S9 - The Acute Systemic and Local Impact of Lactation in a Pelvic Skeletal Muscle Birth Injury Rat 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 acutely following birth injury is vital for developing effective strategies to prevent PFM dysfunction postpartum. We aimed to assess the impact of the immediate 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. However, other hormones, such as prolactin and oxytocin, are known to be high in lactating animals and may contribute to driving a more anti-inflammatory response. Thus, we hypothesized that due to the fluctuation of hormones in lactating rats, there may be a more immediate pro-regenerative environment for PFM injury response than seen in non-lactating rats.

Materials and

Methods: : Late-pregnant (D20/22 gestation) Sprague-Dawley rats underwent simulated birth injury via vaginal balloon distention. Animals were randomized into non-lactating and lactating groups once litter was seen 2-days after injury and animals were then euthanized 1-day postpartum, 3-days after injury, for histology (N=3-4/group). For all animals, the serum and 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, were collected. Harvested pubocaudalis muscles were then snap frozen and cryosectioned for subsequent histological analyses. 1-day postpartum muscle cross-sections were incubated with anti-laminin antibody and anti-embryonic myosin heavy chain (eMyHC) antibody for quantification of newly formed embryonic fibers. Muscle cross-sections were also evaluated via hematoxylin and eosin staining to assess cell infiltration by nuclei density quantification. Serum collected was used to evaluate circulating proteins with a Proteome Profiler Rat XL Cytokine Array kit. Data were compared between groups using Mann-Whitney, unpaired student t-test, or multiple unpaired t-test with false-discovery rate approach.

Results, Conclusions, and Discussions:: Pubocaudalis muscle cross-sections 3-days after injury, 1-day postpartum with animals lactating or not-lactating for 24 hours, were significantly different in eMyHC+ fiber size and nuclei density. eMyHC+ fiber size was significantly larger in lactating animals compared to non-lactating animals (P < 0.0001, Fig.1C). Pubocaudalis had higher nuclei density in lactating animals compared to non-lactating, indicative of increased immune cell infiltration that is occurring immediately postpartum (P=0.03, Fig.1F).
With serum extracted 1-day postpartum we quantified 79 systemic proteins that may influence immediate postpartum PFM regeneration. There was an increase in many tissue remodeling and extracellular matrix proteins in lactating animals that typically increase later in injury response to help break down and rebuild ECM (Fig. 2A). There were higher amounts of galectins, known to have anti-inflammatory properties, and RGM-A, known to reduce pro-inflammatory cytokine production (Fig. 2B). While many growth factors were similar, IGFBP-5 was significantly higher in lactating animals (Fig. 2C). Many soluble receptors and adhesion molecules were higher in lactating animals, better informing the increase in cell infiltration in the injured pubocaudalis muscle (Fig. 2D). Cytokines were generally low, but prolactin, a milk producing hormone known to have anti-inflammatory properties, was in fact higher in lactating animals (Fig. 2E). There were no differences in adipokines, metabolic regulators, and chemokines (Fig. 2F, G).
At 1-day postpartum, lactating animals present more of a systemic immune response, with increases in adhesion molecules, galectins, and tissue remodeling proteins. The increase in VCAM-1 and ICAM-1 demonstrates that lactating animals have an immediate increase in immune cell trafficking markers that may contribute to quicker regeneration seen in the pubocaudalis muscle. ICAM-1 and IGFBP-5 are known to promote myogenesis and aid in muscle development. The larger eMyHC+ myofibers indicate earlier myogenesis and regeneration in lactating animals. We found that lactation had a positive impact on PFM regeneration following birth injury in a pregnant rat model despite the hypoestrogenic state. Lactation supports an immediate pro-regenerative environment that may trigger quicker regeneration than in non-lactating animals. Further understanding the mechanisms by which lactation is pro-regenerative will be critical in identifying novel strategies that promote endogenous muscle regeneration.

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