Biomarker Panel for PCOS-Associated Liver Steatosis in Adolescent Girls (COMPASS-pedPCOS)

Metabolic dysfunction-associated steatotic liver disease (MASLD) is the most common chronic liver condition of childhood and is closely linked to obesity and insulin resistance. Polycystic ovary syndrome (PCOS) frequently co-occurs with obesity in adolescent girls, and hyperandrogenism has been proposed as an additional hepatic insult. Because obesity is a powerful confounder, the independent contribution of hyperandrogenism to hepatic steatosis in adolescents remains poorly defined.

This study will be conducted at the Departments of Pediatric, Departments of Pediatric Endocrinology, Pediatric Gastroenterology and Hepatology, Medical Biochemistry, Medical Genetics, and Pediatric Radiology of Kayseri City Hospital, Kayseri, Türkiye. Participants will be allocated to three predefined groups. Group 1 comprises girls with PCOS and obesity, diagnosed according to adolescent-adapted Rotterdam criteria requiring both hyperandrogenism and oligo-anovulation, with a body mass index at or above the 95th percentile. Group 2 comprises girls with obesity without features of PCOS, matched to Group 1 for age and body mass index. Group 3 comprises healthy normal-weight girls between the 5th and 84th body mass index percentiles.

The central comparison is Group 1 versus Group 2, which equalises adiposity and therefore isolates the contribution of hyperandrogenism. Hierarchical regression models will additionally adjust for body mass index z-score, homeostatic model assessment of insulin resistance, and free androgen index. Hepatic steatosis will be quantified by the controlled attenuation parameter obtained during vibration-controlled transient elastography; liver stiffness will be assessed by vibration-controlled transient elastography and two-dimensional shear wave elastography.

A single venous blood sample will be obtained at the study visit. Serum will be separated and stored at minus 80 degrees Celsius until batch analysis, and genomic DNA will be isolated for TaqMan single nucleotide polymorphism genotyping of PNPLA3 rs738409 and HSD17B13 rs72613567. All enzyme-linked immunosorbent assay kits will be procured as a single lot, and intra-assay and inter-assay coefficients of variation together with spike-recovery validation will be documented for each kit.

Data will be recorded in a REDCap electronic case report form in accordance with ALCOA+ principles. The study is exploratory and is designed to generate effect-size and variance estimates for a subsequent validation study. Reporting will follow the STROBE statement, and non-invasive diagnostic performance analyses will follow the STARD 2015 statement. The study is also referred to as COMPASS-PedPCOS in institutional, ethics committee and funding documents.

• Extended Serum Biomarker and Hormonal Panel (Diagnostic test): A single fasting venous blood sample is obtained at the study visit. Enzyme-linked immunosorbent assay measurement of Fetuin-A, fibroblast growth factor 21, adiponectin, visfatin, cytokeratin-18 M30, cytokeratin-18 M65, soluble CD163, growth differentiation factor 15, 11-ketotestosterone and 11beta-hydroxyandrostenedione is performed. Total testosterone and sex hormone-binding globulin are measured by liquid chromatography-tandem mass spectrometry. Routine biochemistry and hormonal profiling are performed from the same sample. Observational only; no therapeutic agent is administered.
• Liver Elastography with Controlled Attenuation Parameter (Diagnostic test): Hepatic steatosis and liver stiffness are assessed non-invasively at the same study visit. Vibration-controlled transient elastography with controlled attenuation parameter yields attenuation (dB/m) and stiffness (kPa) values; two-dimensional shear wave elastography provides an independent stiffness estimate. Predefined validity criteria are applied to all acquisitions. No sedation, contrast agent or ionizing radiation is used; the procedure is observational.
• Targeted Genotyping of PNPLA3 rs738409 and HSD17B13 rs72613567 (Genetic): Genomic DNA is isolated from the same single venous blood sample; no additional venipuncture is required. TaqMan allelic discrimination assays are used for genotyping of PNPLA3 rs738409 and HSD17B13 rs72613567. Genotype call rate and Hardy-Weinberg equilibrium are reported. Genotyping is performed for research purposes only; results are not used to guide clinical management.

