Test Id : TGRP

This is a second-level test for suspected increases or decreases in physiologically active testosterone. The preferred test for assessment of active testosterone is TTBS / Testosterone, Total and Bioavailable, Serum.

Patient's age and sex are required.

Collection Container/Tube: Red top (serum gel/SST are not acceptable)

Submission Container/Tube: Plastic vial

Specimen Volume: 2.5 mL

Collection Instructions: Centrifuge and aliquot serum into plastic vial

• Steroid Pathways

If not ordering electronically, complete, print, and send a General Request (T239) with the specimen.

1 mL

Alternative, second-level test for suspected increases or decreases in physiologically active testosterone:

-Assessment of androgen status in cases with suspected or known sex hormone-binding globulin-binding abnormalities

-Assessment of functional circulating testosterone in early pubertal boys and older men

-Assessment of functional circulating testosterone in women with symptoms or signs of hyperandrogenism but normal total testosterone levels

-Monitoring of testosterone therapy or antiandrogen therapy in older men and in female patients

Testosterone is the major androgenic hormone. It is responsible for the development of the male external genitalia and secondary sexual characteristics. In female patients, its main role is as an estrogen precursor. In both sexes, it also exerts anabolic effects and influences behavior.

In men, testosterone is secreted by the testicular Leydig cells and, to a minor extent, by the adrenal cortex. In premenopausal women, the ovaries are the main source of testosterone with minor contributions by the adrenals and peripheral tissues. After menopause, ovarian testosterone production is significantly diminished. Testosterone production in testes and ovaries is regulated via pituitary-gonadal feedback involving luteinizing hormone (LH) and, to a lesser degree, inhibins and activins. 

Most circulating testosterone is bound to sex hormone-binding globulin (SHBG), which, in men, also is called testosterone-binding globulin. A lesser fraction is albumin bound and a small proportion exists as free hormone. Historically, only free testosterone was thought to be the biologically active component. However, testosterone is weakly bound to serum albumin and dissociates freely in the capillary bed, thereby becoming readily available for tissue uptake. All non-SHBG-bound testosterone is therefore considered bioavailable.

 During childhood, excessive production of testosterone induces premature puberty in boys and masculinization in girls. In women, excess testosterone production results in varying degrees of virilization, including hirsutism, acne, oligo-amenorrhea, or infertility. Mild-to-moderate testosterone elevations are usually asymptomatic in male patients but can cause distressing symptoms in female patients. The exact causes for mild-to-moderate elevations in testosterone often remain obscure. Common causes of pronounced elevations of testosterone include genetic conditions (eg, congenital adrenal hyperplasia); adrenal, testicular, and ovarian tumors; and abuse of testosterone or gonadotrophins by athletes. 

Decreased testosterone in female patients causes subtle symptoms. These may include some decline in libido and nonspecific mood changes. In male patients, it results in partial or complete degrees of hypogonadism. This is characterized by changes in male secondary sexual characteristics and reproductive function. The cause is either primary or secondary/tertiary (pituitary/hypothalamic) testicular failure. In men, there also is a gradual modest but progressive decline in testosterone production starting between the fourth and sixth decades of life. Since this is associated with a simultaneous increase of SHBG levels, bioavailable testosterone may decline more significantly than apparent total testosterone, causing nonspecific symptoms similar to those observed in testosterone deficient women. However, severe hypogonadism, consequent to aging alone, is rare. 

Measurement of total testosterone (TTST / Testosterone, Total, Mass Spectrometry, Serum) is often sufficient for diagnosis, particularly if it is combined with measurements of LH and follicle-stimulating hormone (FSH) (LH / Luteinizing Hormone [LH], Serum and FSH / Follicle-Stimulating Hormone [FSH], Serum). However, these tests may be insufficient for diagnosis of mild abnormalities of testosterone homeostasis, particularly if abnormalities in SHBG (SHBG1 / Sex Hormone-Binding Globulin, Serum) function or levels are present. Additional measurements of free testosterone or bioavailable testosterone are recommended in this situation; bioavailable (TTBS / Testosterone, Total and Bioavailable, Serum) is the preferred assay. 

