Evaluation of Circadian Hormonal Balance: LC-MS/MS Measurement of Salivary and Serum Steroids in Patients With Congenital Adrenal Hyperplasia (MASSALIVE)

In congenital adrenal hyperplasia (CAH), lifelong hormone replacement therapy is required to treat adrenal insufficiency and to reduce elevated androgen levels. This is essential to ensure "normal" growth and puberty. Replacement therapy includes hydrocortisone and 9α-fludrocortisone acetate (as a mineralocorticoid substitute). Defining appropriate criteria for evaluating therapeutic goals is a key component of patient follow-up.

Currently, monitoring is generally limited to the quantification of serum 17-hydroxyprogesterone (17OHP), testosterone (T) and delta-4 androstenedione (D4), measured in the morning after an overnight fast and before the morning hydrocortisone dose. However, such single-point serum measurements do not take into account the circadian rhythm of these steroids.

The objective of the study is to evaluate correlations between steroid levels (21-deoxycortisol, 17-hydroxyprogesterone, testosterone, delta-4 androstenedione, and cortisol) measured by LC-MS/MS in multiple at-home self-collected saliva samples and those measured in serum during routine monitoring.

Study Overview

• Status: Not yet recruiting
• Conditions: Congenital Adrenal Hyperplasia; Adrenal Insufficiency; Hyperandrogenism; 21-hydroxylase Deficiency

Detailed Description

In this project, in addition to the analysis of circulating steroids routinely performed during semi-annual follow-up, salivary steroid profiling will be performed using mass spectrometry and compared with serum measurements. The study also describe their variations over the nycthemeral cycle in a population of children with CAH. The ability to collect saliva samples at home and throughout the nycthemeral period provides significant advantages. Only five non-invasive salivary self-samples (8 a.m., 12 p.m., 4 p.m., 8 p.m., and 8 a.m.) will be requested as additional collections (1 to 2 times over 12 months). Therefore, this study does not require any additional invasive procedure beyond standard clinical follow-up. Preliminary results have demonstrated the technical feasibility of this approach. The temporal evolution of these profiles may subsequently be modelled using multivariate statistical methods to provide clinicians with innovative tools for improved therapeutic monitoring.

This is a prospective, bicentric, non-interventional observational cohort study conducted in two French pediatric endocrinology centers (Hôpital Armand Trousseau and Hôpital Bicêtre). The study focuses on children with congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency, treated with glucocorticoids according to current clinical practice. CAH is a rare adrenal disorder characterized by adrenal insufficiency and variable degrees of hyperandrogenism. Lifelong hormone replacement with hydrocortisone and 9α-fludrocortisone acetate is required to control androgen excess and ensure normal growth and pubertal development. Therapeutic management must strike a balance between overtreatment (risk of growth retardation, weight gain, osteoporosis, and metabolic and cardiovascular complications) and undertreatment (persistent hyperandrogenism, accelerated growth, menstrual cycle disturbances, and testicular adrenal rest tumors).

Current monitoring relies mainly on single morning serum measurements of steroid profiles, especially 17-hydroxyprogesterone (17OHP), testosterone, and delta-4 androstenedione, generally sampled in the fasting state before the morning hydrocortisone dose. However, several studies have highlighted that these steroids exhibit circadian variation, and a single time-point sample may not adequately reflect overall hormone control or guide treatment optimization.

Advances in liquid chromatography tandem mass spectrometry (LC-MS/MS) now allow highly sensitive and specific profiling of multiple steroids in low-concentration matrices such as saliva. This technique offers improved analytical performance compared with immunoassays, including lower detection limits, better specificity, and the possibility to quantify numerous steroids in a single run. Salivary sampling is non-invasive, feasible at home, and well suited for repeated sampling across the nycthemeral cycle.

