Deterioration of pelvic floor muscle function and prolonged urethral dysfunction in a rat model of simulated birth trauma

Introduction

Female urinary incontinence is a urologic disorder defined as the involuntary leakage of urine, with its overall prevalence ranging from 25% to 45% worldwide (1). Urinary incontinence is categorized into stress urinary incontinence (SUI), urge urinary incontinence, and mixed urinary incontinence, with SUI accounting for 49% of all cases. The majority of SUI is often attributed to pregnancy and childbirth as well as advanced age. Reportedly, 12.2% of women with a history of vaginal delivery develop SUI (2). In addition, it has been shown that pelvic floor damage due to vaginal delivery can have long-term consequences.

Treatment of SUI includes behavioral therapies, such as lifestyle modifications and pelvic floor muscle (PFM) training, pharmacotherapy, and surgical interventions. Behavioral therapy is often recommended as a first-line conservative treatment option (3).

PFM training aims to strengthen the contractility of the PFM through repeated contraction and relaxation. Biopsies have shown that slow-twitch fibers (type I) make up approximately 70% of the human PFM, while fast-twitch fibers (type II) make up 30% of the PFM (4). Type I fibers in the PFM have a slow contraction rate and excellent endurance, and are responsible for supporting the pelvic organs. Meanwhile, type II fibers have a faster contraction rate and superior instantaneous force, and are involved in urethral closure (5). To maximize the muscle-strengthening effects of training, it is extremely important to fully understand the pathophysiology of SUI and to tailor the training to the characteristics of each muscle fiber type.

To elucidate the mechanism of SUI development after vaginal delivery, we investigated the changes in muscle composition for PFM in a rat model of simulated birth trauma via vaginal distention (VD) (5). Our previous study focusing on the subacute phase (4 weeks post-VD) showed that muscle atrophy occurred in the pubococcygeus muscle (Pcm), and a reduction in the proportion of type I fibers was observed (6). With regard to urethral function, urinary incontinence episode during elevated abdominal pressure occurred in the 1 week after VD, but resolved within 2 weeks. These results indicated that SUI after VD may be influenced by muscle composition changes in the PFM and muscle weakness due to muscle atrophy (6).

Investigation of the mid-to long-term effects of simulated birth trauma on the PFM is urgently needed. While our previous study showed that incontinence episode during elevated abdominal pressure had resolved by 2 weeks post-VD, clinical evidence indicates that 2–21% of women experience persistent SUI for over two months after delivery (7,8). Since our earlier study focused on a short period of time, specifically 4 weeks after vaginal delivery, we speculated that a more detailed study of the long-term effects on the PFM would be necessary.

Characterizing mid- to long-term PFM changes after simulated birth trauma has significant clinical implications, as it would enable the development of more targeted and effective PFM training protocols, potentially improving treatment outcomes for prolonged SUI.

The purpose of this study was to investigate the mid- to long-term effects of simulated birth trauma on the muscle composition of the PFM and urethral function using a rat VD model. We present this article in accordance with the ARRIVE reporting checklist (available at https://tau.amegroups.com/article/view/10.21037/tau-2026-0357/rc).

Methods

Animals and experimental design

Eighteen female Sprague-Dawley rats (12 weeks old, purchased from Sankyo Labo Service Corporation, Inc., Sapporo, Japan) were allocated into three groups: sham surgery (sham), 4 weeks post-VD (4W group), and 12 weeks post-VD (12W group) (n=6/group). The rats from the sham and 4W groups were previously utilized in our earlier study (6), while the 12W group comprised newly acquired animals with identical characteristics. To clarify the long-term changes, the functional and histological data from the sham and 4W groups were re-analyzed and compared with the newly obtained data from the 12W group in this study. The animals were given water and rat chow ad libitum throughout the study period. At the end of the experimental period, animals were euthanized by exsanguination under deep anesthesia induced by 3% isoflurane inhalation. All animal experiments were performed under a project license (No. 019-0062) granted by the Animal Experimentation Committee of Hokkaido University, in compliance with national guidelines for the care and use of animals.