For more information, see Steroid Pathways

TESTOSTERONE, FREE

Males (adult):

20-<25 years: 5.25-20.7 ng/dL

25-<30 years: 5.05-19.8 ng/dL

30-<35 years: 4.85-19.0 ng/dL

35-<40 years: 4.65-18.1 ng/dL

40-<45 years: 4.46-17.1 ng/dL

45-<50 years: 4.26-16.4 ng/dL

50-<55 years: 4.06-15.6 ng/dL

55-<60 years: 3.87-14.7 ng/dL

60-<65 years: 3.67-13.9 ng/dL

65-<70 years: 3.47-13.0 ng/dL

70-<75 years: 3.28-12.2 ng/dL

75-<80 years: 3.08-11.3 ng/dL

80-<85 years: 2.88-10.5 ng/dL

85-<90 years: 2.69-9.61 ng/dL

90-<95 years: 2.49-8.76 ng/dL

95-100+ years: 2.29-7.91 ng/dL

Males (children):

<1 year: Term infants 

1-15 days: 0.20-3.10 ng/dL*

16 days-1 year: Values decrease gradually from newborn (0.20-3.10 ng/dL) to prepubertal levels

*J Clin Endocrinol Metab 1973;36(6):1132-1142 

1-8 years: <0.13 ng/dL

9 years: <0.13-0.45 ng/dL

10 years: <0.13-1.26 ng/dL

11 years: <0.13-5.52 ng/dL

12 years: <0.13-9.28 ng/dL

13 years: <0.13-12.6 ng/dL

14 years: 0.48-15.3 ng/dL

15 years: 1.62-17.7 ng/dL

16 years: 2.93-19.5 ng/dL

17 years: 4.28-20.9 ng/dL

18 years: 5.40-21.8 ng/dL

19 years: 5.36-21.2 ng/dL

Females (adult):

20-<25 years: <0.13-1.08 ng/dL

25-<30 years: <0.13-1.06 ng/dL

30-<35 years: <0.13-1.03 ng/dL

35-<40 years: <0.13-1.00 ng/dL

40-<45 years: <0.13-0.98 ng/dL

45-<50 years: <0.13-0.95 ng/dL

50-<55 years: <0.13-0.92 ng/dL

55-<60 years: <0.13-0.90 ng/dL

60-<65 years: <0.13-0.87 ng/dL

65-<70 years: <0.13-0.84 ng/dL

70-<75 years: <0.13-0.82 ng/dL

75-<80 years: <0.13-0.79 ng/dL

80-<85 years: <0.13-0.76 ng/dL

85-<90 years: <0.13-0.73 ng/dL

90-<95 years: <0.13-0.71 ng/dL

95-100+ years: <0.13-0.68 ng/dL

Females (children):

<1 year: Term infants 

1-15 days: <0.13-0.25 ng/dL*

16 days-1 year: Values decrease gradually from newborn (<0.13-0.25 ng/dL) to prepubertal levels

*J Clin Endocrinol Metab, 36(6):1132-1142, 1973 

1-4 years: <0.13 ng/dL

5 years: <0.13 ng/dL

6 years: <0.14 ng/dL

7 years: <0.13-0.23 ng/dL

8 years: <0.13-0.34 ng/dL

9 years: <0.13-0.46 ng/dL

10 years: <0.13-0.59 ng/dL

11 years: <0.13-0.72 ng/dL

12 years: <0.13-0.84 ng/dL

13 years: <0.13-0.96 ng/dL

14 years: <0.13-1.06 ng/dL

15-18 years: <0.13-1.09 ng/dL

19 years: <0.13-1.08 ng/dL

TESTOSTERONE, TOTAL

Males

0-5 months: 75-400 ng/dL

6 months-9 years: <7-20 ng/dL

10-11 years: <7-130 ng/dL

12-13 years: <7-800 ng/dL

14 years: <7-1,200 ng/dL

15-16 years: 100-1,200 ng/dL

17-18 years: 300-1,200 ng/dL

> or =19 years: 240-950 ng/dL

Tanner Stages*

I (prepubertal): <7-20

II: 8-66

III: 26-800

IV: 85-1,200

V (young adult): 300-950

Females

0-5 months: 20-80 ng/dL

6 months-9 years: <7-20 ng/dL

10-11 years: <7-44 ng/dL

12-16 years: <7-75 ng/dL

17-18 years: 20-75 ng/dL

> or =19 years: 8-60 ng/dL

Tanner Stages*

I (prepubertal): <7-20

II: <7-47

III: 17-75

IV: 20-75

V (young adult): 12-60

Total testosterone and general interpretation of testosterone abnormalities:

In male patients:

Decreased testosterone levels indicate partial or complete hypogonadism. Serum testosterone levels are usually below the reference range. The cause is either primary or secondary/tertiary (pituitary/hypothalamic) testicular failure. 

