The Murine Uterosacral Ligament Tensile Response Changes with Pregnancy and Parturition

Objectives: Risk factors for Pelvic Organ Prolapse (POP) include increasing parity and vaginal delivery, yet how these factors contribute to prolapse are unknown. The uterosacral ligament (USL) is the primary structure for apical vaginal support, and damage to the USL may lead to prolapse of the uterus; hence, we focus our study on how POP risk factors affect USL behavior and may lead to POP. The objective of this study is to understand how pregnancy and parturition alters the USL mechanics and microstructure.

Methods: Female C57BL/6 mice (10-15 weeks-old) were sacrificed as nulliparous (NP, n = 7), late pregnancy gestation day 18.5 (E18.5, n = 8), and 1 day postpartum (PP1, n = 6); mice (15-22 weeks-old) were also sacrificed at 4 weeks postpartum (PP4wk, n = 6). USLs were isolated, by tying a suture around the cervical end, while leaving the sacral end attached to the pelvis, and then carefully excised. Each sample was stained with Ghost Dye and Sytox and placed in a custom loading chamber on an upright confocal microscope. Samples were loaded (50 μm/s) in tension to global displacements of 1000, 1500, 2000, and 2500 µm, and held until equilibrium was reached at 5 minutes. The USLs were imaged in their reference (time = 0 minutes with 500 µN preload) and equilibrium states (5 minutes). Macroscopic displacement and force measurements were evaluated to calculate stiffness during loading and percent relaxation (e.g., stress magnitude loss during relaxation). Confocal images were used to map USL deformation using the MATLAB function ‘imregdemons’. Two-way ANOVA evaluated for effects of displacement state and parturition status (α < 0.05).

Results: The stress-relaxation response of the USL changed throughout pregnancy and postpartum (Figure 1). Loading to 2000 µm demonstrated linear-elastic behavior without USL failure across groups. Here, stiffness was greatest in NP and PP4wk (274 ± 125 and 286 ± 227 kPa, respectively), as compared to E18.5 and PP1 (151 ± 115 and 162 ± 94.5 kPa) and was significantly affected by displacement state (p < 0.0001) and parturition status (p < 0.01). Stiffness trends were maintained across all displacement levels. Percent relaxation was greatest in E18.5 (36.6 ± 5.5%; averaged across all displacement states) with significant effects of displacement and parturition status (p < 0.0001). Image-based deformation tracking showed inhomogeneous USL deformation (Figure 2) with axial tension (E₁₁), both tensile and compressive strains in the transverse loading directions (E₂₂), and shear strains throughout the USL (E₁₂).

Conclusions: USLs in pregnancy were less stiff and underwent greater relaxation. Stiffness and relaxation values remained lowered immediately following birth but were recovered by 4 weeks postpartum. Research is ongoing to examine the USL histoarchitecture and to apply mathematical (i.e., constitutive mechanics) models to explain behavior differences between treatment groups.