Measurement of Testosterone in Women


Recent studies from the Women’s Health Secrets research group have shown that there is no relationship between testosterone levels throughout the different decades and libido or responses to a sexual function questionnaire in a community-based sample of women. Whether testosterone levels are decreased in women specifically complaining of low libido has not been clearly documented. There is no established level of free testosterone below which a woman can be said to be deficient, nor any level to which a woman should be restored that determines that she is replete. However, the availability of normal ranges for testosterone in different age groups allows the clinician to determine whether an individual’s level is below the normal range. The diagnosis of diminished sexual function due to low testosterone remains a clinical diagnosis of exclusion. In the absence of a reliable free/total testosterone assay the limitations of the available assays should be understood, and the measurement of testosterone used to exclude use of testosterone in women in whom therapy might result in testosterone excess.

Measuring testosterone in women

Measuring testosterone in the blood of women is not straightforward. Most laboratories use assays that were set up to measure testosterone in men. These can be useful for the measurement of elevated testosterone in women who have conditions of androgen excess. However, these assays lack the sensitivity and specificity to accurately measure the much lower values in women who have normal or low testosterone.

Measuring total testosterone

Automated direct total testosterone immunoassays are limited by ‘noise’ from assay interference and by cross-reactivity with other steroids, which becomes worse at low testosterone concentrations and when women are taking steroids such as conjugated equine oestrogens.  The accuracy of an assay can be improved by the inclusion of steps to remove components that cross react, for example, by organic solvent extraction and then isolation of the testosterone by running each sample down a celite column and measuring testosterone in the eluate. This is clearly labour intensive and expensive. Manual testosterone assays may provide greater sensitivity and we have evaluated the accuracy of the Biosource total testosterone assay against other more rigorous methods. This assay was used by the Dorevitch laboratory in the past. The Biosource assay appeared to provide a reliable estimate of testosterone in the low female range and the result could be used to calculate free testosterone. It has been replaced by another assay which gives comparable results. It requires a specific request for ‘sensitive testosterone’, in conjuction with calculated free testosterone. If an accurate assay is not available it is simply not possible to distinguish low testosterone from normal using these assays.

Total testosterone levels alone are not sufficient to evaluate a woman’s androgen exposure. Testosterone circulates in women about 66 per cent bound to sex hormone binding globulin (SHBG) and 33 per cent bound to albumin with only about one per cent being free in the blood. Thus, free or non-SHBG-bound (sometimes called bioavailable) testosterone measures are believed to be the most reliable indicators of tissue testosterone exposure. The measurement of SHBG is not controversial and is relatively simple to perform with good reproducibility.

Measurement of free testosterone

All methods for measuring free testosterone are dependent on having a precise total testosterone assay.

The gold standard methodology for measurement of free testosterone is considered by many investigators to be equilibrium dialysis. In this method, a sample of serum with radiolabelled testosterone added is placed in a chamber and dialysed across a membrane that blocks the transfer of albumin and SHBG. The percentage of free testosterone that is dialysed across the membrane can then be calculated. Absolute free testosterone can be derived from this and total testosterone measurement. This method is influenced by dilution of the hormone to be measured and also relies on being able to measure total testosterone accurately. Furthermore, it is labour intensive and expensive, and not feasible for clinical practice.

Non-SHBG bound testosterone, which correlates highly with free testosterone quantified by equilibrium dialysis, can be measured by the ammonium sulfate precipitation technique. However, frequently encountered sources of error in this assay include incomplete precipitation of globulins, use of impure tritiated testosterone, and insufficient counting time of the relatively small amount of radiolabelled bioavailable testosterone in the assay. Like the equilibrium dialysis, the ammonium sulfate precipitation method generates a percentage of non-SHBG bound testosterone and this percentage is then multiplied by the concentration of total testosterone to determine the bioavailable testosterone concentration.

It is generally recognised that the Sodergard equation can be reliably used to calculate free testosterone if total testosterone, albumin and SHBG are know. Again, this requires a reliable determination of total testosterone, and SHBG and albumin are quantified by routine methodology. See calculated free testosterone above.

Measurement of free testosterone by analogue assays (direct assays) is widely available but notoriously unreliable, particularly at the lower end of the normal female range and is not recommended.

