(Poster 120) Multi-Photon Imaging of Pelvic Floor Muscle Entheses in a Birth Injury Model

Objectives: Pelvic floor muscle (PFM) injury sustained during vaginal delivery commonly occurs in the pubovisceralis entheses. While computational models indicate that this region experiences large strains during parturition, the mechanisms leading to irreversible PFM avulsions have not been sufficiently delineated. We aimed to assess whether simulated birth injury (SBI) alters collagen architecture in the enthesial region of the rat pubocaudalis (PCa), which experiences strain similar in magnitude to the human pubovisceralis.

Methods: Three-month-old Sprague-Dawley rats were randomly assigned to uninjured controls or SBI. SBI was performed using a well-established vaginal distention protocol, where a balloon with 5mL volume is placed for two hours to simulate the circumferential and downward distention associated with vaginal delivery. Animals were euthanized following SBI, pelvises were fixed in formalin with balloon catheters in situ for eight days, then bisected into hemipelvises. PCa were exposed but left attached to the pubic rami to avoid disruption of the enthesial region during dissection. Second Harmonic Generation (SHG) microscopy was used to assess collagen architecture, specifically fibril curvature (i.e., waviness), in enthesis and muscle belly. This measure can range from 0 to 1, where smaller values indicate wavier and larger values indicate straighter fibrils. Multiple images were captured in both regions. Data were compared between groups for each region independently using Student t-test (SPSS v30).

Results: Images from the bilateral hemipelvises of 3 controls and 4 animals that underwent SBI were quantified using CT-Fire. Our preliminary analysis demonstrates a significant difference in the curvature of the enthesial collagen between the groups (p=0.02, Fig.1A). Collagen in the entheses of the SBI group was significantly straighter (0.890±0.003) than in the control group (0.882±0.004, Fig.1B). No significant differences in collagen curvature were noted in muscle bellies.

Conclusions: This study indicates that strains associated with parturition straighten collagen fibrils in the PFM enthesial region but not in the muscle belly. The above coincides with strain predictions from computational models of human vaginal delivery and injury. These data suggest that collagen fibrils in the entheses experience larger tensile strains and are closer to mechanical failure (i.e., tearing), while muscle belly collagen is seemingly protected against strain-induced injury. Importantly, the preclinical SBI model demonstrates that collagen straightening precedes avulsion, suggesting the entheses may experience damage even in the absence of complete or partial avulsion. Future efforts to understand implications of changes in enthesial collagen architecture include longitudinal assessments to determine whether alterations induced by parturition-related strains persist long-term and their impact on the outcomes of subsequent delivery/SBI.

Figure 1: (A) Example SHG images displaying the enthesial region of each experimental group. (B) Boxplots displaying collagen curvature, which ranges from 0 to 1 where 1 represents a perfectly straight fibril, in the enthesis (left) and muscle belly (right).