Changes in the Pelvic Floor Muscle Stem Cell Phenotype During Pregnancy Francesca Boscolo, PhD1; Alessandra Sacco, PhD2; Marianna Alperin1, MD MS. 1Department of Obstetrics, Gynecology, and Reproductive Sciences, University of California, San Diego. 2Development, Aging and Regeneration Program, Sanford Burnham Prebys Medical Discovery Institute. Pelvic floor disorders (PFDs) are a major public health issue given their high prevalence, negative impact on quality of life of millions of women, and related economic burden. Maternal childbirth injury to the pelvic floor supportive structures, including pelvic floor muscles (PFMs), confers the greatest hazard for subsequent PFDs. Computational models of human parturition demonstrate that PFMs’ elongation up to 300% of resting muscle length is necessary to achieve fetal delivery. Thus, muscle injury would be expected to occur in most, if not all, vaginal deliveries; however, the majority of women do not exhibit PFM injuries after childbirth. We hypothesized that structural and functional adaptations, acquired by the PFMs during pregnancy, may account for the ability of these muscles to withstand “supraphysiologic” strains during parturition without injury. Using the validated rat model, we have previously shown that during pregnancy PFMs undergo fiber elongation via sarcomerogenesis, or serial addition of muscle functional units, known as sarcomeres. This protective adaptation enables PFMs to withstand deformations during parturition without excessive sarcomere hyperelongation, in turn, decreasing PFMs’ susceptibility to mechanical birth injury. Muscle stem cells (MuSC), are responsible for maintenance of muscle homeostasis, response to altered physiological conditions, such as increased load, and regeneration in case of injury. To exert their function, MuSCs, quiescent in unperturbed muscles, become activated, proliferate and differentiate. In this study, we tested the hypothesis that MuSCs are involved in pregnancy-induced adaptations of the rat PFMs. To determine phenotypic changes associated with MuSCs during pregnancy, we first assessed in vivo cells proliferative ability at different time points across the rat gestational period: non-pregnant (NP), mid-pregnant (MP; D11 out of 23-day gestation), and late-pregnant (LP; D21). Through EdU incorporation assay together with Pax7 (MuSC marker) immunohistochemistry, we observed a significant increase in MuSC proliferation in MP animals. LP MuSCs showed reduced proliferation compared to NP and MP cells. These results suggest that MuSCs become activated during the first half of pregnancy, and then return to quiescence by the end of the gestational period. To determine the fate of the activated cells, we assessed the proportion of MuSCs positive for Myogenin (differentiation marker). In MP rats, Myogenin+ MuSCs were significantly increased compared to NP and LP time points. These results were further validated through qRT-PCR performed on freshly isolated cells. To determine whether the observed changes were cell autonomous, we isolated MuSCs from rat PFMs and assessed their ability to divide in vitro through time-lapse microscopy. Consistent with our in vivo data, MuSCs from MP animals entered the cell cycle faster compared to MuSCs procured from NP and LP rats, indicating the increased activation. To determine whether LP MuSCs re-entry into quiescence is comparable to NP quiescent state, we performed RNA sequencing analysis and observed a significant decrease in cell growth, cell proliferation, and cell differentiation pathways in LP cells. To validate these results, we assessed MuSC differentiation in vitro. LP MuSCs exhibited significant decrease in differentiation ability compared to NP cells. Altogether these results suggest that MuSC quiescent state is not comparable in NP and LP animals, and that the observed difference is cell autonomous. In summary, the physiological changes associated with pregnancy have a strong effect on the MuSC autonomous behavior. During the first half of pregnancy, MuSCs become activated, returning to quiescence during the second half of pregnancy. These changes in MuSCs behavior are consistent with pregnancy-induced adaptation timeline, where fiber elongation starts during the first half of pregnancy, aided by activated MuSCs, to be completed by the end of the rat gestation. In late pregnancy, MuSCs return to quiescence. Altogether these results strongly suggest that MuSCs are involved in fiber elongation via sarcomerogenesis during gestation. Thus, PFM resident stem cells contribute to the generation of protective antepartum adaptations that mitigate susceptibility to muscle birth injury. Future directions focus on determining the specific physiological stimuli that govern PFM stem cell changes during pregnancy.