Testosterone Testing

T is the prototypical male hormone, though it is also produced in females. T is the most abundant androgen secreted by the male gonads. In females, 25% of T is produced in the ovaries, 25% in the adrenal glands, and 50% in the peripheral conversion of androgen precursors. T is metabolized to dihydrotestosterone (DHT) and estradiol (E₂) in both males and females. The intracellular effects of T and DHT are mediated by the androgen receptor (AR); the effects of E₂ are mediated by the estrogen receptor (ER).

T circulates in 3 forms: sex hormone-binding globulin (SHBG)-bound T, albumin-bound T, and free T (FT).

• SHBG-bound T accounts for ~44% of circulating T and is not bioavailable (ie, does not reach AR in the target cell) because of high-affinity binding between SHBG and T.
• Albumin-bound T accounts for ~54% of circulating T and is thought to become biologically active when albumin and T dissociate.
• FT, or unbound T, accounts for 1% to 4% of circulating T; FT directly accesses the AR in the target cell, resulting in androgenic effects. 

In clinical practice, 3 forms of T are measured: total T (TT), free T (FT), and bioavailable T. The reference method for measuring TT endorsed by the Endocrine Society is liquid chromatography–tandem mass spectrometry (LC-MS/MS).¹ Direct measurement of FT and bioavailable T is not available, so concentrations are estimated from calculations.

Quest Diagnostics offers the following measurements:

• TT (by LC-MS/MS, test code 15983) includes sex-hormone bound globulin (SHBG)-bound T, albumin-bound T, and FT.
• FT (test code 36170) is calculated by equilibrium dialysis using a modified Vermeulen equation.²
• Bioavailable T (test code 14966) is TT minus SHBG-bound T. Bioavailable T is calculated using the Sodergard equation.³

Males 

During the initial weeks of gestation of a male fetus, the testes release hormones such as T, dihydrotestosterone (DHT), anti-Müllerian hormone (AMH), and the insulin-like factor 3 (INSL3), which lead to fetal virilization, penile development, and testicular descent. Lack of T at this development phase is associated with ambiguous genitalia, which physicians categorize as complete, intermediate, or mild.  

During puberty, T increases and induces further virilization of the body, development of the skeleton and muscles, growth of the larynx followed by deepening of the voice, development of pattern hair loss with receding hairline in the temporal area, full maturation of the sexual organs, and development of sex drive and reproductive functions. The absence of T at this development phase is called prepubertal hypogonadism and is associated with delayed puberty. The patient appears younger than his age and has a small phallus, small testes, a high-pitched voice, and an underdeveloped muscular apparatus. 

During adulthood, T is the primary androgen responsible in men for maintaining muscle and bone mass, strength, fat distribution, sex drive, and sperm production. Lack of androgens in adulthood is associated with gradually decreasing energy, libido, and erectile function. Typically, primary and secondary sexual characteristics do not regress to pre-pubertal levels.

Human illnesses associated with high testosterone in males are very rare; examples of such illnesses include gonadotroph pituitary adenomas and tumors secreting human chorionic gonadotropin (HCG). If they arise before puberty, these conditions are associated with premature puberty. If they arise after puberty, the clinical presentation ranges from minimal manifestations to azoospermia, gynecomastia, and polycythemia.  

Females

The role of T in regulating reproductive and non-reproductive functions in females is only partially understood. However, it is widely recognized that T in females provides the substrate for estrogen synthesis and may play a role in regulating follicular development.⁴

Measuring total T (TT) by liquid chromatography-tandem mass spectrometry (LC-MS/MS) in females is the most useful laboratory test for evaluating polycystic ovary syndrome (PCOS), a common endocrine condition in women of reproductive age. High TT in PCOS is associated with virilizing symptoms, such as acne, hair loss/alopecia, increased body or facial hair, hypertension, irregular or absent menstruation, and insulin resistance. TT levels >150 ng/dL should prompt investigations to rule out the presence of a virilizing tumor of the ovaries or adrenals, which are typically associated with extreme symptoms of virilization, including clitoromegaly, cystic acne, and hirsutism.

Medical conditions associated with low androgen levels are well established and include premature ovarian insufficiency, bilateral oophorectomy, panhypopituitarism, primary adrenal insufficiency, use of oral contraceptives or glucocorticoids, anorexia nervosa, and hypoactive sexual desire disorder (HSDD). However, guidelines published by the Endocrine Society^5,6 in 2014 and 2019 recommend against diagnosing androgen deficiency in healthy women because the syndrome is not well-defined; symptoms are not well characterized; serum androgen levels are not independent predictors of sexual function in women^5,7; and age-based, standardized  low T cutoffs for women are not available.⁵

The US Endocrine Society recommends using LC-MS/MS (test codes 15983) to measure TT.¹ Historically, immune assay (IA) platforms have been the method of choice for measuring serum TT. IA (test code 19958) produces reliable results for normal or elevated levels of TT but lacks sensitivity, specificity, and precision at lower concentrations. Thus, IA is not recommended for testing women, children, transgender women, and hypogonadal men^8-10; in these patients, TT should be measured only using LC-MS/MS. 

