Urinary Steroid Research Articles

The association of infant urinary adrenal steroids with the risk of childhood asthma development

BackgroundAdrenal steroids play important roles in early-life development. However, our understanding of the effects of perinatal adrenal steroids on the development of childhood asthma is incomplete. ObjectiveTo evaluate the associations between early-life adrenal steroid levels and childhood asthma. MethodsParticipants included the Infant Susceptibility to Pulmonary Infections and Asthma following Respiratory Syncytial Virus Exposure birth cohort children with untargeted urinary metabolomics data measured during early infancy (n = 264) and nasal immune mediator data measured concurrently at age 2 to 6 months (n = 76). A total of 11 adrenal steroid compounds were identified using untargeted metabolomics and 6 asthma-relevant nasal immune mediators from multiplex assays were a priori selected. Current asthma at ages 5 and 6 years was ascertained using validated questionnaires. Associations were tested using logistic and linear regression with confounders adjustment. ResultsPregnenetriol disulfate (adjusted odds ratio [aOR] = 0.20, 95% CI = 0.06-0.68) and 3a,21-dihydroxy-5b-pregnane-11,20-dione-21-glucuronide (aOR = 0.34, 95% CI = 0.14-0.75) were inversely associated with childhood asthma at 5 and 6 years after multiple testing adjustment. There was a significant interaction effect of pregnanediol-3-glucuronide by biological sex assigned at birth (aOR = 0.11, 95% CI = 0.02-0.51, for those with female sex) on childhood asthma. Pregnenetriol disulfate was inversely associated with granulocyte-macrophage colony-stimulating factor (β = −0.45, q-value = 0.05). 3a,21-dihydroxy-5b-pregnane-11,20-dione 21-glucuronide was inversely associated with interleukin [IL]-4 (β = −0.29, q-value = 0.02), IL-5 (β = −0.35, q-value = 0.006), IL-13 (β = −0.26, q-value = 0.02), granulocyte-macrophage colony-stimulating factor (β = −0.35, q-value = 0.006), and fibroblast growth factor-β (β = −0.24, q-value = 0.01) after multiple testing adjustment. ConclusionThe inverse association between adrenal steroids downstream of progesterone and 17-hydroxypregnenolone and the odds of childhood asthma and nasopharyngeal type 2 immune biomarkers suggest that increased early-life adrenal steroids may suppress type 2 inflammation and protect against the development of childhood asthma.

Profiling of urinary steroids aided by lithium ion adduction-based ultrahigh-performance liquid chromatography-tandem mass spectrometry.

As 3-OH-containing steroids are prone to dehydration by conventional electrospray ionization, reducing detection sensitivity, Li ion adduction-based ultrahigh-performance liquid chromatography-tandem mass spectrometry (UHPLC/MS/MS), developed to prevent dehydration and effectively detect 3-OH steroids, was applied for profiling total and free steroids in urine. Free urinary steroids were isolated directly from urine by solid-phase extraction (SPE) with 80% acetonitrile. The total steroids were prepared by enzymatic treatment of urine with a cocktail of sulfatase and glucronidase, protein precipitation, and separation with the above SPE. In order to detect as many steroid types as possible, UHPLC/MS/MS (Li method) with Li+ solution added after the column was used for analysis in addition to the conventional method of detecting protonated ions (H method). The 13 3-OH steroids and the remaining 16 steroids were quantified by standard curves prepared using product ion transitions derived from [M + Li]+ and MH+ , respectively. Two groups of human urine, male and female urine, were analyzed. 3-OH steroids could be detected with greater sensitivity using the Li method than the conventional method. The absolute amounts of each steroid were normalized based on creatinine levels. The difference between the male and female groups are clearly attributable to sex steroids. Twenty-nine total steroids and 19 free steroids were identified in a limited volume (240 mL) of urine. Of these, 13 3-OH steroids were better detected by Li+ adduction-based UHPLC/MS/MS.

