Urinary Testosterone Research Articles

Ovarian vein sampling, and serum and urine testosterone monitoring in ovarian Leydig cell tumors: A report of two cases.

BackgroundOvarian Leydig cell tumors are rare, testosterone-producing tumors that pose diagnostic challenges.CasesA 36-year-old woman presented with 10 years of amenorrhea, facial hair growth and clitoromegaly. A 59-year-old woman presented after 2 years of voice deepening and terminal hair growth. Testosterone concentrations were elevated for both patients; however, imaging failed to identify ovarian or adrenal pathology. For the first patient, selective ovarian venous sampling was performed with results suggesting right ovarian testosterone production. Right ovarian Leydig cell tumors were found in both patients after salpingo-oophorectomy. Testosterone levels immediately declined following tumor removal.ConclusionAdditional diagnostic modalities, such as ovarian venous sampling, should be considered when the etiology of hyperandrogenism cannot be identified through lab work or imaging. In addition, sequential post-operative testosterone levels in serum or urine can help confirm adequate removal of the ovarian tumor.

A multichannel system integrating molecularly imprinted conductive polymers for ultrasensitive voltammetric determination of four steroid hormones in urine.

This work reports on a modularized electrochemical method for the determination of the hormones cortisol, progesterone, testosterone and 17β-estradiol in urine. These hormones were employed as templates when generating molecular imprints from aniline and metanilic acid by electropolymerization on the surface of screen-printed electrodes. The electrically conductive imprint was characterized by SEM, AFM and cyclic voltammetry. A four-channel system was then established to enable simultaneous determination of the hormones by cyclic voltammetry. The detection limits for cortisol, progesterone, testosterone and 17β-estradiol are as low as 2, 2.5, 10 and 9 ag·mL-1 (for S/N = 3). Graphical abstract A four-channel system was established to enable simultaneous determination of 4steroid hormones by cyclic voltammetry and by using moleculalry imprinted polymers.

Detection of testosterone doping in female athletes

Testosterone doping remains a prevalent and potent form of drug cheating among elite athletes. In men, the urine testosterone (T) to epitestosterone (E) ratio (T/E ratio) can identify administration of exogenous T by its suppression of endogenous T production through strong negative feedback on endogenous T and E production as well as spill over into urine of extra testosterone. However, this mechanism may be partially inoperative in females whose much lower circulating T derives from three sources, none subject to powerful negative T feedback. Hence, additional methods to detect T doping in females are required. In this study we report two cases of elite female athletes who were sanctioned for T doping proven by measurement of serum T using liquid chromatography-mass spectrometry (LC-MS), when serial urine T and T/E ratio in one were not indicative of T doping, and in the other were nullified by incidental genetic inactivation of T glucuronidation through the uridine diphosphoglucuronosyl transferase 2B17 (UGT2B17) deletion genotype-phenotype. These findings indicate the potential for serum T measurement by LC-MS to detect T doping in female athletes, especially if implemented in the Bayesian format of an athlete biological passport.

Monitoring urinary testosterone and epitestosterone levels, and their ratio, in Korean chemical castration subjects using liquid chromatography-tandem mass spectrometry.

In Europe, chemical castration has been adopted as a treatment for paraphilia since the 1930s. Among the various chemical castration agents, luteinizing hormone-releasing hormone (LHRH) agonists are now used widely because of their effectiveness and safety. In South Korea, a legislation of chemical castration to control the sexual impulses of sexual offenders was enforced in July 2011. Most of these subjects are treated with leuprorelin acetate, an LHRH agonist, for chemical castration. Despite this, there are few studies that address the long-term influence of LHRH agonists on testosterone (T) and epitestosterone (E) levels in chemical castration subjects. In order to analyze the urinary levels of T in chemical castration subjects, whose T levels are extremely low, we developed and validated an analytical method for the detection of both T and E in human urine using a liquid chromatography-tandem mass spectrometry (LC-MS/MS) system. The urine samples were hydrolyzed, extracted, and analyzed by LC-MS/MS with electrospray ionization in the positive-ion mode. The limits of detection were 0.02 ng/mL and the limits of quantitation were 0.05 ng/mL, which provided great sensitivity. The established method was applied to urine samples from chemical castration subjects and healthy male volunteers. The chemical castration subjects showed significantly lower urinary T levels than the control subjects. In addition, the urinary E levels were also lower in the chemical castration subjects; however, the T/E ratios were constant and did not show a notable decrease because of the simultaneous decrease in both urinary T and E. The urinary T levels and T/E ratio did not exceed the doping control criteria for exogenous T ingestion for any subject. This study shows the trend of urinary T and E levels in long-term treated chemical castration subjects by establishing a highly sensitive LC-MS/MS method, that provides useful information for monitoring chemical castration.

