By: Alvin M. Matsumoto, MD
University of Washington School of Medicine
VA Puget Sound Health Care System
The androgen, testosterone, is the major sex steroid hormone in males. Testosterone is produced primarily by Leydig cells of the testes, under the stimulatory action of the pituitary gonadotropin, luteinizing hormone (LH) which in turn is stimulated by the brain (hypothalamic) hormone, gonadotropin-releasing hormone (GnRH). Circulating concentrations of testosterone are regulated and maintained within the normal range as a result of negative feedback suppression of LH (and GnRH) secretion by testosterone.
In females, very small amounts of testosterone are produced by the ovaries, adrenal glands and conversion of circulating adrenal androgens, androstenedione and dehydroepiandrosterone (DHEA), to testosterone. Circulating testosterone concentrations in women are 5% to 10% of those in men.
- During fetal life, testosterone production by the fetal testes is necessary for normal male internal and external genital development. In the absence of testosterone production or action, internal and external genitalia develop as female.
- During puberty, brain activation of LH secretion increases testosterone secretion by over 10-fold. The progressive increase in serum concentrations of testosterone and its active metabolites, estradiol and dihydrotestosterone, are required for normal development of secondary sexual characteristics (growth of the penis, male hair pattern, prostate gland and testes, and sperm production), sexual function (increase in libido and erections), and other body changes (increase in muscle mass and strength and bone mineral density) that are characteristic of an adult male.
- During adulthood, normal adult levels of testosterone are needed to maintain male hair pattern, sperm production, sexual function, muscle mass and strength and bone mineral density, as well as vitality and mood.
- With aging, there is a gradual and progressive decline in testosterone levels that are associated with age-related decline in sexual function, muscle mass and strength, bone mineral density, vitality and mood. However, these changes are also associated with age-associated chronic illnesses and medication use and physiological significance of the decrease in serum testosterone levels with aging is not clear at present.
- In women, the physiological importance of testosterone is also not clear. Testosterone is thought to be important for the maintenance of sexual function (libido) and perhaps bone mineral density and mood in women, but this has not been studied very well.
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What happens if testosterone levels are too low or too high?
- In male neonates, exposure to inadequate amounts of testosterone during fetal development or those with genetic defects in testosterone action on tissues (androgen resistance syndromes) results in varying degrees of ambiguous genitalia (from phenotypic female to nearly-normal male genitalia).
- In female neonates, excessive production of testosterone and other androgens by the adrenal glands or exposure to excessive amounts of testosterone or other androgens in utero results in varying degrees of masculinization and ambiguous genitalia.
- During puberty in boys, lack of hypothalamic activation of LH secretion or testicular disorders resulting in inadequate testosterone production results in significantly, chronologically delayed puberty, characterized by poor male sexual development or eunuchoidism.
- In boys, premature hypothalamic activation of LH secretion, gonadotropin-independent testosterone production by the testes, adrenals or tumors or exposure to excessive amounts of testosterone or other androgens results in precocious puberty.
- In girls, testosterone production by the ovaries, adrenals or tumors, or exposure to excessive amounts of testosterone or other androgens results in varying degrees of virilization.
- In men, inadequate testosterone production results in the androgen deficiency syndrome or male hypogonadism and may be due either to disorders of the testes (primary hypogonadism), or the hypothalamus and/or pituitary gland (secondary hypogonadism).
- In women, inadequate testosterone and other androgen production may cause an androgen deficiency syndrome, manifested primarily by sexual dysfunction, particularly in women with bilateral ovariectomy, primary adrenal insufficiency and hypopituitarism.
- In men, excessive production of testosterone or other androgens by the testes, adrenals or tumors or exposure to excessive amounts of testosterone or other androgens, suppresses sperm production and may cause infertility.
- In women, excessive testosterone or other androgen production by the ovaries, adrenals or tumors, or exposure to excessive amounts of testosterone or other androgens results in varying degrees of hyperandrogenism or frank virilization (acne, hirsutism, oligo/amenorrhea, alopecia or clitoromegaly).
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How is testosterone status determined?
(note – for more information, please see the TESTS page)
In circulation, testosterone is mostly bound to plasma proteins. Testosterone is weakly-bound to albumin and tightly-bound to sex hormone-binding globulin (SHBG), with only 0.5% to 3% of testosterone unbound to plasma proteins, referred to as free testosterone.
