Salivary Testosterone Measurements Research Articles

Assessment of exercise-induced stress via automated measurement of salivary cortisol concentrations and the testosterone-to-cortisol ratio: a preliminary study

In this study, our aim was to validate whether the automated measurement of salivary testosterone and cortisol concentrations and the testosterone-to-cortisol (T/C) ratio, considering their individual circadian rhythms can be used to assess the stress response of male athletes to different exercise intensities accurately and effectively. We measured the salivary testosterone and cortisol concentrations and their respective serum concentrations that were collected from 20 male long-distance runners via passive drooling in the morning and evening for two consecutive days involving different exercise intensities. An electrochemiluminescence immunoassay was performed to evaluate the salivary testosterone and cortisol concentrations. The results showed a positive correlation between the salivary testosterone and cortisol concentrations and their respective serum concentrations. The participants were divided into two groups: with and without interval training. The interval training group showed a significantly higher rate of change in the salivary cortisol concentration and a significantly lower rate of change in the T/C ratio in the evening interval training on day 1 than lower-intensity running on day 2. Our results indicated that the salivary cortisol concentrations and the T/C ratio could distinguish between exercises at different intensities, which may be beneficial for detecting differences in stress responses among athletes.

Measurement of salivary testosterone in adolescents and young men with Duchenne muscular dystrophy

BackgroundMany young adults with Duchenne Muscular Dystrophy (DMD) receive long-term glucocorticoids (GC). GC can cause hypogonadotrophic hypogonadism and adolescents may therefore be candidates for pubertal induction. It is unclear whether men with DMD on or off GC have age-appropriate endogenous testosterone production.MethodsWe undertook a quality improvement project to assess the feasibility of measuring salivary testosterone (SalT) levels in men with DMD at home. A Sal-T sampling kit was sent by post to all patients with DMD, aged 17 and older, registered at the John Walton Muscular Centre in Newcastle (n = 75). Submitted Sal-T samples were collected and submitted for analysis.ResultsTwenty-eight out of seventy-five patients returned samples (age range: 17–34 years). 6/28 samples were unsuitable for analysis. Overall Sal-T levels (n = 22) were significantly lower than in the healthy population (178 ± 107 v 287 ± 109 pmol/l, p = 0.0001). Sal-T was lower in those on GC compared to those off GC (144 ± 81 versus 218 ± 125 pmol/l, p = 0.05). Three patients were unable to collect a sample due to ventilator dependence.ConclusionSal T can provide information about androgen status in DMD patients at home, overcoming barriers such as mobility difficulties and challenging venepuncture. However we only obtained samples in a minority of patients suggesting that Sal-T measurement may not be appropriate or acceptable to everyone. There needs to be a more detailed exploration of the barriers to sample submission.

Salivary testosterone may not serve as a screening test in the diagnosis of biochemical hyperandrogenism.

The diagnosis of biochemical hyperandrogenism is still challenging because a set of appropriate, recommended diagnostic tests has not been established. In our study, we aimed to answer the question of whether salivary testosterone is a reliable test to establish the diagnosis of biochemical hyperandrogenism as compared to serum total testosterone (TT) measured either by liquid chromatography-tandem mass spectrometry (LC-MS/MS) or immunoassay and to assess which set of biochemical tests would be the most appropriate for the identification of biochemical hyperandrogenism. A total of 39 women, aged 18-45 years, with clinical or biochemical hyperandrogenism and 41 healthy individuals, aged 19-45 years, were enrolled in the study. Salivary testosterone was measured using the Salimetrics test. Serum TT was measured either using the LC-MS/MS method or immunoassay, and dehydroepiandrosterone sulphate (DHEA-S) and androstenedione were measured using LC-MS/MS. In 15 of 17 (88%) patients with elevated serum TT measured by LC-MS/MS and in 14 of 16 (87%) measured with immunoassay, salivary testosterone showed normal levels. In 11 of 39 women (28%) with normal serum testosterone levels, DHEA-S was elevated. All patients with elevated androstenedione presented with an elevated concentration of either serum testosterone or DHEA-S. Salivary testosterone measurement may lead to the underdiagnosis of biochemical hyperandrogenism. Both serum testosterone and DHEA-S should be measured in the endocrine work-up toward biochemical hyperandrogenism.