Activity-Based Training Alters Penile Reflex Responses in a Rat Model of Spinal Cord Injury

IntroductionMultisystem functional gains have been reported in males with spinal cord injury (SCI) after undergoing activity-based training (ABT), including increases in scoring of sexual function and reports of improved erectile function. AimThis study aims to examine the effect of daily 60-minute locomotor training and exercise in general on sexual function in a rat SCI contusion model. MethodsMale Wistar rats received a T9 contusion SCI. Animals were randomized into 4 groups: a quadrupedal stepping group (SCI + QT), a forelimb-only exercise group (SCI + FT), a non-trained harnessed group (SCI + NT), and a home cage non-trained group (SCI + HC). The 2 non-trained groups were combined (SCI) post hoc. Daily training sessions were 60 minutes in duration for 8 weeks. Urine samples were collected during bi-weekly 24-hour metabolic cage behavioral testing. Latency, numbers of penile dorsiflexion, and glans cupping were recorded during bi-weekly penile dorsiflexion reflex (PDFR) testing. Terminal electromyography (EMG) recordings of the bulbospongiosus muscle (BSM) were recorded in response to stimulation of the dorsal nerve of the penis (DNP). OutcomesABT after SCI had a significant effect on PDFR, as well as BSM EMG latency and burst duration. ResultsSCI causes a significant decrease in the latency to onset of PDFR. After 8 weeks of ABT, SCI + QT animals had a significantly increased latency relative to the post-SCI baseline. BSM EMG response to DNP stimulation had a significantly decreased latency and increase in average and maximum amplitude in SCI + QT animals. SCI animals had a significantly longer burst duration than trained animals. Time between PDFR events, penile dorsiflexion, glans cupping, and urine testosterone were not affected by ABT. Clinical ImplicationsABT has a positive influence on sexual function and provides a potential therapy to enhance the efficacy of current sexual dysfunction therapies in the male SCI population. Strengths and LimitationsSeveral significant small improvements in sexual function were found in a clinically relevant rat model of SCI using a readily available rehabilitative therapy. The limited findings could reflect insensitivity of the PDFR as a measure of erectile function. ConclusionsThese results indicate that task-specific stepping and/or loading provide sensory input to the spinal cord impacting the neural circuitry responsible for sexual function.Steadman CJ, Hoey RF, Montgomery LR, et al. Activity-Based Training Alters Penile Reflex Responses in a Rat Model of Spinal Cord Injury. J Sex Med 2019; 16:1143–1154.

Effects of Eurycoma Longifolia Jack Supplementation Combined with Resistance Training on Isokinetic Muscular Strength and Power, Anaerobic Power, and Urinary Testosterone: Epitestosterone Ratio in Young Males.

Introduction:This study investigated the effects of resistance training and Eurycoma longifolia Jack supplementation on isokinetic muscular strength and power, Wingate anaerobic power, and testosterone: epitestosterone (T/E) ratio in young males.Methods:Forty young males were weight-matched and assigned into four groups: control (C), Eurycoma longifolia jack (ElJ), resistance training (RT), and Eurycoma longifolia Jack plus resistance training group (ElJ & RT). Participants in ElJ and ElJ & RT groups consumed 200 mg Eurycoma longifolia Jack daily, whereas participants in the C and RT groups consumed placebo capsules daily for 8 weeks. Resistance training program which consisted of 10 different exercises was conducted three times per week for 8 weeks. Participants' isokinetic muscular strength and power, anaerobic power, and urinary TE ratio were measured before and after the intervention period. This is a randomized placebo-controlled intervention study. Paired t-test and one-way analysis of variance were used for statistical analysis.Results:The mean average power of knee flexion at 300°/s in the RT and ElJ & RT groups was significantly (P < 0.05) higher in the posttest compared with pretest. Wingate relative peak power in the RT group increased significantly (P < 0.05) compared with respective pretest value, whereas peak power in the combined ElJ & RT group was significantly (P < 0.05) higher in posttest compared with pretest. There was no significant difference in T/E ratio between pre- and posttests in all the groups.Conclusions:The prescribed resistance training program, either with or without ElJ supplementation, improved isokinetic power of the lower limb. Resistance training alone improved relative anaerobic power, whereas combined Eurycoma longifolia Jack and resistance training improved peak power output. ElJ consumption of 200 mg daily for 8 weeks did not affect the urinary T/E ratio.

