Biological Profile of Primary and Secondary Lymphedema: Inflammatory, Endothelial, Metabolic, Lymphatic, and Fibrotic Markers

This observational, cross-sectional study aims to characterize the biological profile of primary and secondary lymphedema by investigating five interconnected biological domains: Th2-related inflammation, endothelial activation, metabolic dysfunction, lymphatic biology, and tissue fibrosis. Lymphedema is traditionally considered a disorder of impaired lymphatic drainage resulting in the accumulation of interstitial fluid. However, increasing evidence suggests that its development and progression involve chronic inflammation, endothelial dysfunction, metabolic alterations, abnormal lymphatic signaling, adipose tissue accumulation, and progressive tissue fibrosis. While these mechanisms have been investigated predominantly in secondary lymphedema, the systemic biological profile of primary lymphedema remains insufficiently characterized. The study will include approximately 90 participants aged 18-45 years: 30 participants with primary lymphedema, 30 participants with secondary lymphedema, and 30 healthy control participants matched by age and sex. Each participant will attend one study visit lasting approximately 45-60 minutes. Clinical assessment will include medical history, demographic characteristics, blood pressure, body measurements, assessment of lymphedema location and clinical stage, pitting edema, Stemmer sign, skin changes, limb volume measurement by perometry, and assessment of tissue firmness. Venous blood samples will be collected to assess routine laboratory parameters and a panel of inflammatory, endothelial, metabolic, lymphatic, and fibrotic biomarkers. A standardized skin swab will also be collected for exploratory skin microbiome analysis. For the primary analysis, one representative marker will be selected in advance for each of the five biological domains: interleukin-13 (IL-13) for Th2-related inflammatory activity, soluble vascular cell adhesion molecule-1 (sVCAM-1) for endothelial activation, HOMA-IR for metabolic dysfunction, vascular endothelial growth factor C (VEGF-C) for lymphatic biology, and transforming growth factor beta 1 (TGF-β1) for tissue fibrosis. These five variables will constitute the co-primary outcomes. Secondary analyses will evaluate additional biomarkers within each biological domain and their associations with clinical severity, including lymphedema stage, limb volume, pitting edema, Stemmer sign, and skin fibrosis. The study will also investigate relationships between the different biological domains and assess differences between primary and secondary lymphedema. Exploratory analyses will characterize the skin microbiome of affected and standardized comparison sites and investigate associations between microbiome composition, clinical disease characteristics, and systemic biomarkers. In participants with primary lymphedema without a previously established genetic diagnosis, selected genetic variants associated with primary lymphedema will also be investigated. The study will provide a comprehensive assessment of biological alterations associated with lymphedema and may help clarify differences between primary and secondary disease. The findings may contribute to a better understanding of lymphedema as a complex biological and tissue disorder and provide a basis for future studies of more targeted diagnostic and therapeutic approaches.

General inclusion criteria for all participants: * Adults aged 18 to 45 years. * Ability to understand the study information and provide written informed consent. * Willingness and ability to attend one study visit and undergo the planned clinical assessment, blood sampling, and skin swab collection. Additional inclusion criteria for participants with primary lymphedema: * Clinically and, where appropriate, imaging-confirmed primary lymphedema of an upper or lower extremity. * Lymphedema classified as Stage I or II according to the applicable clinical classification. Participants with Stage III disease may be included if disease progression is associated with recurrent cellulitis or erysipelas and this is documented separately. * Current treatment or follow-up at the Department of Dermatovenereology, University Medical Centre Ljubljana. Additional inclusion criteria for participants with secondary lymphedema: * Clearly established acquired cause of lymphedema. * Comparable anatomical location and, where feasible, disease stage to the primary lymphedema group. * Completion of active oncological treatment, where secondary lymphedema is cancer-related. Healthy control participants: * No clinical signs or previous history of lymphedema. * Comparable age and sex distribution to the lymphedema groups. Exclusion Criteria * Other major causes of limb swelling, including heart failure, nephrotic syndrome, clinically significant thyroid disease, or medications likely to cause edema when the effect cannot be adequately accounted for. * Acute systemic infection or cellulitis/erysipelas within 4 weeks before study enrollment. * Pregnancy or breastfeeding. * Active oncological treatment. * Active smoking. * Active inflammatory skin disease that could substantially affect the skin microbiome or systemic inflammatory biomarkers. * Clinically manifest cardiovascular disease or previous cardiovascular event. * Treatment with medications that are expected to substantially affect the selected immunological or metabolic outcomes when their effects cannot be adequately accounted for in the analysis. * Inability to provide valid informed consent. Gender eligibility: \- Eligibility is not restricted by gender identity. Biological sex will be recorded where relevant for clinical and laboratory analyses, including sex-specific calculation of selected metabolic indices. Withdrawal: Participants may withdraw from the study at any time without providing a reason and without any effect on their subsequent medical care.