Purulent pericarditis caused by methicillin-sensitive Staphylococcus aureus bacteriuria

BackgroundPurulent pericarditis (PP)— a purulent infection involving the pericardial space—requires a high index of suspicion for diagnosis as it often lacks characteristic signs of pericarditis and carries a mortality rate as high as 40% even with treatment. Common risk factors include immunosuppression, diabetes mellitus, thoracic surgery, malignancy, and uremia. Most reported cases of PP occur in individuals with predisposing risk factors, such as immunosuppression, and result from more commonly observed preceding infections, such as pneumonia, osteomyelitis, and meningitis. We report a case of PP due to asymptomatic bacteriuria in a previously immunocompetent individual on a short course of high-dose steroids.Case presentationAn 81-year-old male presented for severe epigastric pain that worsened with inspiration. He had been on high-dose prednisone for presumed inflammatory hip pain. History was notable for urinary retention requiring intermittent self-catheterization and asymptomatic bacteriuria and urinary tract infections due to methicillin-sensitive Staphylococcus aureus (MSSA). During the index admission he was found to have a moderate pericardial effusion. Pericardial fluid cultures grew MSSA that had an identical antibiogram to that of the urine cultures. A diagnosis of purulent pericarditis was made.ConclusionPP requires a high index of suspicion, especially in hosts with atypical risk factors. This is the second case of PP occurring as a result of asymptomatic MSSA bacteriuria. Through reporting this case we hope to highlight the importance of early recognition of PP and the clinical implications of asymptomatic MSSA bacteriuria in the setting of urinary instrumentation and steroid use.

Employing 11-Ketotestosterone as a Target Analyte for Adrenosterone (11OXO) Administration in Doping Controls.

Adrenosterone (Androst-4-ene-3,11,17-trione, 11OXO) is forbidden in sports according to the Prohibited List of the World Anti-Doping Agency. The administration of 11OXO may be detected by monitoring the urinary concentrations of its main human metabolites 11β-hydroxy-androsterone and 11β-hydroxy-etiocholanolone. Preliminary urinary concentration and concentration ratio thresholds have been established for sports drug testing purposes, but adaptations are desirable as the suggested limits would result in numerous suspicious findings due to naturally elevated concentrations and ratios. Recently, the metabolism of 11-oxo-testosterone (KT) was investigated in the context of anti-doping research, resulting in a preliminary urinary concentration threshold and a confirmation procedure based on the determination of carbon isotope ratios (CIRs). Gas chromatography coupled to isotope ratio mass spectrometry was employed to investigate the CIRs of selected steroids. As KT is also a metabolite of 11OXO, the developed protocols for KT have been tested to elucidate their potential to detect the administration of 11OXO after a single oral dose of 100 mg. In order to further improve the analytical approach, the threshold for urinary concentrations of KT was re-investigated by employing a reference population of n = 5232 routine doping control samples. Quantification of urinary steroids was conducted by employing gas chromatography coupled to triple quadrupole mass spectrometry. Derived from these, a subset of n = 106 samples showing elevated concentrations of KT was investigated regarding their CIRs. By means of this, potentially positive samples due to the illicit administration of 11OXO or KT could be excluded, and the calculation of reference population-derived thresholds for the concentrations and CIR of KT was possible. Based on the results, the urinary concentration threshold for KT is suggested to be established at 130 ng/mL.

