Which laboratory tests should be obtained to evaluate low testosterone and investigate secondary (hypothalamic‑pituitary) causes?

Laboratory Evaluation for Secondary Causes of Low Testosterone

When low testosterone is confirmed with two morning measurements <300 ng/dL, measure serum LH and FSH to distinguish primary from secondary hypogonadism, then obtain prolactin if gonadotropins are low or low-normal to screen for pituitary pathology. 1, 2

Essential Initial Hormone Panel

Gonadotropins (LH and FSH)

• Measure serum LH and FSH in all patients with confirmed low testosterone to differentiate primary (testicular) from secondary (hypothalamic-pituitary) hypogonadism 1, 3
• Low or inappropriately normal LH/FSH with low testosterone indicates secondary hypogonadism, whereas elevated LH/FSH indicates primary hypogonadism 2, 3
• This distinction is critical because secondary hypogonadism can be treated with gonadotropin therapy to restore both testosterone and fertility, while primary hypogonadism requires testosterone replacement which permanently suppresses fertility 2

Prolactin

• Measure serum prolactin in all patients with low testosterone and low or low-normal LH levels to screen for hyperprolactinemia 1, 3
• If prolactin is elevated, repeat the measurement to confirm it is not spurious 1
• Persistently elevated prolactin (>1.5× upper limit of normal) requires referral to endocrinology and pituitary MRI to evaluate for prolactinoma 1, 2

Free Testosterone and SHBG

• Measure free testosterone by equilibrium dialysis and SHBG in men with total testosterone near the lower limit of normal (231-346 ng/dL) or in obese patients where SHBG abnormalities are common 2, 3, 4
• In men with elevated SHBG (e.g., from liver disease, hyperthyroidism), calculate the free androgen index (total testosterone ÷ SHBG × 100); an FAI <30 indicates true hypogonadism even when total testosterone appears borderline-normal 2
• Avoid direct immunoassays for free testosterone as they are unreliable; use equilibrium dialysis or validated calculation formulas 2, 5

Pituitary Imaging Indications

• Order pituitary MRI when total testosterone is <150 ng/dL with LH/FSH <1.5 IU/L, regardless of prolactin level, as non-secreting adenomas may be present 1, 2
• MRI is also indicated for prolactin >1.5× upper limit of normal, visual field defects (bitemporal hemianopsia), or anosmia 1, 2, 6

Additional Screening for Reversible Causes

Metabolic and Systemic Conditions

• Measure fasting glucose and HbA1c to screen for diabetes, which is strongly associated with secondary hypogonadism 2, 6
• Check TSH to exclude thyroid dysfunction that can mimic hypogonadal symptoms and alter SHBG 2, 3
• Obtain iron saturation and ferritin if hemochromatosis is suspected, as iron overload causes secondary hypogonadism 2

High-Risk Populations Requiring Testing

• Measure testosterone even without symptoms in men with unexplained anemia, bone density loss, diabetes, chemotherapy exposure, testicular radiation, HIV, chronic narcotic use, chronic corticosteroid use, or pituitary disorders 1, 6

Diagnostic Algorithm Summary

1. Confirm hypogonadism: Two separate fasting morning (8-10 AM) total testosterone measurements <300 ng/dL using the same laboratory 1, 2, 3

2. Measure LH and FSH to classify primary vs. secondary hypogonadism 1, 3

3. If LH/FSH are low or low-normal: measure prolactin 1, 3

4. If testosterone <150 ng/dL with LH/FSH <1.5 IU/L or prolactin >1.5× ULN: order pituitary MRI 1, 2

5. If total testosterone is 231-346 ng/dL or patient is obese: measure free testosterone by equilibrium dialysis and SHBG 2, 3, 4

6. Screen for reversible causes: fasting glucose, HbA1c, TSH, and iron studies as clinically indicated 2, 3

Critical Pitfalls to Avoid

• Never diagnose hypogonadism on a single testosterone measurement; two morning values are required due to diurnal variation and assay variability 1, 3, 7
• Never omit LH/FSH testing after confirming low testosterone, as the distinction between primary and secondary hypogonadism directs treatment and fertility counseling 1, 2
• Do not measure testosterone outside the 8-10 AM window; only 9% of tested men have appropriately timed samples, leading to diagnostic errors 7
• Do not rely on direct immunoassays for free testosterone in men with abnormal SHBG; use equilibrium dialysis or validated calculation methods 2, 5
• Do not skip pituitary imaging when testosterone is <150 ng/dL with low gonadotropins, as this may miss treatable pituitary lesions 1, 2