Materials and methods

Study design and setting

This hospital-based comparative observational study was conducted in the Department of Cardiology, Dhaka Medical College Hospital (DMCH), Dhaka, Bangladesh, over a period of 12 months from July 2023 to June 2024.

Study population and sampling

Eligible patients with HFpEF were recruited using purposive fixed-quota sampling. The target sample of 120 participants was prespecified in the original study protocol based on the anticipated availability of eligible patients during the 12-month recruitment period and the feasibility of completing the required clinical, laboratory, and echocardiographic assessments. After eligibility assessment, iron status was determined, and recruitment continued within each iron-status stratum until the prespecified quota of 60 iron-deficient and 60 iron-replete participants was reached. The 1:1 allocation represented a deliberate quota for comparative analysis and did not constitute randomization or individual matching. Therefore, the equal group sizes did not represent the natural distribution of iron deficiency in the HFpEF population, and the study was not designed to estimate its prevalence. This sampling approach may also have introduced selection bias.

Inclusion and exclusion criteria

Patients aged >18 years were included if they fulfilled the predefined diagnostic criteria for HFpEF and provided written informed consent. Patients with significant valvular heart disease, infiltrative cardiomyopathy, hypertrophic cardiomyopathy, congenital heart disease, pericardial disease, atrial fibrillation, chronic obstructive pulmonary disease, renal failure with estimated glomerular filtration rate <60 mL/min/1.73 m², malignancy, chronic blood loss, recent iron therapy, or heart failure with improved ejection fraction were excluded.

Diagnostic criteria for HFpEF

Heart failure with preserved ejection fraction was diagnosed based on symptoms and/or signs of heart failure, left ventricular ejection fraction (LVEF) ≥50%, and objective evidence of elevated cardiac filling pressure, supported by elevated NT-proBNP and/or echocardiographic evidence of diastolic dysfunction or structural heart disease [16]. Echocardiographic evidence included abnormalities in mitral inflow pattern, average E/e′ ratio, left atrial volume index, septal and lateral e′ velocity, or tricuspid regurgitation velocity, assessed according to the 2016 American Society of Echocardiography/European Association of Cardiovascular Imaging (ASE/EACVI) recommendations [17].

Data collection procedure

After enrollment, detailed demographic and clinical information, including age, sex, cardiovascular risk factors, comorbidities, and medication history, were recorded using a semi-structured questionnaire. Symptoms and signs of heart failure were assessed clinically. Blood pressure and pulse were measured in all participants. Laboratory investigations included a complete blood count, a serum iron profile, and an NT-proBNP level. Iron profile assessment included serum iron, serum ferritin, and total iron-binding capacity (TIBC). Transferrin saturation (TSAT) was calculated by dividing serum iron by total iron-binding capacity and multiplying by 100. Hematological and biochemical analyses were performed in the Biochemistry Laboratory of Bangabandhu Sheikh Mujib Medical University using Atellica (Siemens Healthineers, Erlangen, Germany) and Alinity ci (Abbott Diagnostics, Lake County, USA) automated analyzers. Serum iron, ferritin, TIBC, and NT-proBNP were measured according to standard laboratory protocols. Details of analyzer manufacturer, assay platform, and reagent specifications were recorded from the laboratory reports. According to World Health Organization criteria, anaemia was defined as a hemoglobin concentration <12 g/dL in women and <13 g/dL in men. Iron deficiency was defined as serum ferritin <100 ng/mL or ferritin 100-299 ng/mL with transferrin saturation <20%, according to contemporary heart failure guidelines [18].

Echocardiographic assessment

All participants underwent transthoracic Doppler echocardiography using a commercially available ultrasound system with a 3.5 MHz transducer (Affinity 70C, Philips Healthcare, Amsterdam, Netherlands). Echocardiographic examinations were performed in the supine and left lateral decubitus positions using standard parasternal and apical views. Left ventricular ejection fraction was calculated using the modified Simpson’s method. Diastolic dysfunction was assessed according to the 2016 ASE/EACVI recommendations. Parameters evaluated included mitral inflow E/A ratio, average E/e′ ratio, left atrial volume index (LAVI), septal and lateral e′ velocity, and peak tricuspid regurgitation velocity. The key abnormal criteria included septal e′ velocity <7 cm/s, lateral e′ velocity <10 cm/s, average E/e′ ratio >14, LAVI >34 mL/m², and peak tricuspid regurgitation velocity >2.8 m/s. Diastolic dysfunction was graded as Grade I, Grade II, or Grade III according to mitral inflow pattern and supportive evidence of elevated left ventricular filling pressure, as recommended by the 2016 ASE/EACVI guideline [19].

Assessment of heart failure severity

Severity of HFpEF was assessed using New York Heart Association (NYHA) functional classification, NT-proBNP level, and echocardiographic grading of diastolic dysfunction. NYHA functional class was categorized from Class I to Class IV according to symptom severity and limitation of physical activity [20].

Statistical analysis

The Statistical Package for the Social Sciences (SPSS), version 26.0 (IBM Corp., Armonk, USA), was used for data analysis. Data were checked for completeness, consistency, and accuracy before analysis. Continuous variables were expressed as mean ± standard deviation, whereas categorical variables were presented as frequency and percentage. The independent-samples t-test was applied to compare continuous variables between the two groups. Categorical variables were compared using the chi-square test; however, Fisher's exact test was used when expected cell frequencies were less than 5. All between-group comparisons were unadjusted. A two-sided p-value of <0.05 was considered statistically significant.

Ethical assessment

Ethical approval for this study was obtained from the Ethical Committee of Dhaka Medical College, Dhaka, Bangladesh, before commencement of the study (Approval no: ERC-DMC/ECC/2023/203). The study was carried out in accordance with the ethical principles outlined in the Declaration of Helsinki. Written informed consent was obtained from all participants after explaining the objectives, procedures, potential benefits, and possible risks of the study. Privacy, confidentiality, and anonymity of all collected information were carefully preserved throughout the research process. Participants were informed that their participation was entirely voluntary and that they could withdraw from the study at any time without affecting their treatment or standard medical care.