Is total testosterone (TT) or free testosterone (FT) a more accurate measurement of testosterone levels in women?

Free Testosterone is More Accurate Than Total Testosterone for Women

Free testosterone is the most accurate measurement for assessing testosterone levels in women, with calculated free testosterone showing superior sensitivity (89%) compared to total testosterone (74%) for diagnosing hyperandrogenism. 1

Understanding Testosterone Measurements in Women

Free vs. Total Testosterone

• Free testosterone represents the biologically active form that affects tissues and is considered a more sensitive indicator of androgen status in women 2
• Total testosterone measurements can be misleading in women due to:
  • Variations in Sex Hormone-Binding Globulin (SHBG) levels, which strongly bind testosterone 3
  • Low overall testosterone concentrations in women, requiring highly sensitive assays 3
  • SHBG fluctuations that can mask true androgen status (especially in overweight patients where low SHBG may normalize total testosterone despite excess androgens) 3

Diagnostic Performance Comparison

Based on the 2023 International PCOS Evidence-based Guidelines meta-analysis of 2857 participants:

Measurement	Sensitivity	Specificity	AUC
Free Testosterone	0.89 (0.69-0.96)	0.83 (0.79-0.86)	0.85 (0.81-0.88)
Total Testosterone	0.74 (0.63-0.82)	0.86 (0.77-0.91)	0.87 (0.84-0.90)
Free Androgen Index	0.78 (0.70-0.83)	0.85 (0.76-0.90)	0.87 (0.84-0.90)

Best Methods for Free Testosterone Assessment

Recommended Methods (in order of preference):

1. Gold Standard Methods:

   • Equilibrium dialysis
   • Ammonium sulfate precipitation 4, 5

2. Calculated Free Testosterone:

   • Using the Vermeulen equation with high-quality total T and SHBG measurements 2, 6
   • Calculated values correlate extremely well with equilibrium dialysis (r = 0.99) 4

3. Free Androgen Index (FAI):

   • Calculated as 100 × Total T/SHBG
   • Good correlation with equilibrium dialysis (r = 0.93) 4
   • Can be used when more sophisticated methods aren't available 1

Methods to Avoid:

• Direct Free Testosterone Immunoassays (Analog Methods):
  • Show unacceptably high systematic bias and random variability 4
  • Poorer correlation with equilibrium dialysis (r = 0.81) 4
  • Values are only a fraction of true free testosterone, with the fraction varying based on SHBG levels 6

Measurement Considerations

Laboratory Technique

• Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is superior to immunoassay methods 1, 5
• LC-MS/MS provides:
  • Higher sensitivity for measuring free testosterone
  • Better specificity for measuring total testosterone
  • Lower detection limits needed for women's typically low testosterone levels 3

Timing and Sampling

• Morning samples are preferred due to diurnal variation 2
• Multiple samples may improve reliability 5

Clinical Implications

When to Use Free Testosterone

• Women with suspected PCOS or hyperandrogenism 1
• Women with conditions that affect SHBG levels:
  • Obesity (decreases SHBG)
  • Liver disease (increases SHBG)
  • Thyroid disorders (hyperthyroidism increases SHBG)
  • Estrogen therapy (increases SHBG) 2

Pitfalls to Avoid

• Relying solely on total testosterone when SHBG levels may be abnormal 2
• Using direct immunoassay methods without extraction steps 3
• Failing to consider between-kit variability, which is particularly problematic for female samples (57-115% variance between commercial kits) 7

Conclusion

For accurate assessment of testosterone status in women, free testosterone measurement is superior to total testosterone, particularly when using equilibrium dialysis or calculated free testosterone methods. The 2023 International PCOS Guidelines specifically recommend free testosterone as a first-line laboratory test for assessing biochemical hyperandrogenism in women due to its superior sensitivity.