Using salivary testosterone measurements to assess androgen deficiency in adults with Duchenne muscular dystrophy (DMD)

Using salivary testosterone measurements to assess androgen deficiency in adults with Duchenne muscular dystrophy (DMD)

Salivary testosterone measurement in women with and without polycystic ovary syndrome

Clinical and/or biochemical hyperandrogenism is one of the diagnostic criteria for PCOS. An evaluation of the role of salivary testosterone (salT) and androstenedione (salA) for the diagnosis of PCOS was undertaken in a cross sectional study involving 65 women without PCOS and 110 women with PCOS fulfilling all 3 diagnostic Rotterdam criteria. Serum and salivary androgen measurements were determined by LC-MS/MS. salT and salA were significantly elevated in PCOS compared to controls (P < 001). No androgen marker was more predictive than another using ROC curves, but multiple logistic regression suggested salT was more predictive than free androgen index (FAI) (p < 0.01). The combination of salT or FAI identified 100% of PCOS women. PCOS women with both biochemical and clinical hyperandrogenism as opposed to clinical hyperandrogenism alone showed a metabolic phenotype (p < 0.05) and insulin resistance (p < 0.001). PCOS patients with an isolated elevated FAI showed increased insulin resistance compared to those with an isolated salT (P < 0.05). salT appeared to be at least as predictive as FAI for the diagnosis of the classical PCOS phenotype, and the combination of salT or FAI identified 100% of PCOS patients. This suggests that salT measurement by LC-MS/MS holds the promise of complementing existing laboratory tests as a means of assessing hyperandrogenemia.

Distribution of Salivary Testosterone in Men and Women in a British General Population-Based Sample: The Third National Survey of Sexual Attitudes and Lifestyles (Natsal-3).

Introduction:Measurement of salivary testosterone (Sal-T) to assess androgen status offers important potential advantages in epidemiological research. The utility of the method depends on the interpretation of the results against robustly determined population distributions, which are currently lacking.Aim:To determine age-specific Sal-T population distributions for men and women.Methods:Morning saliva samples were obtained from participants in the third National Survey of Sexual Attitudes and Lifestyles, a probability sample survey of the British general population. Sal-T was measured using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Linear and quantile regression analyses were used to determine the age-specific 2.5th and 97.5th percentiles for the general population (1675 men and 2453 women) and the population with health exclusions (1145 men and 1276 women).Results:In the general population, the mean Sal-T level in men decreased from 322.6 pmol/L at 18 years of age to 153.9 pmol/L at 69 years of age. In women, the decrease in the geometric mean Sal-T level was from 39.8 pmol/L at 18 years of age to 19.5 pmol/L at 74 years of age. The annual decrease varied with age, with an average of 1.0% to 1.4% in men and 1.3% to 1.5% in women. For women, the 2.5th percentile fell below the detection limit (<6.5 pmol/L) from age 52 years onward. The mean Sal-T level was approximately 6 times greater in men than in women, and this remained constant over the age range. The Sal-T level was lowest for men and highest for women in the summer. The results were similar for the general population with exclusions.Conclusions:To our knowledge, this is the first study to describe the sex- and age-specific distributions for Sal-T in a large representative population using a specific and sensitive LC-MS/MS technique. The present data can inform future population research by facilitating the interpretation of Sal-T results as a marker of androgen status.

Salivary Testosterone and Cortisol Measurement in Sports Medicine: a Narrative Review and User's Guide for Researchers and Practitioners.