Isotope dilution LC-ESI-MS/MS and low resolution selected reaction monitoring as a tool for the accurate quantification of urinary testosterone

A new analytical method for the quantification of testosterone in human urine samples by isotope dilution mass spectrometry is proposed. A standard solution of 13C2-testosterone is added to the samples at the beginning of the sample preparation procedure and then the measurements are carried out by UHPLC-ESI-MS/MS. In the proposed method, the resolution of the first quadrupole of the tandem MS instrument is reduced to transmit the whole precursor ion cluster to the collision cell and measure the isotopic distribution of the in-cell product ions with a small number of SRM transitions. The construction of a methodological calibration graph is avoided using a labelled analogue previously characterised in terms of concentration and isotopic enrichment in combination with multiple linear regression. In this way, the molar fractions of natural and labelled testosterone are calculated in each sample injection and the amount of endogenous testosterone computed from the known amount of labelled analogue. Recovery values between 97 and 107% and precisions between 0.4 and 3.7% (as %RSD) were obtained for testosterone concentrations in urine in the range of 1 to 8 ng g−1. The proposed low resolution SRM methodology was compared for the analysis of human urine samples with the traditional IDMS method based on a calibration graph and the IDMS method based on multiple linear regression combined with standard resolution SRM. A similar accuracy and precision was obtained by the three tested approaches. However, using the low resolution SRM method there was no need to resort to calibration graphs or to specific dedicated software to calculate isotopic distributions by tandem MS and a higher sensitivity was obtained. The proposed low resolution SRM method was successfully applied to the analysis of the certified freeze-dried human urine NMIA MX005.

Micro-patterned molecularly imprinted polymer structures on functionalized diamond-coated substrates for testosterone detection

Molecularly imprinted polymers (MIPs) can selectively bind target molecules and can therefore be advantageously used as a low-cost and robust alternative to replace fragile and expensive natural receptors. Yet, one major challenge in using MIPs for sensor development is the lack of simple and cost-effective techniques that allow firm fixation as well as controllable and consistent receptor material distribution on the sensor substrate. In this work, a convenient method is presented wherein microfluidic systems in conjunction with in situ photo-polymerization on functionalized diamond substrates are used. This novel strategy is simple, efficient, low-cost and less time consuming. Moreover, the approach ensures a tunable and consistent MIP material amount and distribution between different sensor substrates and thus a controllable active sensing surface. The obtained patterned MIP structures are successfully tested as a selective sensor platform to detect physiological concentrations of the hormone disruptor testosterone in buffer, urine and saliva using electrochemical impedance spectroscopy. The highest added testosterone concentration (500 nM) in buffer resulted in an impedance signal of 10.03 ± 0.19% and the lowest concentration (0.5 nM) led to a measurable signal of 1.8 ± 0.15% for the MIPs. With a detection limit of 0.5 nM, the MIP signals exhibited good linearity between a 0.5 nM and 20 nM concentration range. Apart from the excellent and selective recognition offered by these MIP structures, they are also stable during and after the dynamic sensor measurements. Additionally, the MIPs can be easily regenerated by a simple washing procedure and are successfully tested for their reusability.

Reactions of female cheetahs (Acinonyx jubatus) to urine volatiles from males of varying genetic distance.

Understanding the factors used by female cheetahs (Acinonyx jubatus) to make mate choice decisions could benefit zoo breeding programs, which currently assign mates based primarily on genetic distance. Because transporting animals between institutions is costly and can be stressful, females are often limited in the number of males available for mating. One solution would be to determine if an easily transported substance could be used to gauge interest by a female to a potential mate. Here, we investigate female interest in urine samples from males of different genetic distances. Twelve females at five institutions were offered scents from 17 males of varying genetic relatedness in a pair-wise choice paradigm. Behavioral responses of the females were recorded to determine preference. Results showed that females spent more time sniffing and in proximity to scents from the most distantly related males, but female response was not influenced by male urine testosterone concentration, female parity, age, or estrous cycling. Further research will be necessary to determine whether a female's interest in male urine translates to mate preference and acceptance before this technique can be applied to zoo breeding programs.