FT measurement is helpful when TT concentration is near the decision level or when perturbations in sex hormone-binding globulin (SHBG) are likely (Table 1). The most precise FT assay requires liquid chromatography-tandem mass spectrometry (LC-MS/MS) and equilibrium-based dialysis methods (test code 36170).

The population defining the TT reference range, and thus the T-deficiency cutoff, has been debated.¹¹ The Quest Diagnostics reference range is based on the distribution of results for a healthy population of men up to age 90, obtained using our TT liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay (test codes 15983). The Quest reference range for adult men (age 18 and over) has a lower limit of 250 ng/dL, which is based on the 2.5th percentile of a distribution of results.

For healthy men older than 18, the Endocrine Society recommends using a Centers for Disease Control and Prevention Hormone Standardization Program (CDC HoSt)-certified TT.¹² The Quest assay is certified by the CDC HoSt program, has a reportable range of 1.0 to 10,000 ng/dL, and has no cross-reactivity with 30 testosterone-related compounds.

In adult women, circulating levels of TT are between 2 and 45 ng/dL, with a modest midcycle rise, when measured using the Quest LC-MS/MS assay (test code 15983). Immunoassay is highly unreliable with the low TT concentrations present in women and should not be used.¹³ According to many studies,^14,15 T declines progressively across reproductive years in adult women, with no changes occurring during menopause. 

Table 2 lists the most relevant clinical uses for T testing in males, females, and transgender patients. Test selection and interpretation, diagnosis, and patient management decisions should be based on the physician’s education, clinical expertise, and assessment of the patient.^1,5,10,16

Men

For men started on TRT, the Endocrine Society guidelines of 2018 recommend measuring total testosterone (TT), hematocrit, and prostate-specific antigen (PSA) after 3 and 12 months of therapy.¹ A urological consultation should be obtained for the presence of an unevaluated prostate nodule if PSA increases >1.4 ng/mL within 12 months of initiating TRT, if total PSA is >4 ng/mL, or if >3 ng/mL and there is high risk for prostate cancer (ie, with positive family history for prostate cancer).¹ All patients should be engaged in decisions to monitor for prostate cancer according to the guidelines of the American Urology Association (AUA)¹⁷ and Endocrine Society.¹⁸ Serum T should be measured midway between injections when using intramuscular (IM) T esters (depot) or 2 hours after gel application. Most clinicians target 600 ng/dL as an optimal midcycle level of TT.¹

Whether TRT causes increased cardiovascular (CV) risk has been controversial. According to the 2018 guidelines,¹ TRT should be discontinued in all patients with severe congestive heart failure or who have developed a CV event during TRT. TRT should not be initiated if a CV event has occurred within the last 6 months or if a patient has a history of breast cancer, metastatic prostate cancer, or severe lower urinary tract symptoms (LUTS) score >19, as indicated by the American Urological Association (AUA)/International Prostate Symptom Score (AUA/IPS). However, the Transverse Trial published in June 2023 suggests TRT is safe from a CV point of view, for at least up to 2 years of treatment.¹⁹

According to the 2018 guidelines,¹ the physician should oversee handling other potential medical situations associated with TRT, including erythrocytosis, osteoporosis, suppression of spermatogenesis, thromboembolic events, sleep apnea, and potential transfer of transdermal T to women and children.

Women

Guidelines endorse the use of TRT only in women with hypoactive sexual desire disorder (HSDD).^5,6 No TT concentration correlates with HSDD or its severity⁷; however, research and clinical evidence support a positive effect of T therapy on sexual desire when premenopausal physiologic levels are maintained.²⁰ There is not a T preparation specifically approved for women in the US. Compounded oral preparations are not recommended. Before initiation of therapy, patients should receive informed consent, and shared decision-making should involve a comprehensive discussion of off-label use of male T preparations.¹³  

For women with HSDD, TT levels by liquid chromatography-tandem mass spectrometry (LC-MS/MS) should be monitored every 3 months, with a target level in the high-normal range for premenopausal women. If a supraphysiologic concentration of TT is reached, the most frequent side effects include acne, hirsutism, and androgenic alopecia, and the dose of T should be decreased. If there is no clinical response at the end of the 6 months, TRT should be suspended.

An important limitation of T use in females is the lack of safety data beyond 24 months.

Transgender men

Once a multidisciplinary group has established a diagnosis of gender dysphoria, IM T therapy is usually initiated at lower doses owing to the smaller size of transgender men. Levels are monitored with TT measurement midway through the T cycle (that is, on the seventh day if the cycle is 14 days). Target TT levels are within the mid-normal male range (600 ng/dL), and measurements are done every 3 months for the first year and 1 to 2 times per year thereafter.²¹ Monitoring of E₂ should be requested until there has been no uterine bleeding for 6 months and the desired level of suppression is <50 pg/mL. Clinicians should request routine tests to rule out the development of erythrocytosis as with cisgender males.

Transgender women

Once a multidisciplinary group has established a diagnosis of gender dysphoria, treatment consists of various formulations of 17beta estradiol (preferentially parenteral or transdermal) to obtain serum levels of around 200 pg/mL and suppression of T to the normal female range.²¹

Monitoring is initially done every 3 months for the first year and 1 to 2 times per year thereafter, and the dose is adjusted for supraphysiologic levels of E₂ or insufficient suppression of T.