Since the initial reporting of salivary hormone measurements in marathon runners in the early 1980s, the practice of utilizing salivary testosterone (T) and cortisol (C) to reflect acute and rhythmic changes to their systemic counterparts has gained considerable momentum. However, substantial variability exists between studies with respect to methodological protocols, laboratory techniques, and interpretation of study findings. These differences can directly influence the salivary hormone values, thus hampering interpretation, limiting cross-study comparison, and constraining the generalizability of individual study findings. This article examines the current body of literature before proposing a sequence of practical guidelines to minimize sample variability in salivary hormone research. The guidelines are grouped into 3 major categories that limit comparison between studies; A) study design, B) sample acquisition and biological variation, and C) technical and analytical error. To achieve this, the present article critically appraises research employing salivary T and C measurements, identifies potential sources of error before proposing appropriate methodological considerations for researchers and practitioners wishing to obtain T and C measurement from saliva.

Sex hormone-binding globulin has no effect on salivary testosterone.

We would like to follow up our 2014 publication in this journal by submitting further validation data to support the measurement of salivary testosterone (T), highlighting its independence from variations in circulating sex hormone-binding globulin (SHBG), unlike serum total T which is positively correlated with SHBG. All analyses were performed as previously described. In addition, further samples were collected from, 48 males and 38 females, healthy volunteers (mean age 32 y, range 18–66). Ethical committee approval was granted from Ghent University Hospital. These samples were analysed for salivary T, total T and equilibrium dialysis using liquid chromatography-tandem mass spectrometry. Figure 1 shows that: (i) serum total T is significantly and positively correlated with SHBG in both men (n1⁄4 93) and women (n1⁄4 86); (ii) serum calculated free T is not correlated with SHBG in men but negatively correlated with SHBG in women; (iii) salivary T is not correlated with SHBG in either men or women; (iv) serum-free T measured using equilibrium dialysis is not correlated with SHBG in men (n1⁄4 48) but, like calculated free T, is inversely correlated with SHBG in women (n1⁄4 38). These results demonstrate that salivary T is not influenced by variations in serum SHBG, unlike the analogue serum free T direct assay. The sexually dimorphic relationship between free T, whether calculated or measured by equilibrium dialysis, and SHBG, is an interesting observation which, to the best of our knowledge, has not been previously reported.

88 - Consequences of bilateral cryptorchidism in adults

Summary Bilateral cryptorchidism treatment results are often shadowed by the majority of unilateral cases. We report the long-term follow-up results of boys treated for bilateral cryptorchidism during childhood. Patients treated in two main paediatric surgery centres were selected from medical registries and invited for a clinical examination including scrotal ultrasound, salivary testosterone measurement and a semen sample. Thirty-six men (38.3%) replied to the written invitation, and 21 agreed to be examined. The mean age at orchidopexy was 74 months (range 24–138). Sperm count was 0.42 × 106 (SD ± 0.64 × 106) ml−1. The correlation between total testicular volume and total sperm count was statistically significant (r = 0.481; P = 0.032). These results show that surgical treatment of bilateral cryptorchidism after the age of 2 years does not prevent infertility. Sperm count and endocrine evaluation advocated after the treatment of bilateral cryptorchidism in all adult patients.

A comparison of salivary testosterone measurement using immunoassays and tandem mass spectrometry

Enzyme immunoassays (EIAs) are widely used to measure salivary testosterone. However, little is known about how accurately different EIAs assess testosterone, partially because estimates across various EIAs differ considerably. We compared testosterone concentrations across EIAs of three commonly used manufacturers (DRG International, Salimetrics, and IBL International) to liquid chromatography tandem mass spectrometry (LC–MS/MS). Relative to EIAs from Salimetrics and IBL International, EIAs supplied by DRG International provided the closest approximation to LC–MS/MS testosterone concentrations, followed closely by EIAs from Salimetrics, and then IBL. Additionally, EIAs tended to inflate estimates of lower testosterone concentrations in women. Examining our results and comparing them to existing data revealed that testosterone EIAs had decreased linear correspondence with LC–MS/MS in comparison to cortisol EIAs. Overall, this paper provides researchers with information to better measure testosterone in their research and more accurately compare testosterone measurements across different methods.

Consequences of bilateral cryptorchidism in adults.