Retroperitoneal synovial sarcoma

Synovial sarcoma of the retroperitoneum is a rare and aggressive tumor. In some cases, differential diagnosis between retroperitoneal sarcoma and adrenocortical cancer is difficult due to the similar clinical and intrascopic manifestations of these neoplasms. The final diagnosis is established based on the results of histological, immunohistochemical and genetic studies. The most important step in the treatment of synovial sarcoma is complete removal of the tumor. However, despite the radical nature of the intervention, the incidence of local relapse and metastasis remains high.
 AIM: to conduct a retrospective analysis of the parameters of operated patients with synovial sarcoma of the retroperitoneum.
 This study is aimed at studying the results of examination and surgical treatment of patients with synovial sarcoma of the retroperitoneal space who were hospitalized in the E.E. Eichwald Clinic surgical department of the North-Western State Medical University named after. I.I. Mechnikov.
 The patients were assessed for hormonal status using an immunoassay to determine the levels of adrenocorticotropic hormone, cortisol, aldosterone, renin, normetanephrine and metanephrine in the blood, and a dexamethasone suppression test has been performed at a dose of 1 mg. High-performance liquid chromatography has determined 6 glucocorticoids in the blood serum: cortisol, cortisone, corticosterone, 11-deoxycorticosterone, 11-dehydrocorticosterone, 11-deoxycortisol. The urine steroid profile was studied in all the patients using gas chromatography-mass spectrometry. All the patients underwent computed tomography of the abdominal organs and retroperitoneal space with contrast, morphological and immunohistochemical examination of the removed material. The material was fixed in 10% neutral buffered formalin. The pieces were then subjected to standard wiring followed by embedding in paraffin. Sections 2–3 microns thick were prepared from the resulting blocks and stained with hematoxylin and eosin. Immunohistochemical studies were performed on paraffin sections. A panel of monoclonal antibodies was used: protein S-100 (poly), pancytokeratin (AE1/AE3), EMA, cytokeratin-8, -18, chromogranin A, synaptophysin, α-inhibin, SOX-10, CD99, TLE-1, bcl- 2, GATA-3. Postoperative outcomes and follow-up were assessed according to the information in the medical records and data obtained from a telephone conversation with each patient. Statistical processing of the results has not been out due to rare cases of the disease.

Comparison of analytical approaches for the detection of oral testosterone undecanoate administration in men.

For antidoping laboratories, the determination of an illicit testosterone (T) administration in urine samples remains a difficult process as it requires the determination of the exogenous origin by carbon isotope ratios (CIRs) of testosterone and its metabolites. As a complement to the urinary analysis, targeting testosterone esters (e.g. testosterone undecanoate [TU]) in serum samples by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) could represent a simpler approach compared with isotope ratio mass spectrometry (IRMS). These two approaches both lead to the direct detection of the administration of exogenous T but with a difference in effort and complexity of the analysis. To compare the detection window obtained with the two strategies, serum and the corresponding urine samples collected from an administration study with oral TU were analysed. Results showed that, at all timepoints where the intact TU was detected in serum, the CIRs of urinary steroids were also not in agreement with an endogenous origin. IRMS analysis required more effort but resulted in slightly longer detection windows than the ester analysis. Finally, this comparison study showed that, in the presence of a suspicious urinary steroid profile, the LC-MS/MS steroid esters analysis in the corresponding serum samples can be very helpful. If steroid esters are not detected, the IRMS analysis can then be conducted on the urine sample afterwards. Overall, the combination of matrices might facilitate the detection of prohibited T administration in sports, especially for athletes with naturally low T/E ratios.

Characteristic external genitalia in male neonates with 5α-reductase deficiency.

There have been no reports comparing neonatal external genitalia of 5α-reductase deficiency (5αRD) with those of other 46,XY differences of sex differentiation (DSD). This study enrolled 31 Japanese cases of 46,XY DSD whose external genitalia was examined during the neonatal period; four were diagnosed as 5αRD and 15 were defined as non-5αRD by genetic analysis of SRD5A2 or urinary steroid metabolites. We compared the following characteristics between 5αRD and non-5αRD groups, adjusting the severity of undermasculinization of the external genitalia: stretched penile length (SPL), glans width, location of the external urethral opening, and proportion of undescended testis. The external genitalia of all the 5αRD cases were Quigley classification grade 2 or 3. We compared the phenotypes between the four 5αRD cases and 11 non-5αRD cases with grade 2 or 3. The median (range) of SPL in the 5αRD group (14 mm [11-16]) was significantly lower than that in the non-5αRD group (22 mm [15-29]) (p = 0.003). An SPL cut-off value of <15 mm yielded a sensitivity of 50% (95% confidence interval [CI]; 7-93%) and specificity of 100% (95% CI, 72-100%) for discriminating between the groups. The median glans width, location of the external urethral opening, and proportion of undescended testis were not significantly different between the groups. The SPL of 5αRD in Quigley classification grade 2 or 3 was significantly shorter than that of other 46,XY DSDs with the equivalent grade.

Translating the potential of the urine steroid metabolome to stage NAFLD (TrUSt-NAFLD): study protocol for a multicentre, prospective validation study

IntroductionNon-alcoholic fatty liver disease (NAFLD) affects approximately one in four individuals and its prevalence continues to rise. The advanced stages of NAFLD with significant liver fibrosis are associated with adverse...