Surgical procedure

A 14-Fr Foley catheter (Clinique, Yokohama, Japan), for which the tip had been removed, was inserted into the vagina, and the vaginal opening was sutured to prevent the catheter from dislodging under anesthesia. The balloon was then expanded for 4 hours with 4 mL of water, thus impeding urethral closure (9). For the sham surgery, the catheter was left uninflated after its insertion into the vagina. This procedure was identical to that described in our previous report (6), ensuring consistent experimental conditions across all groups.

Measurement of urethral pressure

Both ureters were tied off to suppress reflex bladder contractions. Vesical branches of the pelvic nerves that were located near the internal iliac vessels were bilaterally severed. A 3.5-Fr nylon catheter (SPR-524; Millar Instruments, Houston, TX, USA), equipped with a microtransducer 1 mm away from the tip and connected to a pressure control unit (PCU-2000; Millar Instruments), was then inserted into the urethra at a depth of 12.5–15 mm from the urethral opening. An analog-to-digital converter (PowerLab^®; AD Instruments, Nagoya, Japan) was used to track the urethral response, with data captured at a sampling rate of 400 Hz on a personal computer. An electrical stimulator (SEN-3301; Nihon Kohden, Tokyo, Japan) and isolator (SS-104J; Nihon Kohden) were utilized to stimulate the exposed bilateral oblique abdominal muscles (0.5 ms duration; 1.8–2.0 V intensity; 20-ms intervals; repeated every 10 s). The amplitude of the urethral response to electrical stimulation (A-URE), defined as the maximum pressure change (cmH₂O) from baseline, and the urethral baseline pressure (UBP; measurements were made at the flat portion immediately before the pressure response to stimulation) were recorded. Electrodes were inserted near the 11th to 13th ribs via bilateral incisions in order to access the oblique abdominal muscles during electrical stimulation (10). The rats were positioned supine under anesthesia for the duration of the measurements. In accordance with our previous study (6), measurements of UBP were taken 4 weeks after VD in the 4W group, 12 weeks after VD in the 12W group, and 2 weeks after sham surgery in the sham group.

Tissue sampling

After UBP and A-URE measurements, under urethane anesthesia, scissors were used to open the iliac crest via insertion through the pubic symphysis, thereby enabling the collection of the target iliococcygeus muscle (Icm) and pubococcygeus muscle (Pcm) from both sides.

Muscle wet weight (MWW) measurement

The harvested bilateral Pcm and Icm were weighed using a digital scale to measure their MWW, and the weight-to-body ratio (MWW/body weight) was calculated.

Histochemistry

All harvested muscles were immersed in isopentane solution cooled with liquid nitrogen for freezing, and stored at −80 ℃ until further analysis. To obtain representative images of the histochemical profiles for the target muscles, central portions of the muscles were selected (11), and 10 µm transverse continuous sections were cut on a cryostat (CM 1510 S; Leica Biosystems, Nussloch, Germany) cooled to −20 ℃ and air-dried. The frozen sections were stained for succinate dehydrogenase (SDH) activity and myosin adenosine triphosphatase (ATPase; pH 10.2) activity (12). Based on a previous protocol (13), after alkaline pre-incubation, consecutive sections were treated for myofibrillar ATPase. For the SDH staining, all sections were incubated for 30 minutes at 37 ℃ with a mixture of nitro blue tetrazolium, phosphate buffer, and sodium succinate.

Morphometry

Three muscle fiber types were identified based on SDH activity and myofibrillar ATPase activity (pH 10.2). Fibers were categorized into three groups: type I (slow-twitch oxidative), type IIa (fast-twitch oxidative glycolytic), and type IIb (fast-twitch glycolytic). Stained sections were observed under a light microscope (BX 43, Olympus, Tokyo, Japan) at a magnification of 100×. Photographs of each muscle section were captured using a digital camera mounted on a microscope (FX 630, Olympus). In this study, the cross-sectional area (CSA) of 100 randomly selected fibers from each type was determined, using the Icm and Pcm samples from the left side. The captured images were then analyzed using image processing software (ImageJ version 1.52a, NIH) for CSA measurements.

