Testosterone (Total and Free) and SHBG

Testosterone is the main circulating androgen. In men it is produced mainly by the testes under luteinising hormone control (95%), and in women by the ovaries and adrenal glands and by peripheral conversion. About 40 to 60% is bound to sex hormone binding globulin (SHBG) with high affinity, 40 to 50% is weakly bound to albumin, and only 1 to 3% is free. Albumin bound plus free testosterone is bioavailable.

Total testosterone is measured by immunoassay or, preferably, liquid chromatography with tandem mass spectrometry. Free testosterone is estimated by calculation (Vermeulen equation, using total testosterone, SHBG and albumin) or by equilibrium dialysis, and direct analogue assays are unreliable.

Late onset hypogonadism affects 2 to 6% of men over 40 years, and low testosterone is common in obesity, type 2 diabetes (25 to 40%) and chronic illness. In women, hyperandrogenism with polycystic ovary syndrome affects 6 to 13% of women of reproductive age. Testosterone testing identifies hypogonadism and its cause in men, hyperandrogenism in women, precocious or delayed puberty in children, and androgen secreting tumours.

Indications. Men: low libido, erectile dysfunction, infertility, reduced muscle mass, osteoporosis, anaemia, gynaecomastia, small testes, delayed puberty, hypopituitarism, and monitoring testosterone therapy. Women: hirsutism, acne, oligomenorrhoea, virilisation, infertility, and suspected androgen secreting tumour. Children: precocious puberty, delayed puberty, disorders of sex development, and adolescent monitoring.

Specimen. Serum 1 to 2 mL (not gel tubes with some assays), taken between 07:00 and 10:00 in men because of the diurnal rhythm (morning values are 20 to 30% higher, and the rhythm blunts after age 50 years). Fasting is preferred because glucose and food lower testosterone by about 25%. In premenopausal women the sample is taken in the early follicular phase (day 3 to 5). Acute illness, high dose glucocorticoids and recent heavy exercise are avoided.

Total testosterone.

• Adult men: 10 to 35 nmol/L (about 290 to 1000 ng/dL), with the lower limit of normal near 10 to 12 nmol/L (about 300 ng/dL), based on the 2.5th percentile in healthy young men.
• Adult women: 0.3 to 1.7 nmol/L (9 to 50 ng/dL) in premenopausal women, lower after menopause. Mass spectrometry is required, since immunoassays are inaccurate at low concentrations.
• Children: under 0.7 nmol/L before puberty, rising with Tanner stage.
• Hypogonadism in men: two morning fasting values under 8 to 10 nmol/L (under 230 to 300 ng/dL) with symptoms. Levels of 8 to 12 nmol/L are borderline, and free testosterone is calculated.

SHBG.

• Men 10 to 57 nmol/L, women 18 to 144 nmol/L (assay dependent).
• Raised by ageing, hyperthyroidism, oestrogen, liver cirrhosis, HIV, anticonvulsants and low calorie intake. Lowered by obesity, insulin resistance, hypothyroidism, nephrotic syndrome, androgens, glucocorticoids and acromegaly.
• Changes in SHBG shift total testosterone without changing the free fraction, so total testosterone can mislead in obesity, ageing and liver disease.

Free testosterone (calculated).

• Adult men: 0.2 to 0.6 nmol/L (about 50 to 170 pg/mL), with lower limit near 0.22 nmol/L (about 65 pg/mL).
• Adult women: 0.003 to 0.03 nmol/L.
• Used when total testosterone is borderline or SHBG is abnormal.

Interpretation in men with low testosterone.

• Low testosterone with high LH and FSH: primary hypogonadism (testicular failure from Klinefelter syndrome, mumps orchitis, trauma, chemotherapy, radiotherapy, torsion, cryptorchidism).
• Low testosterone with low or inappropriately normal LH and FSH: secondary (central) hypogonadism from pituitary or hypothalamic disease, hyperprolactinaemia, haemochromatosis, opioids, anabolic steroids, obesity, severe illness or malnutrition. Prolactin, iron studies and pituitary MRI are done.
• Confirmation requires repeating on a second morning sample at least 2 weeks apart.

Interpretation in women.

• Total testosterone mildly raised (up to 3 nmol/L) with low SHBG, oligomenorrhoea and polycystic ovaries: polycystic ovary syndrome (Rotterdam criteria).
• Total testosterone over 5 nmol/L (over 150 ng/dL), or rapid onset virilisation: androgen secreting ovarian or adrenal tumour, and imaging is needed. DHEA-S over 18 micromol/L suggests adrenal source.
• Mild rise with raised 17-hydroxyprogesterone: non-classic congenital adrenal hyperplasia.
• Other: hyperprolactinaemia, Cushing syndrome, acromegaly and drugs (danazol, valproate, androgens).

Children. Raised testosterone with advanced puberty indicates precocious puberty (gonadotrophin dependent or independent). Low testosterone with absent puberty by 14 years in boys indicates delayed puberty, hypogonadotrophic hypogonadism or primary gonadal failure.

Critical values and findings needing immediate action.

• Total testosterone over 5 nmol/L (or over 10 nmol/L in women) with rapid virilisation: urgent imaging for androgen secreting tumour.
• Newborn with ambiguous genitalia: urgent karyotype, 17-hydroxyprogesterone, electrolytes and glucose, and exclusion of salt wasting adrenal crisis.
• Very low testosterone with headache or visual field loss: pituitary mass.
• Suspected anabolic steroid abuse with low LH and FSH and hepatic injury.
• Testosterone replacement with haematocrit over 54%: hold treatment and evaluate.

Interferences and pitfalls.

• Immunoassays for total testosterone overestimate at female and child concentrations, and mass spectrometry is required there.
• A single low value is repeated before diagnosis, since 30% normalise on repeat.
• Acute illness, obesity, diabetes, opioids and glucocorticoids lower testosterone, and low testosterone in these conditions often improves with treatment of the cause.
• Biotin interferes with some immunoassays.
• Calculated free testosterone depends on accurate SHBG and albumin.
• Testosterone therapy, including gels (with transfer risk) and anabolic steroids, suppresses gonadotrophins and spermatogenesis, and is avoided in men seeking fertility.
• Testosterone is not interpreted without LH, FSH and symptoms. Treatment is for symptomatic men with consistently low values only.
• Monitoring of therapy: total testosterone mid-interval for injections or 2 to 8 hours after gels, haematocrit, PSA and lipids at baseline, 3 to 6 months and then yearly.

Special groups.

• Older men: age related decline of about 1% a year, with SHBG rise. Treatment is considered only for symptomatic men with consistently low values.
• Women: the sample is taken in the early follicular phase, and oral contraceptives raise SHBG and lower free testosterone.
• Transgender patients on hormone therapy: target ranges are tied to the gender affirming plan.
• Athletes: testing for doping uses the urinary testosterone to epitestosterone ratio.
• Regions: higher prevalence of obesity related hypogonadism, and HIV-related hypogonadism in high prevalence areas.

Follow up tests. LH and FSH, prolactin, SHBG and calculated free testosterone, oestradiol, DHEA-S, androstenedione, 17-hydroxyprogesterone, TSH, iron studies and ferritin, HbA1c and lipids, semen analysis, karyotype in suspected Klinefelter syndrome, pituitary MRI, bone densitometry, haematocrit, PSA (for men older than 40 considering therapy), and pelvic ultrasound in women.