Childbirth trauma to the maternal pelvic soft tissues, including pelvic floor muscles (PFMs) and other soft tissues confers the greatest hazard for subsequent pelvic floor disorders, especially pelvic organ prolapse (POP), with attributable risk for POP as high as 46%.Thus, vaginal delivery is the most important modifiable risk factors for these potentially preventable conditions. Throughout adult life, skeletal muscles rely on muscle stem cells (MuSCs) to regenerate in response to injury. The physiological role of MuSCs has been established by seminal studies, mainly conducted in male experimental models, which unequivocally show that MuSCs are indispensable for regeneration of injured limb muscles. However, almost nothing is known about the role of MuSCs in PFM recovery after birth injury and in prevention of PFM dysfunction. To repair injured muscles, normally quiescent MuSCs need to activate, proliferate, and differentiate into new myofibers. Using the rat model, previously validated for the studies of the human PFMs, we aimed to investigate the role of MuSCs in PFM regeneration following simulated birth injury (SBI).

Overall, ten 3-month old female Sprague-Dawley rats were used for this study. PFMs were irradiated with a single dose of 20Gy at 1Gy/min to induce DNA damage within MuSCs and impair cell function. To assess the baseline effect of x-irradiation, PFMs were harvested 1-day post irradiation and prepared for immunohistochemical analyses. MuSC reservoir was compared to age-matched non-irradiated controls. In the following set of experiments, irradiated and non-irradiated rats were subjected to SBI, using vaginal balloon distention with 5mL volume. Animals were allowed to recover for 7 days, after which PFMs were similarly harvested and prepared for immunohistochemical analyses (Figure A). In situ MuSCs were identified with DSHB antibody against Pax7 transcription factor that is specifically expressed in the quiescent and activated MuSCs. To further assure proper identification of MuSCs, we capitalized on their unique location between sarcolemma and the basal lamina. Thus, in addition to DSHB Pax7 antibody coupled to Alexa Fluor 647 (green), PFM cross-sections were labeled with anti-laminin antibody coupled to Alexa Fluor 594 (red), and DAPI nuclei stain. MuSCs were identified with fluorescence microscopy by co-localization of these 3 fluorescent signals (Figure B) and quantified using a custom-written MATLAB program (MathWorks, Natick, MA). PFM regeneration was assessed by the presence of embryonic myosin heavy chain (eMyHC), expressed only in newly formed myofibers. eMyHC+ fibers’ cross-sectional area (CSA) was compared between the groups. Power calculation (G*Power) yielded n=2-3/group (alpha of 0.05; beta of 0.8), based on partial eta squared of 0.94. Data were compared using two-way ANOVA with Sidak’s post-hoc testing (Graph Prism, USA). Values are reported as mean ± SEM.

PFM stem cell reservoir did not differ between irradiated animals (14.7 ± 1.5 cells/mm2) and non-irradiated controls (12.5 ± 2.8 cells/mm2), P=0.9. Seven days after SBI, a dramatic increase in MuSC number was observed in non-irradiated controls (57.5 ± 3.4 cells/mm2). However, this was not recapitulated in irradiated rats, in whom MuSC quantity was almost identical to the non-injured irradiated counterparts (12.2 ± 1.2 cells/mm2), and significantly lower compared to injured non-irradiated animals, P<0.0001 (Figure B). The above denotes impaired proliferative capacity of MuSCs in the irradiated group. Concurrent with the lack of MuSC expansion in response to SBI, basal lamina was highly disorganized in irradiated rats (Figure C). CSA of eMyHC+ fibers was dramatically reduced (189.4 ± 7.8 µm2) in this group, compared to non-irradiated animals (500.0 ± 9.8 µm2), P<0.0001, demonstrating impaired PFM regeneration (Figure C).

Our results indicate that radiation does not impact MuSC number in the unperturbed homeostatic state. Despite normal MuSC reservoir, compromised MuSC function precludes stem cell expansion, in turn, delaying PFM recovery. Future assessment of functional outcomes is needed to confirm the causal link between MuSC deficiency and PFM dysfunction following birth injury.

Endogenous MuSCs appear indispensable for the efficient regeneration of PFMs after birth injury.