Delineating endogenous Cushing’s syndrome by GC-MS urinary steroid metabotyping

BackgroundDiagnosing Cushing’s syndrome (CS) is highly complex. As the diagnostic potential of urinary steroid metabolome analysis by gas chromatography-mass spectrometry (GC-MS) in combination with systems biology has not yet been fully exploited, we studied a large cohort of patients with CS. MethodsWe quantified daily urinary excretion rates of 36 steroid hormone metabolites. Applying cluster analysis, we investigated a control group and 168 patients: 44 with Cushing’s disease (CD) (70% female), 18 with unilateral cortisol-producing adrenal adenoma (83% female), 13 with primary bilateral macronodular adrenal hyperplasia (PBMAH) (77% female), and 93 ruled-out CS (73% female). FindingsCluster-Analysis delineated five urinary steroid metabotypes in CS. Metabotypes 1, 2 and 3 revealing average levels of cortisol and adrenal androgen metabolites included patients with exclusion of CS or and healthy controls. Metabotype 4 reflecting moderately elevated cortisol metabolites but decreased DHEA metabolites characterized the patients with unilateral adrenal CS and PBMAH. Metabotype 5 showing strong increases both in cortisol and DHEA metabolites, as well as overloaded enzymes of cortisol inactivation, was characteristic of CD patients. 11-oxygenated androgens were elevated in all patients with CS. The biomarkers THS, F, THF/THE, and (An + Et)/(11β-OH-An + 11β-OH-Et) correctly classified 97% of patients with CS and 95% of those without CS. An inverse relationship between 11-deoxygenated and 11-oxygenated androgens was typical for the ACTH independent (adrenal) forms of CS with an accuracy of 95%. InterpretationGC-MS based urinary steroid metabotyping allows excellent identification of patients with endogenous CS and differentiation of its subtypes. FundingThe study was funded by the Else-Kröner-Fresenius-Stiftung and the Eva-Luise-und-Horst-Köhler-Stiftung.

Urine steroid metabolomics as a diagnostic tool in primary aldosteronism

Primary aldosteronism (PA) causes 5–10% of hypertension cases, but only a minority of patients are currently diagnosed and treated because of a complex, stepwise, and partly invasive workup. We tested the performance of urine steroid metabolomics, the computational analysis of 24-hour urine steroid metabolome data by machine learning, for the identification and subtyping of PA. Mass spectrometry-based multi-steroid profiling was used to quantify the excretion of 34 steroid metabolites in 24-hour urine samples from 158 adults with PA (88 with unilateral PA [UPA] due to aldosterone-producing adenomas [APAs]; 70 with bilateral PA [BPA]) and 65 sex- and age-matched healthy controls. All APAs were resected and underwent targeted gene sequencing to detect somatic mutations associated with UPA. Patients with PA had increased urinary metabolite excretion of mineralocorticoids, glucocorticoids, and glucocorticoid precursors. Urine steroid metabolomics identified patients with PA with high accuracy, both when applied to all 34 or only the three most discriminative steroid metabolites (average areas under the receiver-operating characteristics curve [AUCs-ROC] 0.95–0.97). Whilst machine learning was suboptimal in differentiating UPA from BPA (average AUCs-ROC 0.65–0.73), it readily identified APA cases harbouring somatic KCNJ5 mutations (average AUCs-ROC 0.79–85). These patients showed a distinctly increased urine excretion of the hybrid steroid 18-hydroxycortisol and its metabolite 18-oxo-tetrahydrocortisol, the latter identified by machine learning as by far the most discriminative steroid. In conclusion, urine steroid metabolomics is a non-invasive candidate test for the accurate identification of PA cases and KCNJ5-mutated APAs.

A novel LC-MS/MS-based assay for the simultaneous quantification of aldosterone-related steroids in human urine.

