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Background: Reference intervals covering the whole life span for all the metabolites in the steroid hormone biosynthesis quantified by sensitive and robust analytical methods are sparse or not existing., Objective: To develop a state-of-the-art LC-MS/MS method for simultaneous quantification of multiple steroid metabolites and to establish detailed sex- and age-specific reference intervals for 16 steroid metabolites., Materials and Method: An isotope diluted LC-MS/MS method was developed for simultaneous quantitation of 16 steroid hormones. Serum samples from cross-sectional cohorts of healthy infants, children, adolescents, and adults aged 0.17 months to 77 years (n = 2458) were analysed., Results: With this novel, specific, and sensitive LC-MS/MS method, it was possible to quantify progesterone, 17-hydroxypregnenolone, 17-hydroxyprogesterone, dehydroepiandrosterone sulfate, androstenedione, testosterone, dihydrotestosterone, 11-deoxycorticosterone, corticosterone, 11-deoxycortisol, cortisol, and cortisone in ≥90 % of the samples, while estrone sulfate, aldosterone and dehydroepiandrosterone were quantified in 77 %, 75 % and 60 % of the samples, respectively. 21-deoxycortisol was only detectable in 2.5 % of samples from healthy subjects. Sex- and age-dependent fluctuations observed in minipuberty, puberty and adulthood including the menopausal transition were modelled. This enabled us to establish valid reference intervals from birth to late adult life for both males and females., Conclusion: Detailed sex- and age-specific reference intervals of multiple, simultaneously quantified steroid metabolites by a novel and specific LC-MS/MS method provides a valuable tool for clinical practice and for future research., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Clinical proteomics aims to deliver cost-effective multiplexing of potentially hundreds of diagnostic proteins, including distinct protein isoforms. The analytical strategy known as targeted proteomics is particularly promising because it is compatible with robust mass spectrometry (MS)-platforms already implemented in many clinical laboratories for routine quantitation of small molecules (i.e. uHPLC coupled to triple-quadrupole MS). Progress in targeted proteomics of circulating insulin-like growth factor 1 (IGF-I) have provided valuable insights about tryptic peptides, transitions, internal standards and calibrants. The present challenge is to examine if targeted proteomics of IGF-I can truly measure up to the routine performance that must be expected from a clinical testing platform., (Copyright © 2017 Elsevier B.V. All rights reserved.)

An isotope-dilution TurboFlow-LC-MS/MS method for simultaneous quantification of the ten steroid metabolites dehydroepiandrosterone sulfate (DHEAS), progesterone, 17α-hydroxyprogesterone (17-OHP), Δ4-androstenedione (Adione), corticosterone, 11-deoxycortisol, cortisol, cortisone, testosterone (T), and estrone 3-sulfate (E1-S) in serum was developed and validated. Limits of quantification, variability (inter- and intra-day), analytical range and linearity were all found to be acceptable for clinical use. Furthermore, sample stability was evaluated including the influence of freeze-thaw cycles and the effects of temperature and storage time. The method was applied to 391 serum samples from healthy, Danish boys 10-18years old. The concentration ranges of the included steroid metabolites for this population are presented. Concentrations of DHEAS, 17-OHP, Adione and T in the 391 serum samples were furthermore compared to results obtained using an existing LC-MS/MS method in our laboratory. Excellent agreement was found between the methods. Furthermore, the improved sensitivity of the new method allowed for quantification of a number of samples found to be below the LOQs of the existing method. Thus, the two instruments and their associated methods were validated as possible back-ups for each other, which we consider an extremely important issue in high-throughput laboratories analyzing clinical samples on a regular basis. The ten analytes included can be analyzed simultaneously but it is also possible only to include some of these analytes for specific diagnostic purposes which make the new method an extremely useful tool in the clinical laboratory., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Sperm concentration is an essential parameter in the diagnostic evaluation of men from infertile couples. It is usually determined by manual counting using a hemocytometer, and is therefore both laborious and subjective. We have earlier shown that a newly developed image cytometry (IC) method may be used to determine sperm concentration. Here we present a validation of the IC method by analysis of 4010 semen samples. There was high agreement between IC and manual counting at sperm concentrations above 3mill/ml and in samples with concentrations above 12mill/ml the two methods can be used interchangeable. However, we found substantial differences in samples below 3mill/ml. We also assessed the accuracy of the two methods by repeated measurements of 248 samples, which revealed that IC measurements seemed more accurate. Moreover, based on ten samples counted by several operators the IC method had a lower coefficient of variation than the manual method (5% vs 10%), indicating a better precision of the IC method. In conclusion, measurement of sperm concentration by IC can be used at concentrations above 3mill/ml and seems more accurate and precise than manual counting, making it an attractive option in the daily clinical practice., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Adrenarche is characterized by steadily rising levels of adrenal androgen metabolites from 4–6 years of age. We recently described marked gender-specific differences in circulating ratios between selected adrenal androgen metabolites in a cross-sectional study. This may suggest gender differences in steroidogenic enzyme activities. We therefore aimed at verifying these findings in a prospective, longitudinal study of healthy boys and girls who were examined during pubertal transition.A longitudinal study of 20 healthy children from the COPENHAGEN Puberty Study, followed every 6 months for 5 years. Clinical examinations were conducted and serum concentrations of Androstenedione (Adione), 17-hydroxyprogesterone (17-OHP), testosterone (T), dehydroepiandrosterone (DHEA) and DHEA sulfate (DHEAS) were quantified by a newly developed LC-MS/MS method. DHEA, DHEAS, Adione, 17-OHP and T increase with age. Boys had higher levels of DHEAS from 10.5 years of age, whereas girls had higher levels of Adione from 13 years of age compared to boys. Interestingly, we observed significantly higher ratios of DHEAS/DHEA (sulfotransferase activity) in boys before and after pubertal onset compared to girls, whereas Adione/17-OHP (CYP 17 activity) appeared to increase more in pubertal girls compared to boys. This suggests that adrenal steroidogenic enzyme activities show developmental as well as gender-specific changes in healthy children.

The influence of sex, age, pubertal development and oral contraceptives on dehydroepiandrosterone (DHEA), DHEA sulfate (DHEAS), 17α-hydroxyprogesterone (17-OHP), Δ4-androstenedione (Adione), testosterone (T), calculated free testosterone (fT), free androgen index (FAI) and selected ratios in 1798 serum samples from healthy children, adolescents and young adults was evaluated. Samples were analyzed by Turboflow-LC-MS/MS. Sex hormone-binding globulin was analyzed by immunoassay. All steroid metabolite concentrations were positively associated with age and pubertal development in both sexes and generally higher in males than in females except for Adione. The pubertal rise in T in males was more pronounced compared to females, reflecting contribution from the testes. Ratios between steroid metabolites varied and depended on sex and age. All ratios were lower during infancy compared to later in life. Use of oral contraceptives significantly lowered serum concentrations of all steroid metabolites, fT, FAI, the 17-OHP/Adione, the Adione/T and the DHEA/Adione ratios, but not the DHEA/DHEAS ratio. We provide reference ranges for DHEA, DHEAS, 17-OHP, Adione, T, fT, FAI and selected ratios in relation to sex, age and pubertal development. Use of oral contraceptives strongly influences adrenal steroidogenesis and should be considered when diagnosing and monitoring treatment of patients with disorders of sex development., (Copyright © 2014 Elsevier B.V. All rights reserved.)