Primary testicular failure is associated with increased luteinizing hormone (LH) and follicle-stimulating hormone (FSH) levels, and decreased total, bioavailable, and free testosterone levels. Causes include:

-Genetic causes (eg, Klinefelter syndrome, XXY males)

-Developmental causes (eg, testicular maldescent)

-Testicular trauma or ischemia (eg, testicular torsion, surgical mishap during hernia operations)

-Infections (eg, mumps)

-Autoimmune diseases (eg, autoimmune polyglandular endocrine failure)

-Metabolic disorders (eg, hemochromatosis, liver failure)

-Orchidectomy 

Secondary/tertiary hypogonadism, also known as hypogonadotropic hypogonadism, shows low testosterone and low, or inappropriately "normal," LH/FSH levels; causes include:

-Inherited or developmental disorders of hypothalamus and pituitary (eg, Kallmann syndrome, congenital hypopituitarism)

-Pituitary or hypothalamic tumors

-Hyperprolactinemia of any cause

-Malnutrition or excessive exercise

-Cranial irradiation

-Head trauma

-Medical or recreational drugs (eg, estrogens, gonadotropin releasing hormone [GnRH] analogs, cannabis) 

Increased testosterone levels:

-In prepubertal boys, increased levels of testosterone are seen in precocious puberty. Further workup is necessary to determine the causes of precocious puberty.

-In adult men, testicular or adrenal tumors or androgen abuse might be suspected if testosterone levels exceed the upper limit of the normal range by more than 50%. 

Monitoring of testosterone replacement therapy:

Aim of treatment is normalization of serum testosterone and LH. During treatment with depot-testosterone preparations, trough levels of serum testosterone should still be within the normal range, while peak levels should not be significantly above the normal young adult range. 

Monitoring of antiandrogen therapy:

Aim is usually to suppress testosterone levels to castrate levels or below (no more than 25% of the lower reference range value). 

In female patients:

Decreased testosterone levels may be observed in primary or secondary ovarian failure, analogous to the situation in men, alongside the more prominent changes in female hormone levels. Most women with oophorectomy have a significant decrease in testosterone levels. 

Increased testosterone levels may be seen in:

-Congenital adrenal hyperplasia: non-classical (mild) variants may not present in childhood but during or after puberty. In addition to testosterone, multiple other androgens or androgen precursors are elevated, such as 17-hydroxyprogesterone (OHPG / 17-Hydroxyprogesterone, Serum), often to a greater degree than testosterone.

-Prepubertal girls: analogous to boys, but at lower levels, increased levels of testosterone are seen in precocious puberty.

-Ovarian or adrenal neoplasms: high estrogen values also may be observed, and LH and FSH are low or "normal." Testosterone-producing ovarian or adrenal neoplasms often produce total testosterone values above 200 ng/dL.

-Polycystic ovarian syndrome: hirsutism, acne, menstrual disturbances, insulin resistance and, frequently, obesity, form part of this syndrome. Total testosterone levels may be normal or mildly elevated and uncommonly above 200 ng/dL. 

Monitoring of testosterone replacement therapy:

The efficacy of testosterone replacement in female patients is under study. If it is used, total testosterone levels should be kept within the normal range for females at all times. Bioavailable or free testosterone levels also should be monitored to avoid over treatment.

 Monitoring of antiandrogen therapy:

Antiandrogen therapy is most commonly employed in the management of mild-to-moderate "idiopathic" female hyperandrogenism, as seen in polycystic ovarian syndrome. Total testosterone levels are a relatively crude guideline for therapy and can be misleading. Therefore, bioavailable or free testosterone also should be monitored to ensure treatment adequacy. However, there are no universally agreed biochemical end points and the primary treatment end point is the clinical response.

For more information, see Steroid Pathways.

Free testosterone:

Usually, bioavailable and free testosterone levels parallel the total testosterone levels. However, a number of conditions and medications are known to increase or decrease the sex hormone-binding globulin (SHBG) (SHBG1 / Sex Hormone Binding Globulin, Serum) concentration, which may cause total testosterone concentration to change without necessarily influencing the bioavailable or free testosterone concentration, or vice versa:

-Treatment with corticosteroids and sex steroids (particularly oral conjugated estrogen) can result in changes in SHBG levels and availability of sex-steroid binding sites on SHBG. This may make diagnosis of subtle testosterone abnormalities difficult.

-Inherited abnormalities in SHBG binding

-Liver disease and severe systemic illness

-In pubertal boys and adult men, mild decreases of total testosterone without LH abnormalities can be associated with delayed puberty or mild hypogonadism. In this case, either bioavailable or free testosterone measurements are better indicators of mild hypogonadism than determination of total testosterone levels.

-In polycystic ovarian syndrome and related conditions, there is often significant insulin resistance, which is associated with low SHBG levels. Consequently, bioavailable or free testosterone levels may be more significantly elevated.