Primary aldosteronism is the most common cause of endocrine hypertension and is associated with significant cardiovascular morbidities. The diagnostic workup depends on determinations of plasma aldosterone and renin which are highly variable and associated with false-positive and false-negative results. Quantification of aldosterone in 24 h urine may provide more reliable results, but the methodology is not well established. We aimed to establish an assay for urinary aldosterone and related steroids with suitability for clinical routine implementation. Here, we report on the development and validation of a quantitative LC-MS/MS method for six urinary steroids: aldosterone, cortisol, 18-hydroxycorticosterone, 18-hydroxycortisol, 18-oxocortisol, tetrahydroaldosterone. After enzymatic deconjugation, total steroids were extracted using SepPak tC18 plates and quantified in positive electrospray ionization mode on a QTRAP 6500+ mass spectrometer. Excellent linearity was demonstrated with R2>0.998 for all analytes. Extraction recoveries were 89.8-98.4 % and intra- and inter-day coefficients of variations were <6.4 and <9.0 %, establishing superb precision. Patients with primary aldosteronism (n=10) had higher mean 24 h excretions of aldosterone-related metabolites than normotensive volunteers (n=20): 3.91 (95 % CI 2.27-5.55) vs. 1.92 (1.16-2.68) µmol/mol for aldosterone/creatinine, 2.57 (1.49-3.66) vs. 0.79 (0.48-1.10) µmol/mol for 18-hydroxycorticosterone/creatinine, 37.4 (13.59-61.2) vs. 11.61 (10.24-12.98) µmol/mol for 18-hydroxycortisol/creatinine, 1.56 (0.34-2.78) vs. 0.13 (0.09-0.17) µmol/mol for 18-oxocortisol/creatinine, and 21.5 (13.4-29.6) vs. 7.21 (4.88-9.54) µmol/mol for tetrahydroaldosterone/creatinine. The reported assay is robust and suitable for routine clinical use. First results in patient samples, though promising, require clinical validation in a larger sample set.

Urinary steroid profiling by ultra-high-performance liquid-chromatography tandem mass spectrometry: Method Validation and comparison to GC-MS

Urinary steroid profiling by ultra-high-performance liquid-chromatography tandem mass spectrometry: Method Validation and comparison to GC-MS

Urine steroid metabolomics as a diagnostic tool in endocrine hypertension

Urine steroid metabolomics as a diagnostic tool in endocrine hypertension

Effectiveness of blood steroidal passport markers for detecting testosterone abuse in Asians.

The World Anti-Doping Agency (WADA) has introduced an Athlete Biological Passport (ABP) with a steroidal module, which is intended for the monitoring of longitudinal profiles of an athlete's steroid variables in urine to identify endogenous anabolic androgenic steroids that are administered exogenously. It has been in use since 2014. The prevalence of UGT2B17 gene deletion with relatively low levels of testosterone (T) glucuronide in urine is high in the Asian region. There are cases in which urinary T is below the detection limit in specific urine samples, for example, diluted urine, urine collected from females, or urine collected from UGT2B17 deletion individuals. Additional steroid markers T, 4-androstenedione (A4), and the T/A4 ratio in serum were newly added to the ABP steroidal module by WADA in 2023 to compensate for the urine steroid profile. In this study, populations of blood steroid markers in Asians (n = 510) were investigated and classified according to UGT2B17 polymorphism to confirm the effectiveness of blood steroid markers in monitoring Asian athletes. No significant difference in the T/A4 ratio was observed between the genotypes. Furthermore, an administration study of T enanthate in females (n = 10) who were classified according to UGT2B17 genotypes was performed. The concentration of T and the T/A4 ratio were found to be significantly increased in all post-administration samples until 15 days after administration (p < 0.01). The overall results supported the high effectiveness of subject-based monitoring for serum T and T/A4 ratio for recently identified shortcomings in the detection of T abuse in Asians.

Adrenocortical carcinoma (ACC) with massive liver metastases in a 4-year-old female patient: the significance of chemotherapy and imaging studies in treatment and diagnosis – a case report

