Background/AimsThe longitudinal relationships of within-individual hormone and anthropometric changes during puberty have not ever been fully described. The objectives of this study were to demonstrate that 3 monthly urine collection was feasible in young adolescents and to utilise liquid chromatography-tandem mass spectrometry assay methods for serum and urine testosterone (T), estradiol (E2) and luteinizing hormone (LH) in adolescents by relating temporal changes in urine and serum hormones over 12 months to standard measures of pubertal development.MethodsA community sample of 104 adolescents (57 female) was studied over 12 months with annual anthropometric assessment, blood sampling and self-rated Tanner staging and urine collected every 3 months. Serum and urine sex steroids (T, E2) were measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and LH by immunoassay.ResultsA high proportion (92%) of scheduled samples were obtained with low attrition rate of 6.7% over the 12 months. Urine hormone measurements correlated cross-sectionally and longitudinally with age, anthropometry and Tanner stage.ConclusionWe have developed a feasible and valid sampling methodology and measurements for puberty hormones in urine, which allows a sampling frequency by which individual pubertal progression in adolescents can be described in depth.

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.

47 Objectives: 48Urinary hormone concentrations are often adjusted to correct for hydration status. We 49 aimed to determine whether first morning void urine hormones in growing 50 adolescents require adjustments and, if so, whether urinary creatinine or specific 51 gravity (SG) are better adjustments. 52 53 Design and Methods: 54The study population was adolescents aged 10.1 to 14.3 years initially who provided 55 fasting morning blood samples at 0 and 12 months (n=343) and first morning urine 56 every three months (n=644). Unadjusted, creatinine and SG-adjusted hormonal 57concentrations were compared by Deming regression and Bland-Altman analysis and 58 grouped according to self-rated Tanner stage or chronological age. F-ratios for self-59 rated Tanner stages and age groups were used to compare unadjusted and adjusted 60 hormonal changes in growing young adolescents. Correlations of paired serum and 61 urinary hormonal concentration of unadjusted and creatinine and SG adjusted were 62 also compared. 63 64 Results: 65Fasting first morning void hormone concentrations correlated well and were unbiased 66 between unadjusted or adjusted by either creatinine or SG. Urine creatinine 67 concentration increases with Tanner stages, age and male gender whereas, urine SG

Urine provides a convenient non-invasive alternative to blood sampling for measurement of certain hormones. Urinary luteinizing hormone (LH) measurements have been used for endocrinology research and anti-doping testing. However, the commercially available LH immunoassays are developed and validated for human blood samples but not urine so that LH assays intended for use with urine samples need thorough validation. Therefore, the present study evaluated the measurement of urinary LH immunoreactivity using previously validated immunofluorometric (IF) and immunochemiluminometric (ICL) LH assays after prolonged frozen storage. LH was measured in serial urine samples following administration of a single injection of one of two doses of recombinant human chorionic hormone (rhCG) with assays run at the end of study (2008) and again after four years of frozen (-20 °C) storage where samples were stored without adding preservatives. The ICL assay showed quantitatively reproducible LH measurements after prolonged -20 °C storage. However, the IF immunoassay gave consistently lower LH levels relative to ICL (2008) with a further proportionate reduction after four years of sample storage (2012). Yet, both the assays displayed similar patterns of the time-course of urine LH measurement both before and after four years of frozen storage. In conclusion, we found that both immunoassays are suitable for urinary LH measurements with ICL assay being more robust for quantitative urinary LH measurement such as for anti-doping purposes, whereas the IF could be applicable for research studies where urine LH levels are compared within-study but not in absolute terms.

This study demonstrates that A) DBS sampling with liquid chromatography mass spectrometry steroid analysis achieves frequent time sampling in the community without requiring clinic visits, venesection, or frozen serum storage, and B) androgen esters in an oil vehicle can be delivered effectively by sc injection, thus avoiding the need for medically supervised deep-im injections.

Context Can injectable testosterone undecanoate (TU) be administered effectively and acceptably by the subcutaneous (SC) route? Objective To investigate the acceptability and pharmacokinetics (PK) of SC injection of TU. Design Randomized sequence, crossover clinical study of SC vs IM TU injections. Setting Ambulatory clinic of an academic andrology center. Participants Twenty men (11 hypogonadal, 9 transgender men) who were long-term users of TU. injections. Intervention: Injection of 1000 mg TU (in 4 mL castor oil vehicle) by SC or IM route. Main Outcome Measures: Patient-reported pain, acceptability, and preference scales. PK by measurement of serum testosterone, dihydrotestosterone (DHT), and estradiol (E2) concentrations with application of population PK methods and dried blood spot (DBS) sampling. Results Pain was greater after SC compared with IM injection 24 hours (but not immediately) after injection but both routes were equally acceptable. Ultimately 11 preferred IM, 6 preferred SC, and 3 had no preference. The DBS-based PK analysis of serum testosterone revealed a later time of peak testosterone concentration after SC vs IM injection (8.0 vs 3.3 days) but no significant route differences in model-predicted peak testosterone concentration (8.4 vs 9.6 ng/mL) or mean resident time (183 vs 110 days). The PK of venous serum testosterone, DHT, and E2 did not differ according to route of injection. Conclusions We conclude that SC TU injection is acceptable but produces greater pain 24 hours after injection that may contribute to the overall majority preference for the IM injection. The PK of testosterone, DHT, or E2 did not differ substantially between SC and IM routes. Hence whereas further studies are required, the SC route represents an alternative to IM injections without a need to change dose for men for whom IM injection is not desired or recommended.

Measurement of faecal steroids as a non-invasive technique is widely used to monitor reproductive hormones in captive and free-ranging wild animals. This method offers a great advantage over invasive techniques like blood collection and deserves to be used in domestic animals. Repeated blood sampling is stressful, having animal welfare issues, difficult to obtain in field condition. In the faeces of cow, estradiol 17-α predominated, whereas in mares and sows, estradiol 17-β and estrone are main estrogens. Steroids are metabolized in liver, excreted in faeces and extraction is done by using a variety of methods such as ethanol, methanol etc. Faecal estrogen and progesterone metabolites evaluations are wel1 established approaches for monitoring of reproductive functions in a variety of mammalian species. So in future this method may be the most suitable for monitoring of reproductive status in farm animals particularly under field conditions.

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