This study clarified the distribution of muscle tissue around the urethra in women and the relationship between EUS and LA. Our research showed that four layers of muscle surround the female urethra: uLM, uCM, skeletal muscle (EUS), and the outermost smooth muscle layer from the inner to the outer layer. The smooth muscle layer was contiguous with the smooth muscles of the vaginal wall. Smooth muscle fibers extending from the vaginal wall were found between the LA and EUS. This finding indicates that the LA muscle influences urinary function by interacting with the smooth muscle between the LA and EUS.

Previous studies have described three muscle layers surrounding the female urethra: uLM, uCM, and striated muscle from the inner to the outer layer. The morphological characteristics of each muscle layer have been previously reported. For example, some studies reported that the uCM is thinner than the uLM, and the striated sphincter layer encircles the urethra, with the thickest muscle layer in the middle [4, 5]. However, its extension into the surrounding muscle layers has not been reported in detail. Our findings revealed four rather than three layers of muscle surrounding the female urethra. In addition to the three layers discussed in previous studies, there is an outermost smooth muscle layer. Previous studies have mainly focused on the sphincteric action of the internal urethral sphincter and EUS in relation to urethral control and contraction in women [5, 21]. Consequently, the EUS in women is widely regarded as a key component in controlling urethral contraction and managing urinary incontinence [22, 23]. Our study highlights the presence of the outermost smooth muscle layer and its connective role within the skeletal muscle groups of the female urethra and its essential role in urinary control.

This study showed that the urethra is surrounded by the outermost smooth muscle layer, which is contiguous with the smooth muscle of the vaginal wall. This smooth muscle is not only attached to the LA muscle, as reported by Kato et al. [4], but is also connected to the EUS. The smooth muscle may likely be involved in elevating the LA, thereby influencing the action of the EUS. When the LA contracts, it pulls this smooth muscle upward and forward, which subsequently supports the posterior and inferior aspects of the urethra. It is hypothesized that urethral fixation by smooth muscles enhances the contractile force of the EUS. The horseshoe-shaped structure of the EUS, which surrounds the anterior and lateral sides of the urethra, is believed to support this function.

Moreover, this smooth muscle layer protruded anterolaterally and extended its muscle fibers toward the pubic bone. In the proximal part of the female urethra, the smooth muscle extending toward the pubic bone is a part of the pubo-urethral ligament [4]. This structure further supports the idea that smooth muscles pull the urethra upward and forward, thereby stabilizing it and aiding the EUS in urethral closure. The anterior curvature of the distal urethra in some specimens may reflect this elevating action of the smooth muscle (Fig. 3a and b). In male individuals, the EUS is continuous with the external anal sphincter (EAS) and LA; therefore, the supportive action of the EAS and LA in EUS contractions is thought to contribute to its urinary function. In addition, smooth muscle tissue is present on the dorsal side of the urethra in male individuals and is believed to facilitate the contractile function of EUS [16]. However, in female individuals, EUS is not directly continuous with the LA or EAS; therefore, urinary function may depend significantly on the balance of the entire pelvic floor, particularly on urethral fixation by the smooth muscle. The uLM and uCM do not form a simple ring around the urethra; instead, these layers extend anteriorly, separating the skeletal muscle layer, which forms a horseshoe shape around the urethra without muscle fibers posteriorly and extends ventrally in a horseshoe shape, spreading side to side.

The relationship between urethral muscles and LA is still debated. In men, a study has shown continuity between the skeletal muscles of the EUS, LA, EAS, and bulbospongiosus. That study also revealed that the smooth muscles of the rectum fit into the spaces created by these muscles [15]. For women, some studies indicated that the LA may not actively participate in urethral control because it does not encircle the ventral side of the urethra [10]. In contrast, others reported that proper LA function is necessary for EUS function since the lower portion of the female EUS is connected to the puborectal portion of the LA by tendons [11]. A previous study by our research group on female urethra showed that vaginal smooth muscle fibers covered the urethra. We found that the LA and smooth muscle fibers were in direct contact and that the LA may have an impact on the function of the female urethra [4]. In addition, our current study clarified that smooth muscle fibers extend from the vagina between the LA and EUS in female individuals. Smooth muscle fibers were in direct contact with the LA and EUS, mediating the relationship between them. Thus, the LA may affect the function of the female urethra even without a direct connection to the urethral sphincter.

Understanding the distribution of the muscles around the urethra in women is clinically significant. To date, attention has primarily been focused on the urethral sphincter muscles when considering urinary control. However, our findings suggest that urinary function depends on the urethral sphincter and the balance with surrounding structures. The pelvic floor is susceptible to aging, and its structure descends with age [24, 25]. Under such conditions, it is necessary to consider both the urethral structure and function and the balance of the pelvic floor.

In addition to the sphincter effect, the striated urethral sphincter acts as a support structure to maintain stability and urinary continence [26]. The inner smooth muscle surrounding the urethra is thought to enhance the EUS force [25]. In particular, uLM contraction can shorten urethral length, lower closure pressure, and strengthen the sphincter mechanism, which is essential for initiating urination [5, 27]. This study revealed that the arrangement of the muscle layers differs depending on the urethral height, which may result in different effects on urination. The discovery that the urethra is not uniform and that its functional roles may vary depending on its height is crucial.

This study had some limitations. First, the cadaver donors were older adults with an average age of over 78 years, which could mean the weaker muscles in the aging female urethra may influence the muscle distribution. Second, the information on BMI, medical history, specifically obstetric history, vaginal deliveries, and history of pelvic/vaginal surgeries (prolapse/incontinence) of the cadavers was not possible because such information was not collected during the body donation process.