Label-free structural and mechanical characterization of rat uterosacral ligaments.

This study presents quantitative applications of label-free imaging methods to characterize the structure of the uterosacral ligaments (USLs) before, during, and after loading. Rat USLs (n=14) were excised with their spinal and cervical attachments, clamped at these attachment sites, and pulled uniaxially in a custom-built tensile testing machine along their main in vivo loading direction. During uniaxial testing, optical coherence tomography (OCT) images were recorded, revealing the re-arrangement and failure of the structural components of the USLs. Before and after uniaxial testing, second harmonic generation (SHG) microscopy was also used to image collagen and smooth muscle within the proximal, intermediate, and distal regions of the USLs. From the OCT images, two metrics, the global depth variation (GDV) and the bundle energy projection (BEP), were extracted to quantify morphological changes as a function of the applied load and displacement. The GDV metric measured the heterogeneity of the USLs, while the BEP metric quantified the re-orientation of fiber bundles under uniaxial testing. SHG images showed that the rat USLs have a complex microstructure with wavy collagen fibers interwoven with smooth muscle bundles. These findings on the structure-function relationship of USLs may have implications for developing non-invasive, label-free imaging modalities suitable for diagnosing conditions such as pelvic organ prolapse (POP) by evaluating the structural integrity of USLs. Statement of Significance: The uterosacral ligaments (USLs), often compromised in pelvic organ prolapse (POP), are the primary support to the uterus and vagina, yet surgeries to restore their function frequently have poor outcomes. Non-invasive diagnostic tools are needed to assess the integrity of the USLs for treatment planning and monitoring. This study examines how the morphology of the USLs changes under mechanical loading, using optical coherence tomography (OCT) for detailed three-dimensional imaging and quantitative optical parameters that correlate morphology with load. Complementary second harmonic generation (SHG) microscopy reveals the organization of smooth muscle and collagen within the tissue structure. These label-free imaging techniques may enable the real-time, noninvasive assessment of tissue integrity and hold potential for future applications in improving the diagnosis and treatment of POP.