Primary outcomes

• Hepatic Steatosis Measured by Controlled Attenuation Parameter (CAP) [Day 1 (single study visit)]: Controlled attenuation parameter obtained during vibration-controlled transient elastography, compared between girls with PCOS and obesity and adiposity-matched girls with obesity, and across all three study groups, with adjustment for body mass index z-score, homeostatic model assessment of insulin resistance, and free androgen index. Unit of measure: dB/m.
• Serum Fetuin-A Concentration [Day 1 (single study visit)]: Serum Fetuin-A measured by enzyme-linked immunosorbent assay and compared across the three study groups with adjustment for body mass index z-score, homeostatic model assessment of insulin resistance, and free androgen index. Unit of measure: µg/mL.
• Serum Fibroblast Growth Factor 21 (FGF-21) Concentration [Day 1 (single study visit)]: Serum FGF-21 measured by enzyme-linked immunosorbent assay and compared across the three study groups with adjustment for body mass index z-score, homeostatic model assessment of insulin resistance, and free androgen index. Unit of measure: pg/mL.
• Serum Adiponectin Concentration [Day 1 (single study visit)]: Serum adiponectin measured by enzyme-linked immunosorbent assay and compared across the three study groups with adjustment for body mass index z-score, homeostatic model assessment of insulin resistance, and free androgen index. Unit of measure: µg/mL.
• Serum Visfatin Concentration [Day 1 (single study visit)]: Serum visfatin measured by enzyme-linked immunosorbent assay and compared across the three study groups with adjustment for body mass index z-score, homeostatic model assessment of insulin resistance, and free androgen index. Unit of measure: ng/mL.

Secondary outcomes

• Serum Soluble CD163 Concentration [Day 1 (single study visit)]: Serum soluble CD163, a marker of macrophage activation, measured by enzyme-linked immunosorbent assay and compared across the three study groups; evaluated as a candidate mediator of the association between hyperandrogenism and hepatic steatosis. Unit of measure: ng/mL.
• Serum Cytokeratin-18 M30 and M65 Concentrations [Day 1 (single study visit)]: Serum cytokeratin-18 M30 (apoptotic fragment) and M65 (total cell death) measured by enzyme-linked immunosorbent assay and compared across the three study groups to characterise the mode of hepatocyte death. Unit of measure: U/L for each analyte.
• Liver Stiffness Measurement [Day 1 (single study visit)]: Liver stiffness assessed by vibration-controlled transient elastography and by two-dimensional shear wave elastography, compared across the three study groups. Unit of measure: kPa.
• Discriminatory Performance of the Biomarker Panel for Hepatic Steatosis [Day 1 (single study visit)]: Receiver operating characteristic analysis of individual biomarkers and of a composite preliminary risk score for the identification of hepatic steatosis. Measures include area under the receiver operating characteristic curve with 95% confidence intervals, sensitivity, and specificity at the Youden index.

Other outcomes

• Serum Growth Differentiation Factor 15 (GDF-15) Concentration [Day 1 (single study visit)]: Serum GDF-15, a marker of mitochondrial stress, measured by enzyme-linked immunosorbent assay and compared across the three study groups. Unit of measure: pg/mL.
• Serum 11-Oxygenated Androgen Concentrations [Day 1 (single study visit)]: Serum 11-ketotestosterone and 11beta-hydroxyandrostenedione measured by enzyme-linked immunosorbent assay and evaluated for association with hepatic steatosis measures independently of body mass index and insulin resistance. Unit of measure: ng/dL for each analyte.
• PNPLA3 rs738409 and HSD17B13 rs72613567 Genotype Distribution and Gene-Gene Interaction [Day 1 (single study visit)]: Genotype frequencies of PNPLA3 rs738409 and HSD17B13 rs72613567 determined by TaqMan assay, and evaluation of gene-gene interaction with respect to hepatic steatosis measures. Measure: genotype counts and interaction estimates; Hardy-Weinberg equilibrium will be assessed.

Sex: Female, ages: 10 Years to 18 Years, accepts healthy volunteers

Inclusion Criteria:

* Female, aged 10 to 18 years, with written informed consent from a parent or legal guardian and written assent from the child.
* Group 1 (PCOS with obesity): both hyperandrogenism and oligo-anovulation required, based on adolescent-adapted Rotterdam criteria; body mass index at or above the 95th percentile.
* Group 2 (Obesity control): body mass index at or above the 95th percentile, without features of PCOS, matched to Group 1 for age and body mass index.
* Group 3 (Healthy control): healthy normal-weight girls (body mass index between the 5th and 84th percentile) without chronic disease, hyperandrogenism, or menstrual irregularity.

Exclusion Criteria:

* Known acute or chronic liver disease (including viral, autoimmune, or Wilson disease) or use of hepatotoxic medication.
* Endocrine disorders or secondary hyperandrogenism, including Cushing syndrome, hypothyroidism, uncontrolled diabetes mellitus, congenital adrenal hyperplasia, androgen-secreting tumour, or hyperprolactinaemia.
* Syndromic or monogenic obesity (including Prader-Willi syndrome, Bardet-Biedl syndrome, Alström syndrome, MC4R or LEP variants) or a known genetic disorder.
* Use of relevant or hepatotoxic medication within the preceding three months, including corticosteroids, metformin, oral contraceptives, valproic acid, or antiandrogens.
* Pregnancy or regular alcohol use.
* Refusal of consent or assent.

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• Agah B ÖZTÜRK, MD, Principal investigator, Kayseri City Hospital, Kayseri, Türkiye