Poster BB20 - Impact of Lactation on Pelvic Skeletal Muscle Regeneration in a Pregnant Birth Injury Model

Introduction: Pelvic floor disorders negatively impact ~30% of women in the United States. Vaginal delivery is the leading risk factor for pelvic floor muscle (PFM) injury and the associated pelvic floor disorders. However, the impact of pregnancy and lactation on PFMs’ response to birth injury remains elusive. Understanding the mechanisms of PFM recovery following birth injury is vital for developing effective 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 pregnant rat model. During breastfeeding estrogen levels remain low (hypoestrogenemia), and estrogen is known to aid in regeneration of appendicular skeletal muscles. Thus, we initially hypothesized that lactation would hinder or delay PFM regeneration due to the accompanying hypoestrogenemia.

Materials and

Methods: Late-pregnant (D20/22) Sprague-Dawley rats underwent simulated birth injury via vaginal balloon distention. Animals were randomized into non-lactating and lactating groups. Animals were then euthanized 1- or 3-weeks after injury for bulk RNA sequencing, or 4- or 8-weeks after injury for histology (N=6/group/timepoint). For all experiments, the pubocaudalis portion of the rat PFM complex, a homolog to the human pubococcygeus muscle which is known to sustain the largest mechanical strains during human vaginal delivery, was harvested. Harvested pubocaudalis was then used for either RNA isolation or was snap frozen and cryosectioned for subsequent histological analyses. RNA quality control was performed followed by bulk RNA sequencing. Genes were then analyzed with an established Bioconductor DESeq2 pipeline with P< 0.05 and log2FoldChange>1 cutoffs to assess differentially expressed genes. Differentially expressed genes underwent gene set enrichment analysis with gene ontology to assess biological pathways. Four and 8-week post-injury muscle cross-sections were incubated with anti-laminin antibody and DAPI for quantification of the proportion of regenerating myofibers exhibiting centrally located nuclei and fiber size. Uninjured non-pregnant and late-pregnant animals served as controls for histological endpoints (N=3-6/group). Data were compared between groups using 1-way ANOVA or Kruskal-Wallis with post-hoc pairwise comparisons when indicated.

Results, Conclusions, and Discussions: At 1-week after injury, non-lactating rats were enriched with genes regulating the immune system (e.g. Clec7a, Irf8, Itgal, Cx3cr1), especially leukocyte migration and adhesion, compared to the lactating rats (Fig. 1A, C). In contrast, lactating rats had notable upregulation of anti-inflammatory genes (e.g. Bmp7, Alox15) compared to non-lactating rats (Fig1A, D). At 3-weeks after injury, non-lactating rats had more of an ECM remodeling (e.g. Col5a3, Col19a1, Mmp12, Fap) and pro-inflammatory (e.g. Slamf9, Irf7, Adam12, Siglec10) response than lactating rats (Fig1B, E). Regulatory T-cell transcription factor Foxp3 was upregulated in 3-week lactating animals compared to non-lactating animals, further supporting a switch to an anti-inflammatory environment in lactating animals.
Fiber cross-sectional area and percent of regenerating fibers did not differ between non-pregnant and late-pregnant uninjured controls. The proportion of regenerating myofibers in the lactating group did not return to the baseline control levels at 8 weeks after injury (6.7±1.04% vs 0.26±0.03%, P=0.0134, Fig.2E). Fiber size was significantly decreased at 4- and 8-weeks after injury in the non-lactating animals compared to the age-matched uninjured non-pregnant (NP) controls (P < 0.0001, Fig.2F). Pubocaudalis was enriched with larger fibers at 4- and 8-weeks after injury in the lactating animals compared to non-lactating counterparts (P < 0.0001, Fig.2F).
At acute and sub-acute time points of 1 and 3-weeks after injury, non-lactating animals present a more pro-inflammatory and extracellular matrix remodeling response than the lactating animals. Having an ECM remodeling response associated with fibroblast activation supports that non-lactating animals have more production of proinflammatory cytokines and collagen-containing ECM compared to lactating animals. At longer-term time points of 4- and 8-weeks after injury, the non-lactating animals demonstrate smaller fibers than the lactating animals. Lactating animals also continue to have significantly more actively regenerating myofibers at 8-weeks after injury. With these results, we found that lactation had an unexpected positive impact on PFM regeneration following simulated birth injury in a pregnant rat model despite the hypoestrogenic state. Elucidating the mechanism by which lactation potentiates PFM recovery and supports an anti-inflammatory environment after birth injury will be critical in identifying novel strategies that promote endogenous muscle regeneration.

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