Total testosterone assays measure both free and protein (albumin and SHBG)-bound testosterone. According to the free hormone hypothesis, only free testosterone is available to all tissues for action and therefore, is biologically active, while testosterone tightly bound to SHBG is not available to tissues for action. Because testosterone is weakly-bound to albumin, albumin-bound testosterone may be available to some tissues for action (such as liver and brain). Together, albumin-bound (or weakly-bound) testosterone plus free testosterone is referred to as bioavailable testosterone.
- Total testosterone assays. Testosterone status is usually determined initially by measurement of serum total testosterone concentrations because it is the most widely available to clinicians in local clinical laboratories. In most local clinical laboratories, total testosterone levels are measured by an automated platform-based immunoassay. In most reference and some research laboratories, total testosterone levels are measured by more sensitive, specific, reliable and accurate mass spectrometry-based assays. For a number of reasons (e.g., high instrumentation and reagent costs, lack of technical expertise, inability to completely automate the assay), mass spectrometry-based testosterone assays have not been adopted in most local clinical laboratories. However, it is likely that they will be used more frequently in the future.
- Free testosterone assays. The gold standard method for measuring free testosterone is by equilibrium dialysis or centrifugal ultrafiltration. However, these methods are generally time-consuming and tedious, not fully automated and may require radioactivity or a very sensitive testosterone assay, so are available only in reference and research laboratories. Free testosterone may be calculated by measuring total testosterone and SHBG levels and using various formulae that assume specific affinity constants for SHBG to calculate free testosterone; use of measured or assumed normal albumin concentrations does not significantly affect calculated free testosterone estimates. Calculated free testosterone levels have been shown to provide clinically reasonable estimates of free testosterone by equilibrium dialysis, except when SHBG concentrations are very high (e.g. in pregnant women) and are available in most reference and some local clinical laboratories. Direct free testosterone measurements using testosterone analog/platform-based immunoassays have been shown to be inaccurate and should not be used for assessment of free testosterone levels.
- Bioavailable testosterone assays. Bioavailable testosterone levels may be measured by ammonium sulfate precipitation of SHBG-bound testosterone or calculated by measuring total testosterone and SHBG levels. These are usually only performed in reference or research laboratories.
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What should you know about testosterone testing?
The clinical use of serum testosterone assays for biochemical assessment of a patient’s testosterone status requires appreciation of several issues that may affect testosterone levels and their interpretation.
- Assay variability. It is essential for practitioners to use the most accurate, precise and standardized assays available in order to make clinical decisions (see Testosterone Test Page). Practitioners should be aware that the accuracy and reference ranges for various testosterone assays differ considerably. For example, a recent comparison of testosterone levels measured in the same external quality control sample using several different assays reported concentrations ranging from 160 ng/dL to 508 ng/dL. This astounding degree of variability in assays is the reason that PATH and the CDC focused their initial efforts on developing an accuracy-based quality control (Hormone Standardization, HoSt) program for testosterone assays.
- Biological variability. In same man, there is considerable day-to-day variability in testosterone levels, such that 30% to 35% of men who are found to have a low testosterone level an initial measurement may have a normal testosterone level on repeat testing. Therefore, it is important that testosterone levels be measured on more than one occasion to confirm an abnormal value. In addition to the day-to-day variability, serum testosterone levels in men exhibit a circadian variability with highest values in the morning (blunted in older men), supporting the recommendation that for evaluation male hypogonadism, serum T levels be measured preferably in the morning. More recent data suggest that testosterone levels may be suppressed by food intake, so fasting measurements may be appropriate depending on the clinical situation.
- Alterations in SHBG levels. Clinical conditions or medications may decrease or increase SHBG concentrations, potentially resulting low or high total testosterone levels in the presence of normal free T levels. Conditions that lower SHBG and may potentially lower total testosterone levels include: moderate obesity, type 2 diabetes mellitus, glucocorticoids, androgens, progestins, nephrotic syndrome, hypothyroidism and acromegaly. Conditions that increase SHBG and may potentially increase total testosterone levels are: advanced age, anticonvulsants, estrogens (pregnancy), liver disease (hepatitis or cirrhosis), hyperthyroidism and HIV disease. If conditions or medications that alter SHBG levels are present or suspected in a patient or if total testosterone levels are found to be only slightly abnormal, it is recommended that an accurate measurement of free testosterone (e.g. free testosterone by equilibrium dialysis or calculated free testosterone) be used to assess testosterone status.
- Transient suppression of T levels. Serum testosterone levels may be suppressed transiently during and for some time after an acute illness or a period of nutritional deficiency, or while taking certain medications, such as opioids or glucocorticoids. Therefore, it is important not to measure testosterone levels until a patient has recovered completely from a recent illness or period of nutritional deficiency and an offending medication has been discontinued.