Statistical analysis

The Kruskal-Wallis test, Welch’s t-test, and one-way analysis of variance (ANOVA) were used to conduct comparisons of UBP, A-URE, MWW/body weight (mg/g), CSA, and myofiber composition types among the three groups. Tukey’s test was utilized for subsequent analyses of parametric variables following ANOVA, while nonparametric variables that were assessed using Kruskal-Wallis were then evaluated using the Steel-Dwass multiple comparison test. Statistical significance was set at P<0.05. The R software package (version 2.8.1) was used to perform statistical analyses.

Results

To investigate the mid- to long-term effects of simulated birth trauma on urethral function and PFM composition, we compared urethral pressure parameters (UBP and A-URE), MWW, histochemical fiber composition, and morphometric data among sham, 4W, and 12W post-VD groups. Overall, urethral function showed a tendency toward partial recovery at 12 weeks, whereas structural alterations in the PFM, including persistent muscle atrophy and reduced type I fiber occupancy, remained evident throughout the observation period.

UBP comparison

The UBP was not significantly different among the sham (22.3±7.6 cmH₂O), 4W (24.7±8.1 cmH₂O), and 12W (24.2±7.9 cmH₂O) groups (P=0.86). The 4W group had a significantly lower A-URE (32.5±5.2 cmH₂O) than the sham group (55.4±17.0 cmH₂O) (P=0.04) (Figure 1).

Figure 1 The UBP was not significantly different among the 4W, 12W, and sham groups. The 4W group had significantly lower A-URE values than the sham group (P=0.04). Values were presented as mean ± standard deviation. ANOVA and Tukey’s test were used for statistical analysis. *, statistically significant difference (P<0.05 compared to the sham group). Sham: sham-operated group; 4W: 4 weeks after vaginal distention; 12W: 12 weeks after vaginal distention. A-URE, amplitude of the urethral response to electrical stimulation; ANOVA, analysis of variance; UBP, urethral baseline pressure.

MWW comparison

The 4W (0.39±0.07 mg/g, P<0.01) and 12W (0.41±0.07 mg/g, P=0.01) groups had significantly lower MWWs for the Pcm than the sham group (0.57±0.08 mg/g).

There was no significant difference in the MWW for the Icm among the 4W (0.50±0.05 mg/g), 12W (0.57±0.06 mg/g), and sham (0.59±0.08 mg/g) groups (P=0.40) (Figure 2).

Figure 2 The muscle wet weight for the Pcm was significantly lower in the 4W (P<0.01) and 12W (P=0.01) groups compared to the sham group. There was no significant difference in the muscle wet weight for the Icm among the three groups. Values were presented as mean ± standard deviation. Tukey’s test was used for statistical analysis. Statistically significant difference: *, P<0.05; **, P<0.01 compared to the sham group. Sham: sham-operated group; 4W: 4 weeks after vaginal distention; 12W: 12 weeks after vaginal distention. Icm, iliococcygeus muscle; Pcm, pubococcygeus muscle.

Histochemical analysis

Pcm

The 4W (3.1%±2.7%, P<0.01) and 12W (6.9%±4.8%, P<0.01) groups had significantly lower type I ratios than the sham group (17.1%±3.9%). There was no significant difference in the type IIa ratio among the three groups (P=0.14). The 4W (63.7%±6.8%, P=0.01) and 12W (65.1%±5.0%, P=0.04) groups had significantly higher type IIb ratios than the sham group (55.2%±3.6%) (Table 1, Figure 3).

Figure 3 Representative ATPase (pH 10.2) staining of the Pcm and ratios of type I, IIa, and IIb fibers in the 4W, 12W, and sham groups. ATPase staining was observed in rat Pcm sections for the sham group (A), 4W group (B), and 12W group (C). Type I and type II fibers were noted in the sections [examples in (A)]. Scale bar =100 µm. Sham: sham-operated group; 4W: 4 weeks after vaginal distention; 12W: 12 weeks after vaginal distention. ATPase, myosin adenosine triphosphatase; Pcm, pubococcygeus muscle.

