Vaginal childbirth is a key risk factor for pelvic floor muscle injury and dysfunction, and subsequent pelvic floor disorders. Multiparity further exacerbates these risks. Using the rat model, validated for the studies of the human pelvic floor muscles, we have previously identified that a single simulated birth injury results in pelvic floor muscle atrophy and fibrosis.

To test the hypothesis that multiple birth injuries would further overwhelm the muscle regenerative capacity, leading to functionally relevant pathological alterations long-term.

Sprague-Dawley rats underwent simulated birth injury and were allowed to recover for 8 weeks before undergoing additional birth injury. Animals were sacrificed at acute (3 and 7 days postinjury), subacute (21, 28, and 35 days postinjury), and long-term (8 and 12 weeks postinjury) time points post second injury (N=3–8/time point), and the pubocaudalis portion of the rat levator ani complex was harvested to assess the impact of repeated birth injuries on muscle mechanical and histomorphological properties. The accompanying transcriptional changes were assessed by a customized NanoString panel. Uninjured animals were used as controls. Data with a parametric distribution were analyzed by a 2-way analysis of variance followed by post hoc pairwise comparisons using Tukey’s or Sidak’s tests; nonparametrically distributed data were compared with Kruskal-Wallis test followed by pairwise comparisons with Dunn’s test. Data, analyzed using GraphPad Prism v8.0, San Diego, CA, are presented as mean ± standard error of the mean or median (range).

Following the first simulated birth injury, active muscle force decreased acutely relative to uninjured controls (12.9±0.9 vs 25.98±2.1 g/mm², P<.01). At 4 weeks, muscle active force production recovered to baseline and remained unchanged at 8 weeks after birth injury (P>.99). Similarly, precipitous decrease in active force was observed immediately after repeated birth injury (18.07±1.2 vs 25.98±2.1 g/mm², P<.05). In contrast to the functional recovery after a single birth injury, a long-term decrease in muscle contractile function was observed up to 12 weeks after repeated birth injuries (18.3±1.6 vs 25.98±2.1 g/mm², P<.05). Fiber size was smaller at the long-term time points after second injury compared to the uninjured group (12 weeks vs uninjured control: 1485 (60.7–5000) vs 1989 (65.6–4702) μm², P<.0001). The proportion of fibers with centralized nuclei, indicating active myofiber regeneration, returned to baseline at 8 weeks post-first birth injury, (P=.95), but remained elevated as far as 12 weeks post-second injury (12 weeks vs uninjured control: 7.1±1.5 vs 0.84±0.13%, P<0.0001). In contrast to the plateauing intramuscular collagen content after 4 weeks post-first injury, fibrotic degeneration increased progressively over 12 weeks after repeated injury (12 weeks vs uninjured control: 6. 7±1.1 vs 2.03±0.2%, P<.001). Prolonged expression of proinflammatory genes accompanied by a greater immune infiltrate was observed after repeated compared to a single birth injury.

Overall, repeated birth injuries lead to a greater magnitude of pathological alterations compared to a single injury, resulting in more pronounced pelvic floor muscle degeneration and muscle dysfunction in the rat model. The above provides a putative mechanistic link between multiparity and the increased risk of pelvic floor dysfunction in women.