Behavioral and endocrine responses to season and social dynamics of captive male southern hairy-nosed wombats (Lasiorhinus latifrons)

Although southern hairy-nosed wombats (SHN wombats; Lasiorhinus latifrons) rarely breed in captivity, further knowledge of their reproductive physiology and behavior is likely to improve their breeding potential. This study examined the effect of seasonal variation and changes in social dynamics on the physiology and behavior of a captive population of male SHN wombats (n = 6). Seasonal changes in urinary testosterone metabolites (UTM), urinary cortisol metabolites (UCM), qualitative estimates of spermatorrhoea (QS), aggressive behavior and reproductive behavior were measured over an 11-month period. While there was no effect of month on QS (GLM ANOVA, P = 0.27), reproductive behavior (GLM ANOVA, P = 0.19) or aggressive behavior (Tukey pairwise comparisons), the secretion of UTM (GLM ANOVA, P = 0.051) was only marginally affected by season, compared to that reported for wild male SHN wombats. Mean UCM concentrations of July and August 2016 were significantly higher than those between October 2015 and January 2016 (Tukey pairwise comparisons). To examine social dynamics, two trials of animal positioning exchange with the enclosure system were implemented and behavioral data were examined for each trial over a six week period; UTM, UCM and general behaviors (n = 27) were measured for each trial. Neither UTM nor UCM concentration varied significantly (P ≥ 0.45) before and after the exchanges. “Scratching” decreased at the group level following the animal exchange in both trials, suggesting reduction in self-grooming may be a behavioral response to novel stimuli. UCM and UTM concentrations were both positively correlated with “standing still” and “body rub” behaviors. This may be evidence of a hormonal control of a “freezing behavioral response” to external stimuli and marking behavior, respectively. As there was no evidence that changing the social dynamics affected reproductive or agonistic behavior or hormone concentrations, it was concluded that captive male wombats in this study showed reduced reproductive seasonality compared to wild wombats and that animal exchange resulted in a behavioral response to novel stimuli but was not sufficient to affect testosterone or cortisol secretion, within the context of our study.

Interrelationship among annual cycles of sex steroids, corticosterone and body condition in Nyctibatrachus humayuni

Synergism between extrinsic and intrinsic factors is crucial for the seasonality of reproduction. Environmental factors such as photoperiod and temperature activate the hypothalamus-pituitary-gonadal axis leading to the secretion of steroid hormones that are crucial for reproduction. Sex steroids are not only essential for the maturation of gonads, but also for development of secondary sexual characters in males and reproductive behaviour of both the sexes. In the present study, we quantified the urinary testosterone (UTM) and corticosterone (UCM) metabolites in males and urinary estradiol metabolites (UEM) and UCM in females of Nyctibatrachus humayuni for two consecutive years to determine annual and seasonal variation in the levels of sex steroids, corticosterone and body condition index (BCI). The results show that sex steroids were highest during the breeding season and lowest during the non-breeding season in both the sexes. An increase in UTM and UEM was observed in males and females respectively during the breeding season. Testicular histology showed the presence of all stages of spermatogenesis throughout the year indicating that spermatogenesis is potentially continuous. Ovarian histology showed the presence of vitellogenic follicles only during the breeding season indicating that oogenesis is strictly seasonal. In males, UCM levels were highest during the breeding season, while in females their levels were highest just prior to the breeding season. In males, BCI was highest during the pre-breeding season, declined during the breeding season to increase again during the post-breeding season. In females, BCI was comparable throughout the year. In males, UTM levels were positively correlated with UCM levels but negatively correlated with BCI. Interestingly, UEM, UCM and BCI were not correlated in females. These results indicate that N. humayuni exhibits an associated pattern of reproduction. Quantification of urinary progesterone metabolites (UPM) during the breeding season showed UPM levels were higher in post-spawning females, suggesting the significance of progesterone in ovulation. Further, non-invasive enzyme immunoassay has been successfully standardized in N. humayuni for the quantification of urinary metabolites of steroid hormones.

Relationship between testosterone in serum, saliva and urine during treatment with intramuscular testosterone undecanoate in gender dysphoria and male hypogonadism.

