Impact of Lactation on Pelvic Skeletal Muscle in a Pregnant Birth Injury Model

Objectives: Pelvic floor muscles (PFMs) are integral to the proper function of the pelvic floor. Vaginal delivery is the leading risk factor for PFM injury and the associated pelvic floor disorders. However, the impact of pregnancy and lactation on PFMs’ response to birth injury remains elusive. Ultimately, understanding the mechanisms of PFM recovery following birth injury is vital for developing scientifically rationale strategies to prevent PFM dysfunction postpartum.
We aimed to assess the impact of the postpartum milieu in the presence and absence of lactation on PFM regeneration following simulated birth injury in a biologically and translationally relevant pregnant rat model. We initially hypothesized that lactation would delay PFM regeneration due to the accompanying hypoestrogenemia, as estrogen is known to aid in regeneration of appendicular skeletal muscles.

Methods: Late-pregnant (D20/22) Sprague-Dawley rats underwent simulated birth injury via vaginal balloon distention per an established protocol. Animals were randomized into non-lactating and lactating groups, with lactation lasting 3-weeks after delivery of pups, and euthanized either 4- or 8-weeks post-injury (N=6/group). Pubocaudalis portion of the rat levator ani complex was harvested, snap frozen, and cryosectioned for subsequent analyses via immunohistochemistry. Muscle cross-sections were incubated with anti-laminin antibody and DAPI for quantification of fiber size (Fig.A) and proportion of regenerating myofibers exhibiting centrally located nuclei (Fig.B). Anti-collagen I antibody conjugated to a fluorophore was used to quantify collagen I – the main component of the intramuscular extracellular matrix - to measure intrafibrillar fibrosis (Fig.C). Uninjured non-pregnant and late-pregnant animals served as controls (N=3-6/group). Data were compared between groups using Kruskal-Wallis or 1-way ANOVA with post-hoc pairwise comparisons when indicated.

Results: Fiber cross-sectional area, percent of fibers with centralized nuclei, and collagen content did not differ between non-pregnant and late-pregnant controls. Fiber size was significantly decreased at 4- and 8-weeks after injury in the non-lactating animals compared to the non-pregnant control (P < 0.0001, Fig.A). The proportion of regenerating myofibers was significantly higher in the lactating group compared to controls 8 weeks after injury (6.7±1.04% vs 0.26±0.03%, P=0.02, Fig.B). Pubocaudalis was enriched with larger fibers at 4- and 8-weeks after injury in the lactating animals compared to non-lactating counterparts that exhibited muscle atrophy (P < 0.0001, Fig.A). Collagen I content did not differ significantly between groups (Fig.C).

Conclusions: Unexpectedly, lactation positively impacts PFM regeneration following simulated birth injury in a translationally relevant pregnant model. Elucidating the mechanisms that potentiate PFM recovery after birth injury is important in identifying novel strategies to promote endogenous muscle regeneration.
Figure Legend: Quantification of muscle fiber cross-sectional area (A), percent of fibers with centralized nuclei (B), and collagen I content (C) of the pubocaudalis portion of the levator ani complex in non-pregnant (NP) controls, non-lactating and lactating groups 4 and 8 weeks after simulated birth injury.