In this study, during routine follow-up visits (1-2 times per year), patients will undergo their usual serum sampling performed as part of standard care. In addition, parents and/or patients will collect five saliva samples at home over a nycthemeral cycle (8:00, 12:00, 16:00, 20:00, and the following morning at 8:00). The last salivary sample at 8:00 a.m. will be collected at the hospital on the day of the routine visit, at the same time as the serum sample and before the morning hydrocortisone dose. Thus, for each evaluation, one serum sample and five saliva samples will be available per patient.

Steroid profiling (21-deoxycortisol, 17-hydroxyprogesterone, testosterone, delta-4 androstenedione and cortisol) will be performed on both serum and saliva using LC-MS/MS in the Department of Clinical Metabolomics (METOMICS) at Hôpital Saint-Antoine. The primary analysis will examine the correlation between steroid concentrations measured in saliva and serum at 8:00 a.m. using intraclass correlation coefficients and their 95% confidence intervals. Secondary analyses will characterize nycthemeral salivary steroid profiles and explore their relationship with hydrocortisone and fludrocortisone dosing regimens (dose and schedule). When repeated measurements are available for a given patient, they will be handled as repeated measures in the statistical models.

The planned sample size is approximately 50 CAH patients (about 25 per center), followed for up to 12 months with 1-2 routine visits during the inclusion period. This sample size provides sufficient precision to estimate an intraclass correlation coefficient of at least 0.85 for the primary endpoint. No additional invasive procedures are required beyond standard care; the only supplementary procedures are non-invasive salivary self-samplings performed at home. Biological samples (serum and saliva) will be stored at -20°C for up to 15 months for the purposes of the study, and remaining material may be used for future research on CAH-related steroid profiles in accordance with French regulations and patient consent.

• Study Type: Observational
• Enrollment (Estimated): 50

Contacts and Locations

• Study Contact: Name: Muriel HOUANG, MD; Phone Number: +33 1 44 73 66 31; Email: muriel.houang@aphp.fr
• Study Contact Backup: Name: Antonin LAMAZIERE, PU-PH; Phone Number: +33 1 40 01 14 90; Email: antonin.lamaziere@aphp.fr

Study Locations

• France
  • Paris, France, 75012
    • Service des Explorations Fonctionnelles Endocriniennes, Hôpital Armand Trousseau
    • Contact: Muriel HOUANG, MD; Phone Number: +33 1 44 73 66 31; Email: muriel.houang@aphp.fr
    • Contact: Claire BOUVATTIER, MD; Phone Number: +33 1 45 21 22 75; Email: claire.bouvattier@aphp.fr

Participation Criteria

Eligibility Criteria

• Ages Eligible for Study: Child; Adult; Older Adult
• Accepts Healthy Volunteers: No

• Sampling Method: Non-Probability Sample

Study Population

Pediatric patients with congenital adrenal hyperplasia followed in the pediatric endocrinology departments of Hôpital Armand Trousseau (Centre de Référence Maladies Endocriniennes Rares de la Croissance) and Hôpital Bicêtre (Service d'Endocrinologie et diabète de l'Enfant, Centre de Référence DevGen), seen during their routine follow-up visits.

Description

Inclusion Criteria:

• Patients aged 6 years or older
• Confirmed diagnosis of congenital adrenal hyperplasia treated with glucocorticoids
• Signed informed consent provided by legal guardians
• Affiliation to a national health insurance system

Exclusion Criteria:

• Patients younger than 6 years of age
• Lesions of the oral mucosa that could interfere with saliva sampling
• Inability to provide the patient or legal representatives with appropriate study information (e.g., poor understanding of French)
• Underage parents
• Lack of affiliation with a social security/health insurance system

Study Plan

How is the study designed?

Number of groups / cohorts

1

Cohorts and Interventions

Group / Cohort
CAH pediatric cohort treated with glucocorticoids; Children and adolescents with congenital adrenal hyperplasia followed in the participating pediatric endocrinology centers, with 1-2 routine follow-up visits per year, during which paired serum and salivary steroid measurements are obtained.

What is the study measuring?