Long-term testosterone replacement therapy is mainly monitored by trough levels of serum testosterone (S-T), while urinary testosterone (U-T) is used by forensic toxicology to evaluate testosterone doping. Testosterone in saliva (Sal-T) may provide additional information and simplify the sample collection. We aimed to investigate the relationships between testosterone measured in saliva, serum and urine during standard treatment with 1,000mg testosterone undecanoate (TU) every 12th week during 1year. This was an observational study. Males with primary and secondary hypogonadism (HG; n=23), subjects with gender dysphoria (GD FtM; n=15) and a healthy control group of men (n=32) were investigated. Sal-T, S-T and U-T were measured before and after TU injections. Sal-T was determined with Salimetrics® enzyme immunoassay, S-T with Roche Elecsys® testosterone II assay and U-T by gas chromatography-mass spectrometry. Sal-T correlated significantly with S-T and calculated free testosterone in both controls and patients (HG men and GD FtM), while Sal-T to U-T showed weaker correlations. Trough values of Sal-T after 12months were significantly higher in the GD FtM group (0.77±0.35nmol/L) compared to HG men (0.53±0.22nmol/L) and controls (0.46±0.15nmol/L), while no differences between S-T and U-T trough values were found. Markedly elevated concentrations of salivary testosterone, 7-14days after injection, were observed, especially in the GD FtM group. This study demonstrates that Sal-T might be a useful clinical tool to monitor long-term testosterone replacement therapy and might give additional information in forensic cases.

The influence of small doses of ethanol on the urinary testosterone to epitestosterone ratio in men and women.

Endogenous steroid use can increase urinary testosterone/epitestosterone (T/E) values. In addition, ethanol in amounts >0.5g per kg of body weight (g/kg) can also increase T/E values. However, the effect of smaller doses of ethanol on T/E values is unknown. The influence of 0.2 and 0.4g/kg of ethanol on baseline T/E values in 20 men and 20 women with low and high baseline T/E values was investigated and correlated with ethyl glucuronide (EtG) and ethyl sulfate (EtS) concentrations. T/E values for 7 of the women were excluded from the study because of undetectable T concentrations or for other reasons. One man and 1 woman with a high T/E baseline value had a significant increase in their T/E value after ingestion of 0.2g/kg of ethanol. One man and 2 women with a high T/E baseline, and 1 woman with a low T/E baseline had significantly increased T/E values after ingestion of 0.4g/kg of ethanol. There was wide variability in peak EtG concentrations and a lack of correlation between ethanol dose and EtG concentrations. Interestingly, 1 man and 2 women with increased T/E values following ethanol ingestion had EtG concentrations below the World Anti-Doping Agency (WADA) cut-off of 5000ng/mL. These findings demonstrate that small amounts of ethanol can elevate T/E values, with women being more susceptible. In addition, consideration should be given to the lowering of the WADA EtG cut-off to detect samples with elevated T/E values from ingestion of low doses of ethanol.

Sex Hormones, Gonadotropins, and Sex Hormone-binding Globulin in Infants Fed Breast Milk, Cow Milk Formula, or Soy Formula

Measurement of endogenous hormones in early life is important to investigate the effects of hormonally active environmental compounds. To assess the possible hormonal effects of different feeding regimens in different sample matrices of infants, 166 infants were enrolled from two U.S hospitals between 2006 and 2009. The children were classified into exclusive soy formula, cow milk formula or breast milk regimens. Urine, saliva and blood samples were collected over the first 12 months of life. Estradiol, estrone, testosterone, luteinizing hormone (LH), follicle-stimulating hormone (FSH) and sex hormone-binding globulin (SHBG) levels were measured in the three matrices. Lower estradiol and LH levels were found in urine and saliva samples of soy formula-fed boys compared to cow formula-fed boys. Higher LH level was found in urine samples of soy formula-fed girls compared to cow formula-fed girls. However, we found neither a neonatal testosterone rise in the boys nor a gender-specific difference in testosterone levels, which suggests that urinary testosterone levels may not accurately reflect blood levels during mini-puberty. Nevertheless, our study shows that blood, urine and saliva samples are readily collectible and suitable for multi-hormone analyses in children and allow examination of hypotheses concerning endocrine effects from dietary compounds.