Either bioavailable (TTBS / Testosterone, Total and Bioavailable, Serum) or free testosterone (this test) should be used as supplemental tests to total testosterone in the above situations. The correlation coefficient between bioavailable and free testosterone (by equilibrium dialysis) is 0.9606. However, bioavailable testosterone is usually the preferred test, as it more closely reflects total bioactive testosterone, particularly in older men. Older men not only have elevated SHBG levels, but albumin levels also may vary due to coexisting illnesses.

Early-morning testosterone levels in young male individuals are, on average, 50% higher than p.m. levels. Reference values were established using specimens collected in the morning.

Testosterone levels can fluctuate substantially between different days, and sometimes even more rapidly. Assessment of androgen status should be based on more than a single measurement.

1. Manni A, Pardridge WM, Cefalu W, et al: Bioavailability of albumin-bound testosterone. J Clin Endocrinol Metab. 1985 Oct;61(4):705-710. doi: 10.1210/jcem-61-4-705

2. New MI, Josso N: Disorders of gonadal differentiation and congenital adrenal hyperplasia. Endocrinol Metab Clin North Am. 1988 Jun;17(2):339-366

3. Morley JE, Perry HM III: Androgen deficiency in aging men: Role of testosterone replacement therapy. J Lab Clin Med. 2000 May;135(5):370-378. doi: 10.1067/mlc.2000.106455

4. Sizonenko PC, Paunier L: Hormonal changes in puberty III: Correlation of plasma dehydroepiandrosterone, testosterone, FSH and LH with stages of puberty and bone age in normal boys and girls and in patients with Addison's disease or hypogonadism or with premature or late adrenarche. J Clin Endocrinol Metab. 1975 Nov;41(5):894-904. doi: 10.1210/jcem-41-5-894

5. Goudas VT, Dumesic DA: Polycystic ovary syndrome. Endocrinol Metab Clin North Am. 1997 Dec;26(4):893-912

6. Braunstein GD: Androgen insufficiency in women: Summary of critical issues. Fertil Steril. 2002 Apr;77 Suppl 4:S94-S99. doi: 10.1016/s0889-8529(05)70286-3

7. Juul A, Skakkebaek NE: Androgens and the aging male. Hum Reprod Update. 2002 Sep-Oct;8(5):423-433. doi: 10.1093/humupd/8.5.423

8. Hackbarth JS, Hoyne JB, Grebe SK, Singh RJ: Accuracy of calculated free testosterone differs between equations and depends on gender and SHBG concentration. Steroids. 2011 Jan;76(1-2):48-55. doi: 10.1016/j.steroids.2010.08.008

9. Goldman AL, Bhasin S, Wu FCW, et al: A reappraisal of testosterone's binding in circulation: Physiological and clinical implications. Endocr Rev. 2017 Aug;38(4):302-324. doi: 10.1210/er.2017-00025

• Steroid Pathways

Free Testosterone:

This method utilizes equilibrium dialysis to analyze and determine the host serum’s binding capacity for testosterone. Patient sample is placed inside a dialysis well which is immersed in dialysis buffer. The sample is dialyzed. During buffered dialysis, any testosterone which is unbound to sex hormone binding globulin or albumin is free to pass through the semi-permeable dialysis membrane while those testosterone molecules bound to the binding proteins will be held inside the membrane. After dialysis, the buffered dialysate is analyzed for free testosterone by liquid chromatography-tandem mass spectrometry.(Bhasin S, Ozimek N: Optimizing diagnostic accuracy and treatment decisions in men with testosterone deficiency. Endocr Pract. 2021 Dec;27(12):1252-1259. doi: 10.1016/j.eprac.2021.08.002)

Total Testosterone:

Deuterated stable isotope (d3-testosterone) is added to a 0.2-mL serum sample as an internal standard. Protein is precipitated from the mixture by the addition of acetonitrile. The testosterone and internal standard are extracted from the resulting supernatant by an online extraction utilizing high-throughput liquid chromatography. This is followed by conventional liquid chromatography and analysis on a tandem mass spectrometer equipped with a heated nebulizer ion source.(Wang C, Catlin DH, Demers LM, et al: Measurement of total testosterone in adult men: comparison of current laboratory methods versus liquid chromatography-tandem mass spectrometry. J Clin Endocrinol Metab. 2004 Feb;89(2):534-543; Taieb J, Mathian B, Millot F, et al: Testosterone measured by 10 immunoassays and by isotope-dilution gas chromatography-mass spectrometry in sera from 116 men, women, and children. Clin Chem. 2003 Aug;49:1381-1395)

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This test was developed, and its performance characteristics determined by Mayo Clinic in a manner consistent with CLIA requirements. This test has not been cleared or approved by the US Food and Drug Administration.

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