Background: Adrenocortical carcinoma (ACC) is a retroperitoneal tumour, accounting for less than 0.2% of all malignancies in children. In pediatric patients, the most common clinical presentation is virilisation, either isolated or in combination with hypercortisolemia.&#x0D; Case report: A 4-year-old girl was admitted to the hospital for diagnostic evaluation of precocious puberty and features of hypercorticism. Ultrasonography revealed a heterogeneous neoplastic lesion between the left kidney and spleen. Computed tomography (CT) scan confirmed a pathological mass in the left adrenal region. The urine steroid profile was suggestive of adrenocortical carcinoma. The patient was qualified for surgery. Histopathological examination confirmed the diagnosis. A PET scan performed a few months later showed a metabolically active lesion in the left lobe of the liver. Due to the suspicion of a metastatic focus, it was proposed to implement systemic treatment, to which the patient's parents did not agree. After 15 months of no treatment and oncological follow-up, the girl was referred to the ED with symptoms of progression of the neoplastic process. In the face of a renewed lack of consent to the inclusion of treatment, the patient was referred to a home hospice, where she died a short time later.&#x0D; Conclusions: In the pediatric population, adrenocortical carcinoma is a rare neoplasm with a very poor prognosis. Prompt diagnosis enables early identification of the primary tumour and implementation of appropriate treatment. Imaging examinations are crucial in order to assess the size of the tumour, the extent of local invasion and the presence of potential metastases.

Assessment of steroid enzymes action in children and adolescents with obesity

Rising prevalence of obesity has become an important impulse to investigate basic mechanisms involved in regulating the energy balance. It is widely accepted that steroids are potent factors affecting glucose, fat, and protein metabolism. Our study was aimed to analyze differences in the total amount of selected enzymes implicated in steroid metabolism in a group of children suffering from obesity and those with normal weight, further subdivided according to sex and pubertal stage. Data were obtained from 187 Caucasian children and adolescents, including 113 patients (63 girls, 50 boys) with obesity and 74 (34 girls, 40 boys) normal weight volunteers. Standard clinical examinations were performed in both groups. To evaluate the impact of puberty, preadolescent children and those with advanced puberty were assessed separately. Urine steroid excretion profiles were analyzed using gas chromatography/mass spectrometry method. Children with obesity revealed several changes in in the total amount of steroid enzymes as assessed by the relevant metabolite proportions, compared to their norm weight peers. Girls showed a significant increase in the activity of 11βHSD1, while boys demonstrated a relevant elevation in 20αHSD action. Regardless of sex, children with obesity showed an increase in the activity of 5β-reductase + 3αHSD complex and a decrease in the involvement of 11βOH-lase. The effect is attenuated when consider pre- and pubertal subgroups. We hypothesize that changes in the activity levels of selected enzymes may be a compensatory mechanism to limit the glucocorticoid exposure of key target tissues as well as to improve metabolic control and reduce long-term complications of obesity.

OR02-02 Pre-operative Blood Pressure Response To Aldosterone Antagonists And Urinary Hybrid Steroid Ratios Predict Clinical Outcomes In Unilateral Primary Aldosteronism For At Least 2 Years Post-adrenalectomy

