Salivary testing opens a Pandora's box of issues surrounding accurate measurement of testosterone in women.

M easurement 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 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. 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. 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. 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 hormoneY binding globulin (SHBG), and 33% more weakly bound to albumin, in nonpregnant women. 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. 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. 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. 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