Measurement of Testosterone by Immunoassays and Mass Spectrometry in Mouse Serum, Testicular, and Ovarian Extracts

Abstract

Accurate measurement of testosterone is important for reproductive endocrinology research, but the validity of direct (nonextraction) testosterone immunoassays, developed and validated for human serum, has not been appraised for application to mouse serum or steroidogenic tissue extracts. Testosterone was measured in serum and extracts of testis or ovary from male and female wild-type mice by 2 commercial direct testosterone immunoassays, with and without preassay extraction, and by the liquid chromatography, tandem mass spectrometry reference method. Results were compared hierarchically by correlation (Kendall's τ), regression (Passing-Bablok), and deviance (Bland-Altman) analysis, under the null hypothesis of perfect agreement between assays (slope = 1, intercept and deviation = 0). For mouse serum, immunoassays displayed an upward bias with performance better for male vs female sera and, within gender, improved by preassay extraction relative to liquid chromatography, tandem mass spectrometry. Testosterone was detectable in all serum samples, but few (male 54%, female 9%) were accurate (within 20% of the reference measurement). For mouse testis extracts, immunoassays were biased upwards, and preassay extraction improved immunoassay performance. Although testosterone was detectable in all extracts, a minority (45%) was accurate. For mouse ovary extracts, all correlations were poor with severe, upward bias, and while testosterone was detectable in all samples, virtually none were accurate. We conclude that these direct testosterone immunoassay kits provide relatively, but not absolutely, accurate results with male mouse serum and testis extracts but not with female mouse serum and ovary extracts, with performance improved by preassay extraction. Whether relative accuracy is fit for purpose depends on the experimental aims, design, and interpretation.

Currently available murine Leydig cell lines can be applied to study early steps of steroidogenesis but not testosterone synthesis

“…Radioimmuno assays (RIA) and enzymes-linked immunosorbent assays (ELISA) are methods that have been used to detect very low amounts of steroids for decades. However, all steroids share a common backbone and, therefore, it is important to assess the cross-reactivity rates between the key steroids produced by the system that is used in a corresponding experiment [ 24 , 25 , 29 ]. Thus, in the present study, the recovery rates of eight relevant androgenic steroids were tested by applying the T detection EIA kit (Cayman Chemical, Ann Arbor, MI, USA) that has been used in the earlier study reporting T production by BLTK1 cells [20] .…”

“…Many studies have chosen a single steroid as a read-out, mostly T, and using antibody-based quantification methods. Such methods often suffer from limited specificity [ 23 , 24 , 25 , 26 ], and it cannot be excluded that other steroid metabolites might interfere with the read-out due to the inability of antibodies to distinguish between structurally very similar steroid metabolites.…”

Sex hormone-binding globulin regulation of androgen bioactivity in vivo: validation of the free hormone hypothesis

“…The results in SHBG-Tg mice also unambiguously demonstrate the presence of circulating precursor androgens like DHEA, despite lower adrenal sex steroid production in rodents compared to humans 42 43 . Further misinterpretation of rodent endocrinology derives from reliance on immunoassays 30 rather than mass spectrometry to measure very low concentrated analytes 27 28 . Thirdly, we show that even in SHBG-Tg male mice circulating E2 is very low (compared to humans or female mice).…”

“…There is increasing awareness that reliable sex steroid measurements require mass spectrometry rather than immunoassays, not only in humans but also in rodent models 26 27 28 . This is especially the case for E2, the serum level of which has even been reported to be undetectable in male mice in recent mass spectrometry-based studies 26 28 .…”

Fifty years of reproductive biology in Australia: highlights from the 50th Annual Meeting of the Society for Reproductive Biology (SRB)

“…Understanding the HPG axis has allowed both effective treatment of HPG disorders and the proposal of multiple contraceptive strategies, which take advantage of the testosterone negative feedback loop to induce reliable and reversible azoospermia (for a review, see Khourdaji et al 2018). Looking forward, there are concerns about the current state of steroid immunoassays, because increasing drive towards greater throughput of assays has detracted from their reliability, resulting in a loss of accuracy in testosterone measurement in clinical and experimental settings (Handelsman et al 2015), and the wider use of steroid mass spectrometry is needed. Although there are important opportunities for androgen therapy to improve quality of life in certain pathologies that warrant exploration, there is also a large and growing tide of androgen misuse and abuse for medical and cosmetic misapplications respectively that needs to be curbed.…”