Temporal Effects of Exogenous Testosterone on the Circulating Small Noncoding RNA Landscape in a Placebo‑Controlled Human Study

BACKGROUND: Anabolic-androgenic steroids (AAS) are widely misused, but molecular markers serving as physiological readouts as well as biomarkers of misuse remain poorly defined. Circulating small noncoding RNAs (sncRNAs) are plausible endocrine-responsive messengers and candidate biomarkers. OBJECTIVE: To determine whether exogenous testosterone administration acutely and persistently alters circulating sncRNAs. MATERIALS AND METHODS: Randomized, double-blinded, placebo-controlled trial in healthy eugonadal men (n = 19; 20-30 years), receiving intramuscular injections of 250 mg mixed testosterone esters as Sustanon 250 or placebo on Day 0 and 21. Serum was collected at Day 0, 1, and 42, and the isolated RNA was subjected to small RNA sequencing using the RealSeq-Biofluids library kit. A link between miR-214-3p and testosterone concentrations was tested on longitudinal serum samples from 29 peri-pubertal boys with RT-qPCR, and its expression in testis tissue was investigated by in situ hybridization. RESULTS: Twenty-four hours after testosterone administration, we identified a distinct signature of 19 Sustanon-responsive sncRNAs (12 upregulated, 7 downregulated), with piRNAs as the most represented class. One of the Sustanon-specific sncRNAs, miR-214-3p, was found to correlate positively with circulating testosterone in peri-pubertal boys and showed expression in testicular tissue, including testosterone-producing Leydig cells. Despite testosterone having returned to baseline concentrations at Day 42, 160 sncRNAs remained altered, indicating sustained effects of testosterone use. Intersecting short- and long-term analyses revealed 43 Sustanon-specific sncRNAs, including the small nucleolar RNA U3. DISCUSSION: Despite the modest sample size, this study highlights that multiple sncRNAs respond to testosterone exposure. Further studies are needed to develop a functional, tissue-specific understanding of the identified sncRNAs. CONCLUSIONS: Brief supraphysiological testosterone exposure remodels circulating sncRNAs both acutely and over weeks, implicating androgens in systemic RNA-based communication. Several responsive sncRNAs, notably miR-214-3p and snoRNA U3, show potential as biomarkers of androgen misuse.