutic response, and potential for both refinement of clinical decision-making in HH, and application within other testicular disorders. Funding: MRC, NIHR Biomedical Research Centre Funding Scheme and the NIHR/Imperial Clinical Research Facility, Diabetes UK, BBSRC, MRC, Imperial Private Healthcare Clinical Research Fellowship Scheme, NWLP Research Grant.