Icm

The 4W (4.5%±3.1%) and 12W (6.9%±3.1%) groups had significantly lower type I ratios than the sham group (15.6%±3.7%) (all P<0.01). There were no significant differences in the type IIa (P=0.23) and type IIb (P=0.05) ratios among the three groups (Table 2, Figure 4).

Figure 4 Representative SDH staining of the Icm and ratios of type I, IIa, and IIb fibers in the 4W, 12W, and sham groups. SDH staining was observed in rat Icm sections for the sham group (A), 4W group (B), and 12W group (C). Type I, type IIa, and type IIb fibers were noted in the sections [examples in (A)]. Scale bar =100 µm. Sham: sham-operated group; 4W: 4 weeks after vaginal distention; 12W: 12 weeks after vaginal distention. Icm, iliococcygeus muscle; SDH, succinate dehydrogenase.

Morphometric analysis

Pcm

The 4W (1,183.9±126.7 µm²) group had a significantly lower CSA for Pcm type I fibers than the 12W (1,507.5±213.7 µm²) group (P=0.04). There were no significant differences in the CSA for type IIa (P=0.21) and type IIb (P=0.13) fibers among the three groups (Figure 5).

Figure 5 The 4W group had a significantly lower CSA for type I fibers in the Pcm than the 12W group (P=0.04). There was no significant difference in the CSA for type IIa and IIb fibers in the Pcm among the three groups. Values were presented as mean ± standard deviation. ANOVA and Tukey’s test were used for statistical analysis. *, statistically significant difference (P<0.05 compared to the 12W group). Sham: sham-operated group; 4W: 4 weeks after vaginal distention; 12W: 12 weeks after vaginal distention. ANOVA, analysis of variance; CSA, cross-sectional area; n.s., not significant; Pcm, pubococcygeus muscle.

Icm

The 4W (1,238.9±179.4 µm²) group had a significantly lower CSA for Icm type I fibers than the sham group (1,947.8±235.5 µm²) (P=0.01). The 12W (1,883.8±180.7 µm²) group had a significantly lower CSA for type IIa fibers than the sham (2,246.5±265.1 µm²) group (P=0.04). There was no significant difference in the CSA for type IIb fibers among the three groups (P=0.07) (Figure 6).

Figure 6 The 4W group had a significantly lower CSA for type I fibers in the Icm than the sham group (P=0.01). The 12W group had a significantly lower CSA for type IIa fibers in the Icm than the sham group (P=0.04). There was no significant difference in the CSA for type IIb fibers in the Icm among the three groups. Values were presented as mean ± standard deviation. ANOVA and Tukey’s test were used for statistical analysis. *, statistically significant difference (P<0.05 compared to the sham group). Sham: sham-operated group; 4W: 4 weeks after vaginal distention; 12W: 12 weeks after vaginal distention. ANOVA, analysis of variance; CSA, cross-sectional area; Icm, iliococcygeus muscle; n.s., not significant.

Discussion

This study aimed to investigate the mid- to long-term effects of simulated birth trauma on urethral function and PFM composition in a rat model simulating birth trauma.

Regarding urethral function, there was no significant difference in UBP among the three groups, and the A-URE was significantly lower in the 4W group than in the sham group. The A-URE is measured as the maximum pressure change (cmH₂O) from UBP to evaluate urethral function at elevated abdominal pressure. Clinically, a decrease in A-URE in VD model rats is thought to reflect the dysfunction of the external urethral sphincter (14). Our current results confirm the presence of SUI in the rat model 4 weeks post-VD. Importantly, while the A-URE in the 12W group showed a tendency toward recovery, the morphological changes in the PFM persisted. To our knowledge, the present study is the first report examining urethral function after VD over a mid- to long-term period of 12 weeks.