Online turbulent flow extraction coupled with liquid chromatography–tandem mass spectrometry for high throughput screening of anabolic steroids in horse urine

A high throughput method for simultaneous screening of anabolic steroids and their metabolites (4-esterendione, trenbolone, boldenone, oxandrolone, nandrolone, methandrostenolone, testosterone, 1-androstendione, ethisterone, normethandrolone, methyltestosterone, 16β-Hydroxystanozolol, epitestosterone, bolasterone, norethandrolone, danazol, stanozolol and androstadienone) in equine urine by online turbulent flow extraction coupled with liquid chromatography-tandem mass spectrometry was developed. The use of turbulent flow chromatography could simplify pretreatment of horse urine, which has complex matrices as well as high viscosity. The urine was extracted by mixed-mode cation exchange solid phase extraction, and hydrolyzed using β-glucuronidase/arylsulfatase. Then, the sample was automatically loaded on the TurboFlow Cyclone extraction column for removal of further matrix, followed by separation on a fused core C18 column before MS/MS detection. Optimization and validation of the method were discussed in detail. All analytes were rapidly detected within 10min with high sensitivity (picogram to nanogram per milliliter level), and no interference was observed. The linearity range was from 0.1–10ng/mL for nine steroids and 1.0–50ng/mL for the others, with correlation of coefficient values over 0.995. Precision and accuracy ranged from 0.1 to 14.5% and 1.7 to 12.4%, respectively. The developed method was successfully applied to the analysis of anabolic steroids in horse urine after administration of a model drug.

Measurement of testosterone and cortisol metabolites and luteinising hormone in captive southern hairy-nosed wombat (Lasiorhinus latifrons) urine

This study reports the validation and use of enzyme immunoassays (EIA) to measure changes in plasma and urinary luteinizing hormone, testosterone metabolites (UTM) and cortisol metabolites (UCM) in captive southern hairy-nosed wombats (Lasiorhinus latifrons). GnRH agonist and ACTH agonist challenges were conducted to validate urinary testosterone (male wombat only) and cortisol (male and female wombats) EIAs. Following intra-muscular injection of 8–12μg buserelin (n=4 males), there was a significant increase in both plasma (P<0.001) and urinary testosterone concentrations (P<0.001) 60min and 21h after administration, respectively. Plasma LH levels were elevated (p<0.05) at 20min but there was no significant increase found in urinary LH concentrations after injection. Intra-muscular injection of Synacthen® Depot (250μg) (n=3 males, 3 females) resulted in a significant increase (p<0.05) in plasma cortisol secretion 15min and in urinary cortisol concentrations 3h post injection, respectively. Sex-related differences in cortisol secretion were also reported in this study. These findings indicate that (1) urinary LH might not be an appropriate index for describing the reproductive status in captive male L. latifrons, and (2) the UTM and UCM assays appear to be suitable for the assessment of the testicular steroidogenic capacity and the adrenocortical activity in captive southern hairy-nosed wombats, respectively.

Evaluation of uncertainty sources in the determination of testosterone in urine by calibration-based and isotope dilution quantification using ultra high performance liquid chromatography tandem mass spectrometry

Three quantification methodologies, namely calibration with internal standard (Cal-IS, non-weighted), weighted calibration with internal standard (wCal-IS) and isotope pattern deconvolution (IPD) have been used for the determination of testosterone in urine by LC–MS/MS. Uncertainty has been calculated and compared for the three methodologies through intra- and inter-laboratory reproducibility assays. IPD showed the best performance for the intra-laboratory reproducibility, with RSD and combined uncertainty values below 4% and 9% respectively. wCal-IS showed similar performance, while Cal-IS where not constant and clearly worse at the lowest concentration assayed (2ng/mL) reaching RSD values up to 16%. The inter-laboratory assay indicated similar results although wCal-IS RSD (20%) was higher than IPD (10%) and Cal-IS get worse with RSD higher than 40% for the lowest concentration level. Uncertainty budgets calculated for the three procedures revealed that intercept and slope were the most important factors contributing to uncertainty for Cal-IS. The main factors for wCal-IS and IPD were the volumes of sample and/or standard measured.

Interrelationship among steroid hormones, energetics and vocalisation in the Bombay night frog (Nyctibatrachus humayuni)

