Microstructure and mechanics of the rat uterosacral ligaments

Introduction:: Pelvic organ prolapse, which affects half of people who have given birth, is a physically and psychologically painful condition in which the pelvic organs descend from their normal anatomical positions [1]. In severe cases of prolapse, surgery is the only avenue for correction; these surgeries often involve using sutures or mesh to secure the prolapsed organs to native support tissues. The uterosacral ligaments (USLs) are highly complex supportive structures for the uterus and apical vagina which are often used to restore pelvic organ support in prolapse corrective surgeries [2]. However, these surgeries continue to have high rates of complications and recurrence of prolapse [3]. The heterogeneity of the USLs likely plays a significant role in their supportive function. The USLs extend from the cervix to the sacral spine and are considered to have three physiological regions. Tissue content varies between these regions, with the cervical third being comprised primarily of smooth muscle and collagen, the sacral third made up of collagenous tissue with almost no smooth muscle, and the intermediate region containing the transition between the other two regions [2]. The USLs have been studied from anatomical and mechanical perspectives, but little is known about the USLs’ microstructure and how it relates to their mechanical function. This study presents new applications of imaging methods to characterize the evolution of the USLs’ microstructures during mechanical loading.


Materials and Methods:: The USLs were excised from n=14 rats using the methods described by Donaldson et al. [4], preserving their sacral and cervical attachments. While kept hydrated in phosphate buffered saline (PBS), n=8 USLs were placed under a Zeiss LSM 880 multiphoton confocal microscope for second harmonic generation (SHG) imaging to visualize the collagen and muscle in their cervical, intermediate, and sacral regions. Then they were clamped at their attachment sites and loaded into a custom built uniaxial tensile testing apparatus consisting of an 8.8 N-capacity load cell, two linear actuators, and a PBS bath. The USLs (n=14) were pulled along their main in vivo loading direction (MD) at 15 μm/s until ultimate failure. During loading, a spectral domain optical coherence tomography (SD-OCT) system consisting of a superluminescent diode (λ = 837 nm, ∆λ = 54 nm) and a custom spectrometer using a linescan camera (1024 pixel, 78 kHz) was used to take 3D volume scans of the USLs in their cervical/intermediate regions. The SD-OCT system was mounted 150 mm above the specimens with a focused beam diameter of 60 μm. The beam was raster scanned in two dimensions across a 15 mm × 7.5 mm area. The secant modulus for each specimen was calculated as the slope of a line drawn from the origin to the end of the toe region on the load-displacement curve, and the tangent modulus was the slope of a line drawn tangent to the linear-elastic region. Following mechanical testing, the USLs were imaged again using SHG.


Results, Conclusions, and Discussions:: The SHG images taken both before and after mechanical testing showed rich collagen and muscle content in each region of the USLs. With some regional variation, the collagen was largely organized in wavy bundles oriented in many different directions. Little muscle was observed in the cervical regions of the USLs, but plentiful curly collagen bundles were consistently found (Figure 1.A.). In the proximal regions, collagen generally appeared to be less dense and was often woven between layers of muscle (Figure 1.C.), though densely organized curly collagen bundles sometimes appeared in the post-test images, indicating that the tissue contents may have reorganized during loading. The intermediate regions contained characteristics of the other two regions (Figure 1.B.).

The load-displacement data from the uniaxial tests were characterized by an initial toe region followed by a near-linear elastic region and a protracted non-linear failure region, reflecting many discrete breakages within the tissue. The specimens consistently experienced ultimate failure in their intermediate regions. The mean (± S.D.) maximum load achieved by the USLs was 1.157 ± 0.311 N, and the mean displacement at the maximum load 15.042 ± 2.453 mm. The mean secant moduli of USLs across their toe regions was 0.040±0.018 N/mm. The mean tangent moduli of USLs across their linear regions was 0.132 ± 0.038 N/mm.

The SD-OCT 3D volume scans showed that at the millimeter scale, the cervical and intermediate regions of the USLs were composed of tissue masses interwoven with fibers of collagenous tissue (Figure 2.). These structural components moved relatively to each other during mechanical loading with the fibers straightening and reorienting along the MD, and they appeared to experience delamination during tissue failure. In the failure regions, these structural fibers were observed to break at time points corresponding with local minima on the specimens’ load-displacement curves.

Together, these findings provide context to previous mechanical characterization of the USLs, which may be used to drive the development of new surgical meshes that reflect the complexity of the native tissues to which they are applied.


Acknowledgements (Optional): : Funding was provided by NSF Grant No. 1804432.


References (Optional): : 1. Barber, M. D., and C. Maher. Epidemiology and outcome assessment of pelvic organ prolapse. Int. Urogynecol. J. 24(11): 1783–1790, 2013.

2. Donaldson, K., Huntington, A., De Vita, R.: Mechanics of uterosacral ligaments: current knowledge, existing gaps, and future directions. Ann. Biomed. Eng. 49(8), 1788–1804, 2021.

3. Bowen, S.T., Moalli, P.A., Abramowitch, S.D., Lockhart, M.E., Weidner, A.C., Ferrando, C.A., Nager, C.W., Richter, H.E., Rardin, C.R., Komesu, Y.M., et al.: Defining mechanisms of recurrence following apical prolapse repair based on imaging criteria. Am. J. Obstet. Gynecol. 225(5), 506–e1, 2021.

4. Donaldson, K. and De Vita, R.: Ex vivo uniaxial tensile properties of rat uterosacral ligaments. Ann. Biomed. Eng. 51, 702-714, 2023.