UBP and A-URE reflect different components of urethral continence function. As demonstrated in a previous study using the VD rat model, UBP likely represents the baseline coapting urethral function including urethral smooth muscle activity, which is mediated by α-adrenoceptors and hypogastric nerve innervation, whereas A-URE reflects striated muscle-mediated continence mechanisms dependent on pudendal nerve activation of the external urethral sphincter and PFMs (14). Consistent with this interpretation, our previous study also demonstrated that UBP remained stable across all time points following single VD, while A-URE was significantly reduced (6). These findings suggest that single VD preferentially impairs striated muscle-mediated active urethral closure mechanisms while leaving smooth muscle-dependent baseline urethral tone relatively intact.

We next examined structural changes in the PFM, including MWW and fiber-type composition, to further characterize the mid- to long-term effects of simulated birth trauma. The MWW/body weight in the Pcm was significantly lower in the 4W and 12W groups compared with the sham group. Previous research suggests that muscle atrophy is present 1 month after denervation and can progress for the next 6 months, which aligns with the findings of this study (15). The apparent paradox can be explained by fiber type specific differences in the atrophic response to denervation-like injury. In fast muscles of the rat, type II fibers undergo rapid atrophy, whereas type I fibers retain their CSAs for at least two months, and type I fibers continue to be more resistant to atrophy than type II fibers even in later post-denervation stages (15). Similar fiber type specific patterns have also been reported in other rat muscles, where denervation selectively induced atrophy of type IIb fibers while type I fiber CSA remained unchanged or slightly hypertrophied (16). These findings are consistent with our results: the increase in type I fiber CSA observed between 4W and 12W likely reflects the relative resistance of slow-twitch fibers to atrophy, or a compensatory hypertrophic response in residual type I fibers, while the continued reduction in overall MWW reflects the dominant and progressive atrophy of the numerically predominant type IIb fibers in the Pcm. Taken together, the net reduction in overall muscle mass and the relative preservation—or partial compensatory enlargement—of type I fiber CSA are not contradictory, and may instead suggest differential temporal dynamics of fiber type specific atrophy following simulated birth trauma.

The more pronounced and persistent atrophy observed in the Pcm compared with the Icm may be attributable to anatomical differences between the two muscles. The Pcm is located in closer proximity to the urethra and vagina, rendering it more susceptible to direct mechanical injury during VD, whereas the Icm is positioned more laterally and may be less directly exposed to the compressive forces generated by vaginal balloon distension. These anatomical considerations may also be relevant to the differences observed in CSA between the two muscles. This anatomical vulnerability of the Pcm is consistent with clinical imaging studies in humans, which have demonstrated that the Pcm undergoes the greatest elongation during vaginal delivery and is therefore at the highest risk for stretch-related injury (17).

In contrast, while the 4W group had a significantly lower CSA for type I fibers in the Icm than the sham group, there were no significant differences in the CSA for type I fibers in the Pcm between the sham group and the VD groups.

A study investigating histological changes in denervated muscles in rats over a 2-month period (18) reported a significant decrease in CSA for type II fibers in the extensor digitorum longus muscle, while no changes were observed in type I fibers. Additionally, in the tibialis anterior muscle, a significant reduction in CSA for type II fibers was observed, whereas a significant increase in CSA for type I fibers was noted during the first 1–2 months after denervation. These findings highlight the heterogeneity in CSA responses to denervation across several studies. Furthermore, previous investigations have shown varied responses to denervation beyond any changes in CSA. For instance, a previous report showed decline in the total number of muscle fibers 4 months after denervation (19). In our current study, atrophy of the Pcm persisted up to 12 weeks after simulated birth trauma. While we did not measure the total fiber count, the persistent atrophy might involve multiple mechanisms, including potential changes in fiber number, in addition to the documented CSA changes. The observation that Pcm atrophy persisted into the chronic phase (12 weeks), despite partial functional recovery, underscores the importance of long-term histological monitoring to fully understand the impact of simulated birth trauma on PFM integrity. Future studies that directly quantify both fiber size and number in these specific muscles would help clarify the complete nature of trauma-induced atrophy in PFM.

