Collagen behavior in the human broad ligament under biaxial tensile testing with small-angle X-ray scattering

The human broad ligament connects the uterus laterally to the pelvis as part of the uterine support system. Although being an integral part of this system, its role in the context of pelvic organ prolapse remains elusive. There is still limited knowledge if aging affects the composition and biomechanics of the broad ligament as observed in other uterine ligaments. Here, the mesometrium, the largest portion of the broad ligament, from young (n = 7) and aged female individuals (n = 19) was investigated ex vivo. First, extracellular matrix composition and collagen orientation were assessed using histology and immunofluorescence labeling. Second, nanoscale collagen deformation was quantified using small-angle X-ray scattering (SAXS) during macroscale biaxial tensile testing within physiological ranges. Muscle and collagen type I and III content varied between individuals but not with age. Glycosaminoglycan content was not significantly different between age groups. Histology and SAXS demonstrated two orthogonal collagen fiber/fibril families. Under SAXS, unloaded mesometrium samples showed high collagen fibril dispersion (82-121°) and no difference in median d-spacing (young: 64.96 nm (interquartile range, IQR, 64.94-64.98 nm); aged: 64.96 nm (IQR 64.92-64.97 nm)). Young samples showed no significant variance in nanoscale fibril strain, while aged samples varied notably in fibril strain response. The observed orthogonal fibril families with high dispersion underline the mesometrium's adaptation to multiaxial loading. While no age-related difference in median nanoscale collagen deformation response was observed, the variable response within aged samples indicates additional influencing factors beyond aging such as AGE accumulation. This highlights the need to examine how clinical factors and diseases impact collagen in the uterine support system. STATEMENT OF SIGNIFICANCE: The mesometrium, part of the broad ligament, supports the uterus and may be affected by aging, yet its mechanical properties remain under-researched. This research used advanced imaging (SAXS) and biaxial tension to explore how collagen, a key structural protein, responds to load in young and aged samples and mapped ECM composition using histology. Unlike other uterine ligaments, the broad ligament showed no major age-related differences in ECM composition or structure. Aged samples demonstrated a variable collagen nanoscale response suggesting changes in load transfer in the ECM co-occurring with age. This study advances understanding of the uterine support system and provides a foundation for future research on aging and pelvic health.