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

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.