Abstract Disclosure: X. Wu: None. E. Goodchild: None. R. Senanayake: None. W. Bashari: None. J. Salsbury: None. C.P. Cabrera: None. G. Argentesi: None. S.M. O’Toole: None. J. McFarlane: None. M. Matson: None. L. Parvanta: None. N. Hilliard: None. V. Kosmoliaptsis: None. A. Marker: None. D.M. Berney: None. W. Tan: None. R. Foo: None. C.A. Mein: None. E. Wozniak: None. A. Sahdev: None. N. Bird: None. K. Laycock: None. E. Adeyeye: None. A. Dawnay: None. D. Gillett: None. A. Prete: None. A.E. Taylor: None. W. Arlt: None. A.N. Bhuva: None. C. Manisty: None. K.J. Cruickshank: None. H. Cheow: None. G. Mark: None. W. Drake: None. M.J. Brown: None. Introduction: A recent prospective, within-patient study (MATCH) demonstrated 11C-metomidate PET-CT is non-inferior to Adrenal Vein Sampling in accurately detecting unilateral Primary Aldosteronism (PA)1. At 6 months post-adrenalectomy 79.5% and 28.2% of patients achieved, respectively, partial/complete or complete clinical success (by PASO consensus). These outcomes reiterate the need for careful selection of patients for surgery, especially when a widely available non-invasive test could increase, by many-fold, the number of patients diagnosed with unilateral PA. In MATCH, age, sex, genotype of tumour and systolic blood pressure (SBP) response (reduction to &amp;lt;135 mmHg) after 4 weeks of Spironolactone therapy all predicted clinical success at 6 month-adrenalectomy. Aims and Methods: To determine whether these short-term clinical outcomes and the above predictors of success are sustained at 2-year follow-up; and establish whether baseline urinary hybrid steroid (18-OH cortisol/cortisol) ratio could assist clinical decision making by providing surrogate evidence of genotype and probability of clinical success. We report outcomes for all patients who had undergone adrenalectomy with ­­&amp;gt;2-year follow-up on 20th December 2022, including all 78/142 surgical patients in the original study, in addition to 18/40 in the study extension. Results: Partial/complete or complete clinical success were achieved in 78/96 (81%) and 24/96 (25%) of patients respectively. The mean defined daily dose of antihypertensives at 2 years was 1.32 (SD 1.76), comparable to 1.34 (1.90) at 6 months, and significantly lower than at baseline: 3.86 (2.47), t = 7.70, p = 0.0001. Younger age and female sex were associated with higher likelihood of complete clinical success (Fisher’s Exact test p = 0.0084 and p = 0.0001 respectively). Pre-operatively, SBP reduction to &amp;lt;135 mmHg after 4 weeks of spironolactone was seen in 10/23 patients and associated with higher likelihood of complete clinical success at 2 years (versus 6/39 complete clinical success in the SBP &amp;gt;135 mmHg group, Fisher’s exact test p = 0.0135). 13/18 (72%) of patients harbouring KCNJ5 mutations achieved complete clinical success at 2 years, compared to 1/20 (5%) with CACNA1D mutations. A baseline hybrid steroid ratio &amp;gt;2 was seen in 13/14 (93%) patients with KCNJ5 mutations. Absence of clinical success in the KCNJ5 cohort was seen in the only patient with a ratio &amp;lt;2. Conclusion: Favorable clinical outcomes seen at 6 months post-adrenalectomy were sustained at 2-year follow-up. SBP response to spironolactone and KCNJ5 genotype are both potential predictors of ongoing clinical success at 2 years. The presence of high urinary hybrid steroid ratios in patients with KCNJ5 mutations could highlight, pre-operatively, those likely to derive the most clinical benefit from surgery. 1. Nature Medicine, in press, https://doi.org/10.1038/s41591-022-02114-5 Presentation: Thursday, June 15, 2023

THU208 Predicting Treatment Quality Assessment Of Children With Congenital Adrenal Hyperplasia Using 24h Urine Metabolomics Profiling And A Machine Learning-assisted Approach

Abstract Disclosure: O. Abawi: None. G. Sommer: None. M. Groessl: None. U. Halbsguth: None. S.E. Hannema: None. C. de Bruin: None. E. Charmandari: None. E.L. van den Akker: None. A.B. Leichtle: None. C.E. Flueck: None. Introduction: Current treatment monitoring of children with congenital adrenal hyperplasia (CAH) relies on specialist’s interpretation of clinical and biochemical parameters, but remains dissatisfactory. Comprehensive 24h urine steroid profiling provides detailed insight into adrenal steroid pathways, but its merit in routine treatment monitoring of CAH is not yet established. Aim: To investigate whether 24h urine steroid profiling can predict treatment quality assessment in children with CAH using machine learning (ML). Methods: This prospective observational cohort study included children with genetically confirmed 21-hydroxylase deficiency. Children collected 24h urine at 2 outpatient clinic visits (mean 4.1 ± 0.7 months apart). Using gas chromatography-mass spectrometry, 40 adrenal steroids and metabolites from the classic, backdoor and 11-oxygenated pathways were analysed. Patients were classified as undertreated, optimally treated or overtreated by the pediatric endocrinologist based on detailed clinical and endocrinological evaluation including serum 17-hydroxyprogesterone and androstenedione. We used sparse partial least-squares discriminant analysis (sPLS-DA) to investigate optimal prediction of treatment quality assessment. This ML method computes components (combinations of all input variables) and selects the most discriminative parameters to classify samples (in our case optimally treated vs undertreated) by maximizing between-class variance. We computed area under the ROC-curve (AUC) of two sPLS-DA models: 1. using only 24h urine metabolites; 2. adding also clinical variables age, sex, pubertal status, CAH subtype (classic vs non-classic), medication (hydrocortisone [HC] vs prednisolone), daily HC-equivalent dose, Δbone age minus chronological age, ΔBMI-z, and Δheight-z. Results: We included 112 visits (68 [61%] optimally treated, 44 [39%] undertreated) of 59 patients: 27 (46%) girls, 46 (78%) classic CAH, 19 (32%) prepubertal. Mean age at first visit was 11.9 ± 4.0 years and mean BMI SDS 0.6 ± 1.1. SPLS-DA using 24h urine metabolites showed clear clustering of optimally treated patients on two components, while undertreated patients were more heterogenous (AUC 0.88; 95% CI 0.81-0.94). The model selected pregnanetriol and hydroxypregnanolon contributing to excluding undertreatment and 7 metabolites contributing to excluding optimal treatment: estradiol, cortison, tetrahydroaldosterone, androstenetriol, etiocholanolone, androstenediol, and α-dihydrocortison. Addition of clinical variables did not improve classification (AUC 0.90, 95% CI 0.84-0.96, P=0.59). Discussion: Using ML on 24h urine steroid profiling predicted treatment quality assessment in children with CAH even in absence of clinical data, suggesting that comprehensive 24h urine steroid profiling could improve treatment monitoring in children with CAH. Presentation: Thursday, June 15, 2023

