Female patients admitted to the CASCO Centre (High Specialization Centre for the Care of Obesity), Polyclinic Umberto I, Sapienza University of Rome, from 2014 to 2021, were screened during their first admission. Medical history, physical exam and laboratory assays were performed for all patients, as part of routine diagnostic workup. The complete assessment, including biochemical parameters, epicardial fat thickness, and DXA-scan were performed within a time frame of 2 months.

Menopause was assessed biochemically with the assay of FSH (follicle-stimulating hormone) LH (luteinizing hormone) and oestradiol, and according to clinical findings and the hormonal results patients were divided into 2 groups: menopausal patients and non-menopausal patients.

A written informed consent was obtained from all participants. The patients who did not give their informed consent were excluded. All procedures performed were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards. The study was reviewed and approved by the Ethics Committee of Sapienza University of Rome (protocol code CE5475, approved the 24th October 2019, and amendment protocol n. 0513/2024, approved the 6th June 2024).

Anthropometric parameters were obtained at 9 a.m., in fasting conditions, with subjects wearing light clothing and no shoes. Body weight and height were obtained using the same stadiometer and calibrated scale for all patients. Body Mass Index (BMI) was obtained by dividing weight by squared height (kg/m2). Waist circumference was measured at the iliac crest, and hip circumference was measured around the pelvis at the widest point. The tape was parallel to the floor and did not compress the skin. The waist-to-hip ratio (WHP) was obtained by dividing waist circumference by hip circumference.

Blood samples were collected from fasting patients by venipuncture between 8 and 9 a.m. Samples were then transferred to the local laboratory and handled according to the local standards of practice. A complete metabolic assessment was performed, with the measurement of fasting glucose and insulin, cholesterol and triglycerides. Insulin resistance was assessed with the HOMA-IR, calculated as fasting insulin (UI/L) x fasting glucose (mg/dL)/405 and patients were defined as insulin resistant when the value exceeds 2.5.

Evaluation of hepatic steatosis was obtained with non-invasive methods, based on laboratory and anthropometric measurements, including AST, ALT, insulin and BMI. The formulas used were: hepatic steatosis index (HIS), NAFLD liver fat score (NAFLD -LFS) and Fatty Liver Index (FLI).

The HSI was calculated with the formula HSI = 8 × ALT/AST ratio + BMI (+ 2, in case of diabetic patient; + 2, because of female sex female). The diagnosis of diabetes mellitus was based on a fasting glucose of ≥ 126 mg/dL, HbA1c ≥ 6,5% or therapy with anti-diabetic medication. The cut-off value used to discriminate NAFLD was 35.

FLI was calculated with the formula FLI = (e 0.953 x loge (triglycerides) + 0.139 x BMI + 0.718 x loge (GGT) + 0.053 x waist circumference − 15.745)/ (1 + e 0. 953 x loge (triglycerides) + 0 .139 x BMI + 0.718 x loge (GGT) + 0.053 x waist circumference − 15.745) × 100. The cut-off value used to discriminate NAFLD was 60.

NAFLD-LFS was calculated with the formula.

A NAFD-LFS value ≥ 1.257 indicated the presence of steatosis.

All patients underwent DXA (Hologic-Discovery A, software version 12.5.3:2) to evaluate the mineral bone density of the hip and lumbar spine from L2 to L4, and total body composition. DXA was performed with subjects wearing light clothing and no shoes. Bone values that we evaluated included lumbar spine and hip T-score, lumbar spine and hip bone mineral density (BMD), expressed in g/cm2. Osteoporosis was diagnosed when the T-score was equal to or below -2,5. Trabecular Bone Score (TBS) was calculated with the software TBS iNsight. Bone microarchitecture was considered normal when TBS ≥ 1.35, partially degraded when 1.2 < TBS < 1.35 and degraded when TBS ≤ 1.2 [22].

For the total body composition, we considered the values of Body Fat or Fat Mass (FM), Trunk Fat, and Fat-Free Mass or Lean Mass (FFM), expressed in absolute value in Kg and percentage.

All patients underwent an ultrasound evaluation of the epicardial fat thickness, identified at the interface of the external myocardium wall and visceral pericardium, expressed in mm. The evaluation was carried out by the same expert cardiologist, to minimize interobserver variability. Intima media thickness (IMT) was also evaluated.

The statistical analysis was performed using the software Statistica, version 14 StatSoft Inc. and MedCalc® Statistical Software version 20.111 (MedCalc Software Ltd., Ostend, Belgium; https://www.medcalc.org; 2022). Descriptive statistics (n, mean, SD) were calculated for continuous variables. The distribution of continuous variables was tested with the Shapiro–Wilk test and variables were expressed as mean ± standard deviation (SD). Independent samples Student’s t-test was used to assess differences between groups, that were considered statistically significant when p < 0.05.