Primary Outcome Measures

Outcome Measure	Measure Description	Time Frame
Correlation between salivary and serum steroid levels at 8:00 a.m.	Intraclass correlation coefficient between 21-deoxycortisol, 17-hydroxyprogesterone, testosterone, delta-4 androstenedione and cortisol measured by LC-MS/MS in paired salivary and serum samples collected at 8:00 a.m. before the morning hydrocortisone dose.	At the routine follow-up visit within 12 months after inclusion

Secondary Outcome Measures

Outcome Measure	Measure Description	Time Frame
Nycthemeral profile of salivary steroid levels	Description and quantitative characterization of circadian variation in salivary 21-deoxycortisol, 17-hydroxyprogesterone, testosterone, delta-4 androstenedione and cortisol measured by LC-MS/MS in CAH patients treated with glucocorticoids.	Over a 24-hour nycthemeral cycle (8:00, 12:00, 16:00, 20:00, and 8:00 the next morning) within 12 months after inclusion
Correlation between quantitative salivary circadian steroid profiles (ng/mL) obtained by mass spectrometry and hydrocortisone/fludrocortisone dosing (mg/m2 and microg/day respectively)	Evaluation of the relationship between salivary steroid levels (21-deoxycortisol, 17-hydroxyprogesterone, testosterone, delta-4 androstenedione and cortisol) over the nycthemeral cycle and the type, dose and schedule of hydrocortisone and fludrocortisone treatment.	Over a 24-hour circadian cycle during the 12-month observation period

Collaborators and Investigators

• Sponsor: Assistance Publique - Hôpitaux de Paris
• Investigators: Principal Investigator: Muriel HOUANG, MD, Assistance Publique - Hôpitaux de Paris

Study record dates

Study Major Dates

• Study Start (Estimated): September 1, 2026
• Primary Completion (Estimated): September 1, 2027
• Study Completion (Estimated): September 1, 2028

Study Registration Dates

• First Submitted: May 18, 2026
• First Submitted That Met QC Criteria: July 17, 2026
• First Posted (Actual): July 21, 2026

Study Record Updates

• Last Update Posted (Actual): July 21, 2026
• Last Update Submitted That Met QC Criteria: July 17, 2026
• Last Verified: July 1, 2026

More Information

Terms related to this study

• Keywords: circadian rhythm; LC-MS/MS; hydrocortisone; 21-hydroxylase deficiency; fludrocortisone; Congenital adrenal hyperplasia; pediatric endocrinology; steroid profiling; salivary steroids; nycthemeral profile
• Additional Relevant MeSH Terms: Urogenital Diseases; Endocrine System Diseases; Male Urogenital Diseases; Female Urogenital Diseases; Female Urogenital Diseases and Pregnancy Complications; Metabolism, Inborn Errors; Genetic Diseases, Inborn; Metabolic Diseases; Gonadal Disorders; Congenital Abnormalities; Adrenal Gland Diseases; Disorders of Sex Development; Urogenital Abnormalities; Steroid Metabolism, Inborn Errors; 46, XX Disorders of Sex Development; Adrenogenital Syndrome; Congenital, Hereditary, and Neonatal Diseases and Abnormalities; Nutritional and Metabolic Diseases; Adrenal Hyperplasia, Congenital; Adrenal Insufficiency; Hyperandrogenism; Congenital adrenal hyperplasia due to 21 hydroxylase deficiency
• Other Study ID Numbers: APHP240719; IDRCB 2024-A01224-43 (Other Identifier: ANSM)

Plan for Individual participant data (IPD)

• Plan to Share Individual Participant Data (IPD)?: NO
• IPD Plan Description: Individual participant data (IPD) will not be shared outside the study team. This is a non-interventional observational study in a small pediatric cohort, and no external data-sharing plan has been defined in the protocol or by the sponsor.

Drug and device information, study documents

• Studies a U.S. FDA-regulated drug product: No
• Studies a U.S. FDA-regulated device product: No