Influence of multiple human chorionic gonadotropin administrations on serum and urinary steroid Athlete Biological Passport profiles in males.

The Athlete Biological Passport (ABP) is a longitudinal tool used in anti-doping to monitor biological parameters known to change with performance-enhancing drug use. The ABP consists of multiple modules, including two aimed at detecting the use of endogenous anabolic androgenic steroids: the urinary and serum steroid modules. Human chorionic gonadotropin (hCG) is a protein hormone potentially abused by male athletes to increase the production of endogenous testosterone. To date, no studies have investigated the impact of extended hCG administration on the urinary and serum steroid modules of the ABP. The goal of this study was to identify the impact of multiple hCG administrations on the parameters tracked as part of the urinary and serum steroid modules of the ABP. Ten recreationally active, healthy male individuals self-administered seven 250 μg hCG injections over 3weeks. Serum and urine samples were collected before, during, and 2 weeks following the final injection. All ABP parameters were quantified in the respective matrix, and steroid profiles were created with Anti-Doping Administration and Management System adaptive model upper and lower limits for both matrices. In both serum and urine profiles, testosterone increased; however, the testosterone/epitestosterone ratio in urine and the testosterone/androstenedione ratio in serum showed minimal changes. Additionally, serum luteinizing hormone (LH) was quantified using an immunoassay, and a serum testosterone/LH ratio was generated. Serum LH values decreased during administration causing large increases in the serum T/LH ratio, indicating this ratio may be a more sensitive parameter for detecting hCG abuse than urinary testosterone/epitestosterone or serum testosterone/androstenedione.

Biomarkers in congenital adrenal hyperplasia.

Monitoring of hormone replacement therapy represents a major challenge in the management of congenital adrenal hyperplasia (CAH). In the absence of clear guidance and standardised monitoring strategies, there is no consensus among clinicians regarding the relevance of various biochemical markers used in practice, leading to wide variability in their application and interpretation. In this review, we summarise the published evidence on biochemical monitoring of CAH. We discuss temporal variations of the most commonly measured biomarkers throughout the day, the interrelationship between different biomarkers, as well as their relationship with different glucocorticoid and mineralocorticoid treatment regimens and clinical outcomes. Our review highlights significant heterogeneity across studies in both aims and methodology. However, we identified key messages for the management of patients with CAH. The approach to hormone replacement therapy should be individualised, based on the individual hormonal profile throughout the day in relation to medication. There are limitations to using 17-hydroxyprogesterone, androstenedione and testosterone, and the role of additional biomarkers such 11-oxygenated androgens which are more disease specific should be further established. Noninvasive monitoring via salivary and urinary steroid measurements is becoming increasingly available and should be considered, especially in the management of children with CAH. Additionally, this review indicates the need for large scale longitudinal studies analysing the interrelation between different monitoring strategies used in clinical practice and health outcomes in children and adults with CAH.