Bilateral cryptorchidism treatment results are often shadowed by the majority of unilateral cases. We report the long-term follow-up results of boys treated for bilateral cryptorchidism during childhood. Patients treated in two main paediatric surgery centres were selected from medical registries and invited for a clinical examination including scrotal ultrasound, salivary testosterone measurement and a semen sample. Thirty-six men (38.3%) replied to the written invitation, and 21 agreed to be examined. The mean age at orchidopexywas 74months (range 24-138). Sperm count was 0.42×106 (SD±0.64×106 )ml-1 . The correlation between total testicular volume and total sperm count was statistically significant (r=0.481; P=0.032). These results show that surgical treatment of bilateral cryptorchidism after the age of 2years does not prevent infertility. Sperm count and endocrine evaluation advocated after the treatment of bilateral cryptorchidism in all adult patients.

Salivary testosterone measurements in growing pigs: validation of an automated chemiluminescent immunoassay and its possible use as an acute stress marker

This study aimed to validate the use of an automated chemiluminescent immunoassay analyser for salivary testosterone measurements in growing pigs and study how circadian pattern during daytime and stress can influence its values. The test method had intra- and inter-assay coefficient of variation lower than 10%. The method showed good linearity and recovery, and detection limits were low enough to detect salivary testosterone levels. No significant differences were observed in testosterone concentrations at different sampling time, and age and gender did not influence circadian pattern. In addition, this assay was used to quantify testosterone in two models of acute stress and, in both cases, significant increases (P < 0.01) in salivary testosterone were detected. Therefore, the automated assay system tested for porcine testosterone determinations would be suitable for its use in saliva samples and, furthermore, salivary testosterone levels could be used as a possible marker of acute stress in pigs.

Salivary testosterone measurement by liquid chromatography tandem mass spectrometry in adult males and females

Salivary testosterone (Sal-T) may be a useful surrogate of serum free testosterone. The study aims were to use a novel liquid chromatography tandem mass spectrometry (LC-MS/MS) assay to determine whether Sal-T concentrations accurately reflect Sal-T concentrations in both sexes and to investigate practical aspects of sample collection. Saliva and serum samples were collected in 104 male and 91 female subjects. A more sensitive LC-MS/MS assay was developed to enable Sal-T quantitation in the low concentrations found in females. Saliva (200 µL) was extracted with 1 mL of methyl-tert-butyl ether following the addition of D5-testosterone. Quantitation was performed using a Waters TQ-S mass spectrometer. The assay achieved a lower limit of quantification of 5 pmol/L, sufficiently sensitive to measure testosterone in female saliva. Sal-T showed a diurnal variation but samples taken at weekly and monthly intervals showed no significant differences. Sal-T was stable at ambient temperature for up to 5 days, after freeze-thawing and 3 years frozen storage. Reference intervals for Sal-T were 93-378 pmol/L in males and 5-46 pmol/L in females. Sal-T correlated significantly with serum calculated free-T in males (r = 0.71, P < 0.001) and in females (r = 0.39, P < 0.001). These results confirm that testosterone can be reliably and accurately measured by LC-MS/MS in both adult male and female saliva samples. These results lay the foundation for further exploration of the clinical application of Sal- T as a reliable alternative to serum testosterone in the diagnosis and management of androgen disorders and assessment of androgen status in clinical research.

The effects of saliva collection, handling and storage on salivary testosterone measurement

Several endocrine parameters commonly measured in plasma, such as steroid hormones, can be measured in the oral fluid. However, there are several technical aspects of saliva sampling and processing that can potentially bias the validity of salivary testosterone measurement. The aim of this study was to evaluate the effects caused by repeated sampling; 5min centrifugation (at 2000, 6000 or 10,000g); the stimulation of saliva flow by a cotton swab soaked in 2% citric acid touching the tongue; different storage times and conditions as well as the impact of blood contamination on salivary testosterone concentration measured using a commercially available ELISA kit. Fresh, unprocessed, unstimulated saliva samples served as a control. Salivary testosterone concentrations were influenced neither by repeated sampling nor by stimulation of salivary flow. Testosterone levels determined in samples stored in various laboratory conditions for time periods up to 1month did not differ in comparison with controls. For both genders, salivary testosterone levels were substantially reduced after centrifugation (men F=29.1; women F=56.17, p<0.0001). Blood contamination decreased salivary testosterone levels in a dose-dependent manner (men F=6.54, p<0.01, F=5.01, p<0.05). Salivary testosterone can be considered A robust and stable marker. However, saliva processing and blood leakage can introduce bias into measurements of salivary testoterone using ELISA. Our observations should be considered in studies focusing on salivary testosterone.

