Pelvic floor disorders (PFDs), which are present in ~25% of women, profoundly impact quality of life. During childbirth, pelvic floor muscles (PFMs) endure substantial mechanical strain, which can lead to PFM dysfunction. As such, vaginal childbirth is a major risk factor for PFDs. Previous studies in rats demonstrate important structural adaptations in the PFMs that help to withstand the mechanical strain of childbirth, including muscle fiber lengthening by sarcomerogenesis. However, it is diffcult to establish a comprehensive understanding of the molecular mechanisms underlying PFM dysfunction during childbirth in rat models. Here, our objective was to determine if mouse PFMs undergo similar adaptations to rats during pregnancy and vaginal distension. We hypothesized that, akin to rats, PFMs in mice would undergo muscle fiber elongation through sarcomerogenesis during pregnancy. The PFMs (coccygeus [C], iliocaudalis [ICa], pubocaudalis [PCa]) and a non-pelvic floor muscle (tibialis anterior [TA]) were collected from late-pregnant (E16.5) and non-pregnant C57BL/6NJ (3 months old) mice. These animals were subjected or not subjected to physiological vaginal distension to mimic vaginal parturition (N = 4/group); in anesthetized mice, a 6F transurethral catheter was inserted into the vagina, and it was inflated with 0.3 mL (which best approximated fetal head circumference), and a 13 g weight was attached to the catheter creating circumferential and downward strains similar to parturition. Following this, the animals were euthanized, and the pelvis was fixed in situ for assessment of muscle architectural parameters; muscle fibers length was determined using digital calipers and sarcomere length was measured by light microscopy. Pregnancy induced a significant increase in normalized muscle fiber length in all PFMs compared to non-pregnant animals (Pregnant vs. not pregnant — C: 4.71±0.06 mm vs. 3.37±0.15 mm, p<0.0001; ICa 8.74±0.14 mm vs. 7.50±0.27 mm, p=0.004; PCa 7.13±0.09 mm vs. 6.28±0.34 mm, p=0.04); TA muscle fiber length was unchanged (6.92±0.17 mm vs. 6.73 ± 0.19 mm, p=0.28). Sarcomere length was not affected by pregnancy in any muscle (C: 2.44±0.002 μm vs. 2.45 ± 0.003 μm, p=0.55; ICa: 2.26±0.003 μm vs. 2.26±0.004 μm, p=0.64; PCa: 2.16±0.01 μm vs. 2.16±0.003 μm, p=0.74; TA: 2.55±0.004 μm vs. 2.54±0.003 μm, p=0.28). Balloon-mediated vaginal distention resulted in significantly longer sarcomere lengths in non-pregnant compared to pregnant animals in all PFMs (C: 2.98±0.01 μm vs. 2.43±0.01 μm, p<0.0001; ICa: 2.54±0.004 μm vs. 2.24±0.005 μm, p<0.0001; PCa: 2.85±0.001 μm vs. 2.17±0.001 μm, p<0.0001), indicating protection against sarcomere elongation in pelvic floor muscles of pregnant animals. This study shows that, similar to rats, pregnancy causes architectural changes in mouse pelvic floor muscles, which prevent excessive sarcomere stretching during physiologic vaginal distension that is comparable to fetal delivery. This work was funded by NIH grant #K12 HD000849 (Reproductive Scientist Development Program) to LAB and UC San Diego Senate Grant to LAB. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.