In vertebrates, the increase in plasma androgens and corticosteroids is essential for the expression of reproductive behaviour. In male anurans, the interaction between hypothalamus-pituitary-gonadal and hypothalamus-pituitary-interrenal axes plays a pivotal role in calling behaviour and energy mobilisation through the secretion of testosterone and corticosterone respectively. To explain the association among body condition, testosterone, corticosterone and calling behaviour the energetic-hormone-vocalisation (EHV) model has been proposed. The model predicts that with continued participation in chorus activity within and across nights, levels of circulating androgens, corticosterone and vocal effort tend to increase and should be positively correlated in calling males. Consequently, decreasing energy reserves should be inversely correlated with corticosterone level in calling males. Depleted energy reserves lead to the peaking of circulating corticosterone, which suppresses androgen production and calling behaviour. In the present study, we used Nyctibatrachus humayuni with unique reproductive behaviour to test the model by quantifying calling behaviour and urinary metabolites of testosterone and corticosterone. We also computed the body condition index (BCI) to assess the association among energetics, levels of testosterone, corticosterone and calling behaviour. The results show that calling males had higher levels of urinary testosterone metabolites (UTM) than non-calling males indicating the importance of testosterone in controlling the calling behaviour. Surprisingly, urinary corticosterone metabolite (UCM) levels were comparable between calling and non-calling males. Further, calling males had higher body condition estimates than non-calling males. The vocal effort was neither associated with UTM, UCM nor BCI. However, a positive association was observed between UTM and UCM levels in calling males indicating the requirement of higher energy for advertisement. Analysis of UTM and UCM levels throughout the breeding season revealed that breeding basal of UTM was significantly higher than that of UCM. Interestingly, UCM levels were maintained at a lower threshold during the breeding season. These observations are in line with some of the predictions of EHV model.

Application of testosterone to epitestosterone ratio to horse urine – a complementary approach to detect the administrations of testosterone and its pro‐drugs in Thoroughbred geldings

Detection of testosterone and/or its pro-drugs in the gelding is currently regulated by the application of an international threshold for urinary testosterone of 20 ng/mL. The use of steroid ratios may provide a useful supplementary approach to aid in differentiating between the administration of these steroids and unusual physiological conditions that may result in atypically high testosterone concentrations. In the current study, an ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) method was developed to quantify testosterone (T) and epitestosterone (E). The method was used to analyze 200 post-race urine samples from geldings in order to generate the ratios for the reference population. Following statistical analysis of the data, an upper limit of 5 for T:E ratio in geldings is proposed. Samples collected from 15 geldings with atypical urinary testosterone concentrations (>15 ng/mL) but otherwise normal steroid profile, had T:E ratios within those observed for the reference population. The applicability of an upper T:E ratio to detect an administration was demonstrated by the analysis of a selection of incurred samples from testosterone propionate, dehydroepiandrosterone (DHEA), and a mixture of DHEA and pregnenolone (Equi-Bolic®) administrations. These produced testosterone concentrations above the threshold of 20 ng/mL, but also T:E ratios above the proposed limit of 5. In conclusion, consideration of the T:E ratio appears to be a valuable complementary aid to evaluate whether an atypical testosterone concentration could be caused by a natural biological outlier as opposed to the administration of these steroids. Copyright © 2016 John Wiley & Sons, Ltd.

Associations between male testosterone and immune function in a pathogenically stressed forager-horticultural population.

Despite well-known fitness advantages to males who produce and maintain high endogenous testosterone levels, such phenotypes may be costly if testosterone-mediated investment in reproductive effort trade-off against investment in somatic maintenance. Previous studies of androgen-mediated trade-offs in human immune function find mixed results, in part because most studies either focus on a few indicators of immunity, are confounded by phenotypic correlation, or are observational. Here the association between male endogenous testosterone and 13 circulating cytokines are examined before and after ex vivo antigen stimulation with phytohemagglutinin (PHA) and lipopolysaccharides (LPS) in a high pathogen population of Bolivian forager-horticulturalists. A Milliplex 13-plex cytokine panel measured cytokine concentration in whole blood samples from 109 Tsimane men aged 40-89 (median = 50 years) before and after antigen stimulation with PHA and LPS. Urinary testosterone was measured via enzyme immunoassay, demographic, and anthropometric data were collected as part of the Tsimane Health and Life History Project. Higher endogenous testosterone was associated with down-regulated responses in all cytokines after PHA stimulation (but significantly in only 2/13 cytokines), controlling for age and body mass index. In contrast, testosterone was not significantly associated with down-regulation of cytokines after LPS stimulation. MANOVAs indicate that men with higher testosterone showed reduced cytokine responses to PHA compared with LPS (p = 0.0098). Endogenous testosterone appears to be immunomodulatory rather than immunosuppressive. Potentially costlier forms of immune activation like those induced by PHA (largely T-cell biased immune activation) are down-regulated in men with higher testosterone, but testosterone has less impact on potentially less costly immune activation following LPS stimulation (largely B-cell mediated immunity).