Salivary testosterone in female-to-male transgender adolescents during treatment with intra-muscular injectable testosterone esters

IntroductionIn our hospital, female-to-male (FtM) transgender adolescents from the age of 16 are treated with two- or four-weekly intra-muscular injections of testosterone-esters. Some patients treated with four-weekly injections have complaints of fatigue and experience mood swings towards the end of the inter-injection period, which calls for an evaluation of the time-course of testosterone levels between injections. Evaluation of salivary testosterone is a practical approach for sequential measurements. Since only ∼2% of total serum testosterone is present in saliva, a sensitive assay is necessary. The objective was to develop an isotope dilution-liquid chromatography–tandem mass spectrometry method (ID-LC–MS/MS) for salivary testosterone measurements and to evaluate the testosterone profiles after testosterone-ester mixture injections in FtM-adolescents. ExperimentalFtM treated with 125mg/2weeks or with 250mg/4weeks depots of testosterone-ester mixture collected saliva at different time intervals. Salivary testosterone was measured by a thoroughly validated ID-LC–MS/MS assay. ResultsAn ID-LC–MS/MS method for measuring salivary testosterone was developed with adequate accuracy and specificity. The reference range was established at 135–400pmol/L. Testosterone levels peaked supra-physiologically immediately post-injection, and decreased to levels within the male reference range after nine days in all patients. 250mg/4weeks depots resulted in values below the reference range at the end of the 4weeks. DiscussionThe development of an adequate ID-LC–MS/MS method for measuring salivary testosterone allowed us to investigate the testosterone profile in FtM-adolescents after testosterone-esters mixture injections. These injections lead to extreme concentrations which may affect the wellbeing of the patients.

Salivary testosterone concentrations in pubertal ICSI boys compared with spontaneously conceived boys

BACKGROUND To date, no data exist about Leydig cell function of pubertal boys born after ICSI. To evaluate a potential risk of gonadal dysfunction in children born from fathers with compromised fertility, testicular function was assessed by the measurement of salivary testosterone. METHODS Morning salivary testosterone levels at the age of 14 years were compared between 58 ICSI teenagers who are part of the oldest ICSI cohort, and 62 boys born after spontaneous conception (SC). RESULTS Salivary testosterone levels were comparable between ICSI (113 ± 42 pg/ml) and SC (123 ± 56 pg/ml) teenagers at the age of 14 years. In the ICSI group, testosterone levels in boys from fathers with severe oligozoospermia were not different from concentrations in boys from fathers without severe oligozoospermia (115.5 ± 43 and 109 ± 41 pg/ml, respectively). CONCLUSIONS At the age of 14 years, pubertal ICSI boys show testosterone levels comparable to their peers born after SC. ICSI adolescents fathered from men with severely compromised spermatogenesis show testosterone levels comparable to those from fathers with normal spermatogenesis. This notwithstanding, further follow-up of ICSI teenagers into adulthood is mandatory to confirm a normal gonadal function.

Quantitative measurement of salivary testosterone in Korean adults by stable isotope-dilution liquid chromatographyelectrospray-tandem mass spectrometry

Salivary testosterone levels in Korean adults were quantitatively measured for the first time by liquid chromatography-electrospray-tandem mass spectrometry (LC ESI MS/MS). Salivary testosterone was separated on a multiple reaction monitoring (MRM) chromatogram within 7 min. The LC ESI MS/MS assay was validated over the linearity range of 0.01-2.00 ng/ml (r=0.99987) using testosterone-d(3) as an internal standard. The lower limit of quantification (LOQ) was 0.01 ng/ml. The intra- and inter-assay precisions were 1.54% to 4.09% and 0.96% to 4.29%, respectively. The mean recovery was 93.32% (range 88.43-98.05%). The validated assay was then applied to measure the salivary testosterone levels of Korean adults. In men, the salivary testosterone level collected between 9:00-11:00 am was approximately 2.8 times higher than that in women (P < 0.0001). Salivary testosterone levels in both sexes negatively correlated with age. The present assay would also be useful in measuring salivary testosterone levels in clinical laboratories.

