What laboratory tests should be ordered to evaluate hypogonadism in an adult male?

Laboratory Evaluation for Hypogonadism in Adult Males

Core Diagnostic Tests

Measure morning total testosterone (8–10 AM) on two separate occasions; both values must be <300 ng/dL to confirm biochemical hypogonadism. 1, 2 Single measurements are insufficient due to diurnal variation and significant intra-individual variability. 1, 3

Essential Hormone Panel

• Serum luteinizing hormone (LH) – mandatory after confirming low testosterone to distinguish primary (elevated LH) from secondary (low/normal LH) hypogonadism 1, 2
• Serum follicle-stimulating hormone (FSH) – required alongside LH to complete the differentiation between testicular failure and hypothalamic-pituitary dysfunction 1, 2
• Serum prolactin – measure in all men with low testosterone and low/normal LH-FSH to screen for hyperprolactinemia; if elevated >1.5× upper limit of normal, repeat to confirm and order pituitary MRI 1, 2

Free Testosterone Assessment (When Indicated)

Measure free testosterone by equilibrium dialysis (gold standard) or calculate using validated formulas in men with:

• Total testosterone 231–346 ng/dL (borderline range) 2, 4
• Obesity (low SHBG may artificially lower total testosterone while free testosterone remains normal) 2, 4
• Diabetes 2
• Suspected SHBG abnormalities 1, 4

Sex hormone-binding globulin (SHBG) should be measured alongside free testosterone to calculate the free androgen index (total testosterone ÷ SHBG × 100); an FAI <30 indicates true hypogonadism even when total testosterone is borderline-normal. 2, 5

Baseline Safety Assessments

Before Initiating Testosterone Therapy

• Hematocrit/hemoglobin – document baseline; hematocrit >54% is an absolute contraindication to starting therapy 1, 2
• Prostate-specific antigen (PSA) – required in all men ≥40 years; PSA >4.0 ng/mL mandates urologic evaluation and negative prostate biopsy before treatment 1, 2
• Digital rectal examination – assess for palpable prostate nodules or induration 1, 2
• Fasting glucose and HbA1c – screen for diabetes 1, 2
• Lipid profile – baseline metabolic assessment 2
• Thyroid-stimulating hormone (TSH) – exclude thyroid dysfunction that can mimic hypogonadal symptoms 2

Estradiol Measurement (Selective)

Measure serum estradiol only in men presenting with breast symptoms or gynecomastia prior to starting testosterone therapy. 1 This is not a routine screening test.

Pituitary Imaging Indications

Order pituitary MRI when any of the following are present:

• Total testosterone <150 ng/dL with LH and FSH both <1.5 IU/L (regardless of prolactin level) – non-secreting adenomas may be present 1, 2
• Persistently elevated prolactin >1.5× upper limit of normal – screen for prolactinoma 1, 2
• Visual field defects (bitemporal hemianopsia) or anosmia – suggest sellar mass 1, 2

High-Risk Populations Requiring Screening

Measure testosterone even without classic symptoms in men with:

• Unexplained anemia 1
• Decreased bone mineral density 1
• Diabetes mellitus 1, 2
• Recent chemotherapy or testicular radiation exposure 1
• HIV infection 1
• Chronic narcotic use 1
• Chronic corticosteroid therapy 1
• Known pituitary disorders 1
• Infertility 1

Tests NOT Routinely Required

• Vitamin D – not part of standard hypogonadism work-up 2
• C-reactive protein (CRP) – not indicated for hypogonadism evaluation 2
• Scrotal ultrasound – reserved only for abnormal testicular examination findings (nodules, masses), not routine screening 2
• DHEA-S – does not aid in diagnosing hypogonadism or distinguishing primary from secondary forms 2

Critical Diagnostic Algorithm

1. Obtain two fasting morning total testosterone measurements (8–10 AM) on separate days 1, 2
2. If both values <300 ng/dL: measure LH, FSH, and prolactin 1, 2
3. If total testosterone 231–346 ng/dL (gray zone): add free testosterone by equilibrium dialysis and SHBG 2, 4
4. If LH/FSH low or low-normal: measure prolactin; if prolactin elevated or testosterone <150 ng/dL with LH/FSH <1.5 IU/L, order pituitary MRI 1, 2
5. Before starting therapy: obtain hematocrit, PSA (if ≥40 years), digital rectal exam, fasting glucose, HbA1c, lipid profile, and TSH 1, 2

Common Pitfalls to Avoid

• Never diagnose hypogonadism on a single testosterone measurement – two morning values are mandatory 1, 2, 3
• Never test outside 8–10 AM window – later measurements risk false-positive diagnoses due to diurnal variation 1, 2, 3
• Never omit LH/FSH testing after confirming low testosterone – the primary vs. secondary distinction directs treatment and fertility counseling 1, 2
• Never skip pituitary imaging when testosterone <150 ng/dL with low gonadotropins – treatable pituitary lesions may be missed 1, 2
• Never rely on direct immunoassays for free testosterone in men with abnormal SHBG – use equilibrium dialysis or calculate free androgen index 2, 5
• Never diagnose based on symptoms alone without biochemical confirmation – symptoms are nonspecific and overlap with many conditions 1, 2

Laboratory Variability Considerations

Reference ranges vary widely across laboratories, with lower limits ranging from 130–450 ng/dL and upper limits from 486–1,593 ng/dL. 2 This variability directly influences clinical decision-making and explains why approximately 25% of men receiving testosterone therapy do not actually meet diagnostic criteria for hypogonadism. 2 Always use the same laboratory with the same assay method for repeat measurements. 1