Salivary testosterone has been used reliably in studies of women with hyperandrogenism, but has never gained wide support because the normal range seems excessively large and also has questionable accuracy in the lower ranges. It is important to realise that salivary testosterone levels should not be equated to levels of free testosterone.

The free androgen index (FAI) [nmol/L total testosteronex100/nmol/L SHBG ] has been used as a surrogate for free testosterone, but it is unreliable when SHBG levels are low.

Timing of measurement to prevent misdiagnosis of low testosterone

Ideally blood should be drawn between 8:00am and 10:00am as testosterone levels are usually higher in the early morning and lower in the afternoon. In premenopausal women, testosterone levels are lowest during the first week of the menstrual cycle (i.e. during a woman’s period or the early follicular phase) with small but less significant variation across the rest of the cycle. Thus, blood should be drawn after day eight of the cycle, and preferably before day 20. A serum sample is preferred over plasma.

Measuring other androgens

The chief androgen precursor in the adrenal gland is DHEA. It is usually measured in the sulphated form, DHEAS, because the half-life is much longer, resulting in more stable levels. The immunoassay for DHEAS is relatively stable, gives consistent results, and is simple to perform. Several companies provide kits for this clinical assay, and their performance is generally considered to be acceptable. There is a consensus that DHEAS does not vary in concentration within the various phases of the menstrual cycle, and that it is not bound to SHBG. It also does not seem to be affected by oestrogen therapy at standard doses. A number of authors have shown normal, age-related decline curves for DHEAS, which are all quite compatible. If very low levels are found, a morning cortisol level should be drawn to rule out adrenal insufficiency. This hormone is primarily measured to detect androgen excess in women with polycystic ovarian syndrome. There is no evidence that low DHEAS levels should be supplemented and indeed recent research suggests no benefit on a range of health outcomes with two years of DHEAS therapy in men and women.

Summary of androgen measures

  1. Testosterone levels are of limited usefulness in the assessment of libido or sexual dysfunction in women. Low levels do not necessarily correlate with symptoms. Therefore levels should be performed if suspecting androgen excess, or if a clinician is considering androgen therapy based on clinical assessment and has made a diagnosis of exclusion of as yet poorly defined female androgen insufficiency, to exclude high levels prior to considering any testosterone therapy. Free testosterone and/or non-SHBG bound testosterone are felt to be most reflective of the hormone available to tissues. Free testosterone should be measured by equilibrium dialysis or calculated using the Sodergard equation from total testosterone, SHBG, and albumin. Since total testosterone levels play an important role in determining free testosterone concentrations in those methods, a reliable and sensitive total testosterone assay is essential.
  2. SHBG should always be measured so that the testosterone profile can be accurately interpreted and as a guide to therapy (see 4).
  3. The majority of the direct commercial immunoassays cannot be recommended for reliably determining low total testosterone in women.
  4. SHBG provides additional information regarding overall androgen exposure, as it is sensitive to total body androgen status. Low SHBG levels indicate a considerable increase in risk of androgen excess with testosterone therapy, whereas high levels indicate a reduced metabolic clearance of testosterone. SHBG provides additional information regarding overall androgen exposure, as it is sensitive to total body androgen status. Low SHBG levels indicate a considerable increase in risk of androgen excess with testosterone therapy, whereas high levels indicate a reduced metabolic clearance of testosterone.
  5. For either research or diagnostic purposes, serum for testosterone measurement should be drawn between 8:00am and 10:00am, and not during the early follicular phase in premenopausal women.
  6. In the absence of a reliable free/total testosterone assay, the limitations of the available assays should be understood, and the measurement of testosterone used to exclude women in whom testosterone therapy might be dangerous. Other factors must be used as guides such as a low SHBG indicating increased treatment risk and clinical presentation.
  7. DHEAS measurements are primarily useful in diagnosing androgen excess. Evidence that the use of DHEA in women with even low normal levels is beneficial or safe is lacking and current literature suggests that two years therapy has little benefit but also causes little harm. Further research is needed in this area. DHEAS is not approved for use in Australia and indeed is a banned substance and cannot be imported.

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