Which laboratory values indicate exogenous testosterone use or anabolic steroid abuse?

Laboratory Indicators of Exogenous Testosterone or Anabolic Steroid Abuse

The most reliable laboratory indicators of exogenous testosterone or anabolic steroid abuse are suppressed LH and FSH levels, elevated testosterone-to-epitestosterone (T/E) ratio >4, and direct detection of synthetic testosterone esters in blood or urine.

Primary Laboratory Markers

Hormonal Suppression Patterns

The hallmark of exogenous testosterone use is suppression of the hypothalamic-pituitary-gonadal (HPG) axis 1, 2. When synthetic testosterone is administered, it triggers negative feedback that suppresses:

• Luteinizing hormone (LH) - markedly decreased or undetectable
• Follicle-stimulating hormone (FSH) - markedly decreased or undetectable

This suppression pattern is critical because endogenous testosterone production is inhibited through feedback inhibition of pituitary LH, and at large doses, spermatogenesis is also suppressed through FSH inhibition 1. The testosterone-to-LH ratio (T/LH) becomes abnormally elevated, with T/LH >400 nmol/IU in urine considered evidence of exogenous administration in forensic contexts 3.

Testosterone-to-Epitestosterone Ratio (T/E)

The T/E ratio is a cornerstone marker:

• T/E ratio >4 is considered suspicious and warrants further investigation 3
• T/E ratio ≥12 combined with T/LH >400 nmol/IU is used as proof in forensic cases 3

However, this marker has significant limitations. A 2021 Swedish forensic study found that using T/E ≥12 combined with T/LH >400 nmol/IU had only 55% sensitivity, missing 21 of 47 confirmed positive cases 3. This high false-negative rate (32% negative predictive value) indicates these criteria alone are insufficient.

Direct Detection of Synthetic Esters

The most definitive evidence is direct detection of synthetic testosterone esters (testosterone propionate, phenylpropionate, isocaproate, decanoate, undecanoate, cypionate, enanthate) in blood or dried blood spots 4. These compounds are unequivocally exogenous and can be detected at sub-ng/mL levels using liquid chromatography-mass spectrometry (LC-MS), with detection limits below 0.1 ng/mL 4.

Secondary Laboratory Markers

Serum Testosterone Levels

• Elevated serum testosterone measured by LC-MS is particularly useful in females, where the T/E ratio may be unreliable 5
• The testosterone-to-androstenedione ratio (T/A4) in serum shows significant increases after testosterone administration and remains elevated up to 15 days post-administration 6
• Serum testosterone and dihydrotestosterone (DHT) levels both increase with exogenous administration 7

Hematological Changes

Testosterone stimulates erythropoiesis 1, leading to:

• Elevated hematocrit and hemoglobin 8
• Increased reticulocyte count (modest increase) 7
• Polycythemia in some cases 1

Conjugated Steroid Metabolites

Analysis of both sulfate and glucuronide conjugates reveals disruption patterns 9:

• The ratio (epitestosterone sulfate/epitestosterone glucuronide) to (testosterone sulfate/testosterone glucuronide) - expressed as (ES/EG)/(TS/TG) - shows promise as a complementary biomarker independent of traditional steroid passport markers 9

Isotope Ratio Analysis

Gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS) confirms the synthetic origin of testosterone and its metabolites by detecting differences in carbon isotope ratios between endogenous and pharmaceutical testosterone 3.

Critical Pitfalls and Genetic Considerations

UGT2B17 Gene Deletion

A major confounding factor, particularly in Asian populations, is UGT2B17 gene deletion polymorphism 6, 5. This genetic variant:

• Results in extremely low or undetectable urinary testosterone glucuronide
• Makes T/E ratio unreliable or impossible to calculate
• Affects up to 67% of some Asian populations
• Can completely nullify traditional urine-based detection methods 5

In individuals with UGT2B17 deletion, serum testosterone measurement by LC-MS becomes essential 5.

Sex-Specific Differences

In females, detection is more challenging because:

• Endogenous testosterone derives from three sources (ovaries, adrenal glands, peripheral conversion)
• None are subject to powerful negative feedback like in males
• T/E ratio mechanisms may be partially inoperative 5
• Serum testosterone by LC-MS is more reliable than urine markers 5

Practical Algorithm for Detection

1. First-line screening:

   • Measure serum LH and FSH (suppressed levels highly suspicious)
   • Calculate urinary T/E ratio (>4 warrants further investigation)
   • Measure serum testosterone by LC-MS (not immunoassay)

2. If initial screening suspicious:

   • Calculate T/LH ratio in urine (>400 nmol/IU supports abuse)
   • Measure serum T/A4 ratio
   • Check hematocrit/hemoglobin for polycythemia

3. Confirmatory testing:

   • GC-C-IRMS for isotope ratio analysis of testosterone metabolites
   • Direct detection of synthetic testosterone esters by LC-MS in blood/dried blood spots
   • Analysis of conjugated steroid metabolites (ES/EG)/(TS/TG) ratio

4. Consider genetic factors:

   • In cases with unexpectedly low urinary testosterone, consider UGT2B17 genotyping
   • Rely more heavily on serum markers in Asian populations or confirmed UGT2B17 deletion carriers

The combination of suppressed gonadotropins (LH/FSH), elevated T/E ratio, and direct ester detection provides the most robust evidence, while single markers alone have unacceptably high false-negative rates 3.