Measurement of hormone levels by salivary testing is currently in vogue, yet for now, the enthusiasm to embrace this approach far exceeds its capacity to reliably achieve precise and accurate estimates of testosterone at low levels. In this issue of Menopause, Flyckt et al,(1) directly compare measurement of testosterone levels in postmenopausal women using salivary versus serum testing. In doing so, they open a virtual Pandora’s box of issues surrounding measurement of testosterone in general and particularly at low levels, such as those seen in women and children.
Salivary testing of hormones is a valid approach, effectively representing an ultrafiltrate of blood, and has the benefits of being less invasive and potentially less expensive than the serum approach. Salivary cortisol, for example, has been described as having high diagnostic accuracy for the screening of Cushing syndrome and has been recommended in clinical practice guidelines for screening for this condition.(2)
However, as Flyckt et al report, the current technology is such that the accuracy of
testosterone levels achieved by the salivary method is questionable in individuals whose actual testosterone levels are low.
The authors directly compare salivary versus serum testosterone levels in 56 postmenopausal women who provided multiple samples for hormone analysis both before and during treatment with a 300-Kg transdermal testosterone patch versus placebo.
Direct correlation between the methods was poor, although improved somewhat after log transformation of non-normally distributed data. The authors concede that the very low levels of testosterone in their study sample of postmenopausal women were likely to provide a challenge for the salivary approach to measurement. The other issue is that different immunoassays were used to compare testosterone levels as collected by salivary versus the serum approach; others have reported the variation between results from testosterone immunoassays to be broad.(3) The most concerning aspect of the findings from this
study, however, was that there was no increase in testosterone levels 12, 24, or 52 weeks after testosterone treatment, as measured by the salivary approach, even though posttreatment levels were seen to be in the physiological range for premenopausal women as per serum analysis. There was also no consistent difference between salivary testosterone levels in testosterone-treated women at any time point versus those on placebo, and although a potential explanation for these findings is a lack of sensitivity of the salivary testosterone assay used in
this study at low levels of testosterone, the findings stimulate further debate about the best approach to testosterone measurement when levels are likely to be low.
This article serves as a timely reminder that accurate assessment of testosterone and other sex steroid hormone levels is challenging. Pathology laboratories in general invest in expensive biochemical analyzers that effectively deliver a commercially viable approach to biochemical analysis by having the capacity to measure many different biochemical analytes in the one automated machine, hence saving time, money, and staff costs. However, measurement of sex steroid hormones is biochemically a complicated process, and the ability of automated
systems to accurately measure low levels of sex steroids with reliability is limited.(4)
Sex steroids are lipid soluble and protein bound, and there are issues with crossreactivity, whereby similar sex steroid hormones, such as testosterone and dihydrotestosterone, for example, may both be detected by a particular assay as being Btestosterone, hence leading to inaccurate estimates of actual testosterone levels.
Testosterone is highly protein bound, 66% to sex hormone binding globulin (SHBG), and 33% more weakly bound to albumin, in nonpregnant women.(5) The debate continues as to which is the active moiety of testosterone, which some consider to be free testosterone, only comprising 1% to 2% of circulating total testosterone in women, whereby others consider Bbioavailable testosterone to be the combination of free and albumin-bound testosterone.(6) Regardless of which is the active form of testosterone, the issue of protein binding is important because the protein needs to be removed from the testosterone before analysis of levels, adding a further step to the process and hence further potential for error. Finally, it is
common for commercial laboratories to use radioimmunoassays to assess testosterone levels. Radioimmunoassays do not represent the criterion standard technique for testosterone measurement; however, they are cheaper, more easily accessible, and therefore more clinically relevant for assessment of testosterone levels outside the research setting. Various radioimmunoassays have been validated against criterion standard techniques and have performed variably.(3,7,8) Radioimmunoassays for testosterone involve tubes internally coated with antibodies to testosterone bound to radiolabeled testosterone, and serum testosterone added to this environment displaces the radiolabeled testosterone, with measurement of radioactivity from the displaced testosterone then equating to serum testosterone levels. Issues with radioimmunoassays involve quality and affinity of antibodies for testosterone and limited performance at extremes of SHBG.(9,10)
An additional issue in women is that these assays were in general designed to perform at the 10-fold higher levels of testosterone seen in men and, therefore, are limited in the accurate assessment of low levels of testosterone such as those in women and children.(12)
An additional concern with measurement of testosterone levels in women is how best to estimate levels of free testosterone, considering this is thought to be the active testosterone moiety.
Direct assays for free testosterone estimation are extremely limited in terms of delivering an accurate and precise result, mainly due to the very small circulating levels of free testosterone present, especially in women. Calculated free testosterone levels, based on various techniques, have been found to correlate well
with criterion standard techniques of measuring free testosterone, (13) and some use the free testosterone index (total testosterone/SHBG) as a substitute for absolute free testosterone levels. Flyckt et al(1) have approached the measurement of serum free testosterone in their study in this issue of Menopause using equilibrium dialysis, considered to be the criterion standard technique for assessment of free testosterone, hence reinforcing that their approach to serum analysis was robust.
Aside from the issues surrounding the challenges associated with testosterone measurement, important physiological considerations exist with regard to endogenous testosterone in women. Androgen levels decline with age, even from young reproductive age14; testosterone levels are at least 50% lower after oophorectomy (14,15) and are affected by hormonal treatments such as the oral contraceptive pill and postmenopausal hormone therapy.(16,17) Testosterone undergoes both diurnal(18) and menstrual cycle variation,19,20 and blood sampling should be performed in the morning and, for cycling women, in the midphase to late follicular phase of the cycle. Given all of these collective challenges, it is essential to approach measurement of testosterone levels in the community with the most accurate, precise, cost-effective, and valid approach that will enable diagnosis of conditions characterized by testosterone deficiency and excess and be able to ensure that testosterone treatment is targeted to achieve the physiological range.
In this issue of Menopause, Flyckt et al(1) have directly compared testosterone levels measured after salivary collection, as compared with those from serum. In an area of bio chemistry that is already fraught with challenges, the salivary
approach to measurement of testosterone at low levels has a number of issues to be dealt with before this approach can be routinely recommended outside the research setting.
Financial disclosure/conflicts of interest: Dr. Davison has received research grant support from Acrux.
Sonia Davison, MBBS, FRACP, PhD
Women’s Health Program
Department of Medicine
Monash University
Alfred Hospital
Victoria, Australia
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EDITORIAL