Reliability of salivary testosterone measurements in diagnosis of Polycystic Ovarian Syndrome

The aim of this study was to evaluate the reliability of a non-invasive method (saliva) for determination of testosterone level in diagnosing cases of Polycystic Ovarian Syndrome (PCOS), and comparing it with serum free testosterone. Prospective clinical controlled trial. Fifty women with PCOS and 20 normal women as control group were selected for the study. They all aged between 23 and 35year. For all patients serum follicle stimulating hormone (FSH) and luteinizing hormone (LH) were assessed by chemiluminescent assay. Serum free testosterone (FT) was assayed by radioimmunoassay (RIA) and salivary testosterone (ST) by ELISA technique. BMI was calculated for all participants and transvaginal sonography to determine ovarian morphology and ovarian volume. The present study showed a significant positive correlation between salivary testosterone, as measured by ELISA, and serum free testosterone measured by RIA ( p =0.001 and r =0.52). Using the receiver operator curve, salivary testosterone was found to be more sensitive than serum (FT) (84% vs. 66%) in diagnosing PCOS patients. Determination of salivary testosterone is a reliable method to detect changes in the concentration of available biologically active testosterone in the serum. Salivary testosterone provides a sensitive, simple, reliable, non-invasive and uncomplicated diagnostic approach for PCOS.

Bone and Men's Health. Measurement of salivary testosterone

Testosterone is secreted into saliva. In saliva, testosterone is not bound to proteins. Thus concentration of testosterone in saliva is quite close to the serum free testosterone level. Currently serum free testosterone is measured by a convenient method in Japan, that reflects only 10% of the correct values. However, the correct measurement of serum free testosterone is quite costly. Collection of saliva is not invasive to subjects, and it can be done without paramedicals. Besides it is relatively cheaper. Acquisition of samples in multiple occasions is possible. These advantages of utilizing saliva will introduce the measurements of salivary testosterone in cohort studies or screening of low testosterone levels in the community.

Liquid Chromatography-tandem Mass Spectrometry (LC-MS/MS) Assay for Simultaneous Measurement of Salivary Testosterone and Cortisol in Healthy Men for Utilization in the Diagnosis of Late-onset Hypogonadism in Males

It is well known that late-onset hypogonadism in males can cause a variety of symptoms, and the differential diagnosis is relatively difficult, including psychological disorders, stress, and mood disturbances. The level of serum cortisol can be measured to reflect a patient's level of stress. Salivary hormones facilitate the evaluation of physiological hormonal actions based on free hormone assay. For the simultaneous measurement of testosterone and cortisol levels in saliva, we validate a sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay. Concerning accuracy and precision, the lower limit of quantification of salivary testosterone and cortisol were established as 5 and 10 pg/mL, respectively. Testosterone and cortisol in saliva is stable for 2 days, 14 days, and 28 days at room temperature, refrigeration and frozen, respectively. Freezing and thawing for 3 cycles and stimulation of salivation with gum chewing do not alter the measured values of testosterone and cortisol. Total, bioavailable, and free serum testosterone showed slight diurnal changes, but total and bioavailable serum cortisol showed marked diurnal changes. Salivary testosterone levels negatively correlate with age, regardless of the time of saliva collection (r=0.64, p<0.05). However, there is no relationship between salivary cortisol and age (r=0033, p>0.05). LC-MS/MS allows rapid, simultaneous, sensitive, and accurate quantification of testosterone and cortisol in saliva for the diagnosis late-onset hypogonadism or other hormone related disease.