Regarding the muscle fiber composition, our 12-week data revealed a critical finding: the occupancy of type I fibers in both the Icm and Pcm remained significantly lower than in the sham group, even after the subacute phase. Given that pelvic floor injury due to simulated birth trauma has been shown to have long-term effects (20,21), these results may be explained by the fact that urethral function shows a tendency toward recovery at 12 weeks after VD, whereas structural alterations in PFM composition—including persistent reductions in type I fiber occupancy and muscle mass—continue to persist beyond the subacute phase. Muscle atrophy affects type I fibers of slow-twitch muscles to a greater extent than type II fibers of fast-twitch muscles, leading to a shift to fast-twitch muscle and a decrease in duration of muscle contraction (22). In addition, the mechanisms of muscle atrophy are related to changes in composition of myofiber types and decreased rates of muscle protein reduction and synthesis (23,24).

A study that compared the electromyographic characteristics of PFM in women with SUI following vaginal delivery with those of healthy nulliparous women (25) reported a delay in muscle activation, a reduction in activation duration, and an absence of appropriate responses with SUI. Based on these results, simulated birth trauma may be associated with the prolonged persistence of SUI, potentially through sustained impairment of the contractile strength and endurance of type I fibers due to PFM damage. This suggests that while the urethral sphincter itself may show functional improvement, the underlying weakness of the PFM support remains a potential risk factor for chronic SUI.

In summary, our findings suggest that PFM damage associated with simulated birth trauma may alter muscle composition, which could be consistent with a decrease in PFM endurance and supportive strength, potentially contributing to the development and prolongation of SUI. From a clinical perspective, PFM training for SUI has traditionally tended to prioritize fast-twitch (type II) fiber training, given the role of these fibers in rapid urethral closure during sudden increases in abdominal pressure. However, the present findings demonstrate a sustained reduction in slow-twitch (type I) fiber occupancy in both the Pcm and Icm persisting up to 12 weeks after simulated birth trauma. Given that type I fibers are primarily responsible for tonic contraction and sustained support of the pelvic organs and maintaining baseline urethral closure, these results suggest that rehabilitation protocols targeting slow-twitch fiber endurance in addition to fast-twitch fiber strengthening may warrant consideration in the long-term management of birth trauma-related SUI.

However, several limitations should be acknowledged in interpreting these findings. First, rats and humans have different PFM compositions. While slow-twitch muscles are more common in humans, fast-twitch muscles are more common in rats (26). This species difference may limit the direct extrapolation of our results to human physiology. Second, the total number of muscle fibers was not measured in this study, which may have limited our understanding of the mechanisms underlying muscle compositional changes following birth trauma.

Conclusions

In this study, the VD rat model exhibited persistent compositional changes in the PFM for up to 12 weeks post-injury, beyond the initial resolution of incontinence episode during elevated abdominal pressure. These findings suggest that VD associated alterations in muscle fiber composition and reduced muscle endurance may suggest a potential mechanism contributing to the long-term persistence of SUI in clinical settings.

Acknowledgments

The authors are grateful to all members of the Department of Renal and Genitourinary Surgery, Graduate School of Medicine, Hokkaido University for their advice and constructive criticism of this project.

Footnote

Reporting Checklist: The authors have completed the ARRIVE reporting checklist. Available at https://tau.amegroups.com/article/view/10.21037/tau-2026-0357/rc

Data Sharing Statement: Available at https://tau.amegroups.com/article/view/10.21037/tau-2026-0357/dss

Peer Review File: Available at https://tau.amegroups.com/article/view/10.21037/tau-2026-0357/prf

Funding: None.

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tau.amegroups.com/article/view/10.21037/tau-2026-0357/coif). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. All animal experiments were performed under a project license (No. 019-0062) granted by the Animal Experimentation Committee of Hokkaido University, in compliance with national guidelines for the care and use of animals.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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