What labs should be ordered to diagnose low testosterone?

Laboratory Testing for Low Testosterone

Order a morning (8-10 AM) total testosterone level on two separate days, and if both are <300 ng/dL with consistent symptoms, measure serum LH to determine the etiology. 1, 2, 3

Initial Diagnostic Testing

Total Testosterone Measurement

• Obtain two separate morning measurements between 8:00-10:00 AM to confirm the diagnosis, as testosterone levels can be 20-25% lower in the afternoon in younger men (30-40 years old) and 10% lower even at age 70. 2, 3, 4
• Both measurements must be <300 ng/dL to meet diagnostic criteria for testosterone deficiency. 1, 3
• Use the same laboratory and methodology for both measurements to ensure consistency. 3
• No fasting is required for testosterone testing, despite common misconceptions. 2
• Avoid testing during acute illness, as this can artificially suppress testosterone levels. 1

Important Technical Considerations

• Ideally, use liquid chromatography-tandem mass spectrometry in a laboratory certified by the CDC's Hormone Standardization (HoST) Program for the most accurate results. 5
• Patient should be rested and not immediately post-exercise or stressed during blood draw. 2

Adjunctive Hormone Testing

Luteinizing Hormone (LH)

• Measure serum LH in all patients with confirmed low testosterone to distinguish primary hypogonadism (testicular failure) from secondary hypogonadism (pituitary-hypothalamic dysfunction). 1, 3
• Low or low-normal LH with low testosterone indicates secondary hypogonadism. 1
• Elevated LH with low testosterone indicates primary hypogonadism. 3
• This distinction is critical because secondary hypogonadism patients can potentially achieve fertility with gonadotropin therapy, while primary hypogonadism patients require testosterone replacement which suppresses fertility. 1

Prolactin

• Measure serum prolactin if testosterone is low AND LH is low or low-normal to screen for hyperprolactinemia. 1, 3
• Prolactin can be measured at any time of day (no morning requirement). 2
• If prolactin is elevated, repeat the measurement to confirm it wasn't spurious. 1
• Persistently elevated prolactin (>25 ng/mL) warrants endocrinology referral and consideration of pituitary MRI. 1
• Never measure prolactin immediately after a seizure, as post-ictal states artificially elevate levels. 2
• Be aware that proton pump inhibitors and impaired renal/hepatic function can cause artifactual prolactin elevations. 2

Follicle-Stimulating Hormone (FSH)

• Measure FSH in men interested in fertility to assess underlying reproductive health status. 1, 3
• Elevated FSH with low testosterone (hypergonadotropic hypogonadism) indicates impaired spermatogenesis and warrants consideration of semen analysis. 1
• Among men with normal sperm concentration, 10.8% still have elevated FSH, making universal screening valuable. 6

Additional Testing Based on Clinical Context

Free Testosterone

• Measure free testosterone by equilibrium dialysis in obese patients where total testosterone may be falsely low due to decreased sex hormone-binding globulin (SHBG). 3
• Also measure SHBG simultaneously in obese patients to interpret total testosterone accurately. 3
• If free testosterone is also low on at least 2 separate assessments, proceed with hypogonadism workup. 3

Estradiol

• Measure serum estradiol only if the patient presents with breast symptoms or gynecomastia prior to starting testosterone therapy. 1
• Elevated baseline estradiol requires endocrinology referral. 1

Pre-Treatment Safety Labs

• Measure hemoglobin/hematocrit before initiating testosterone therapy to establish baseline. 1
• Assess cardiovascular risk factors including lipid panel if not recently checked. 1

Special Population Considerations

Men Seeking Fertility

• Perform testicular examination to evaluate size, consistency, and descent. 1
• Measure FSH as noted above. 1
• If severe oligospermia (<5 million sperm/mL) or azoospermia is present, offer karyotype testing and Y-chromosome microdeletion analysis. 1

Men with Total Testosterone <150 ng/dL

• Obtain pituitary MRI regardless of prolactin levels if LH is low or low-normal, as non-secreting adenomas may be present. 1

High-Risk Populations Requiring Screening

• Measure testosterone in all patients with unexplained anemia, even without typical hypogonadism symptoms. 3
• Screen patients with bone density loss, diabetes, or HIV/AIDS. 3

Common Pitfalls to Avoid

• Never measure testosterone in the afternoon or evening, as 44.1% of men with normal sperm concentration have low testosterone when measured properly in the morning, and afternoon testing will dramatically increase false-positive diagnoses. 4, 6
• Don't rely on screening questionnaires alone—they lack specificity and cannot substitute for laboratory confirmation. 1
• Don't test during acute illness, as this suppresses the hypothalamic-pituitary-gonadal axis. 1
• Don't assume normal sperm concentration excludes hormonal abnormalities—43.6% of men with normal sperm concentration have at least one hormonal abnormality (low testosterone or elevated FSH). 6