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Appearance of the Levator Ani Muscle Subdivisions in Endovaginal Three-Dimensional Ultrasonography

Shobeiri, S Abbas MD1; LeClaire, Edgar MD2; Nihira, Mikio A. MD, MPH1; Quiroz, Lieschen H. MD1; O’Donoghue, Daniel PA-C, PhD1

doi: 10.1097/AOG.0b013e3181aa2c89
Original Research

OBJECTIVE: To formulate an evaluation system and authenticate the levator ani subdivisions visible on endovaginal three-dimensional ultrasonography.

METHODS: A three-dimensional endovaginal ultrasound examination was performed on five fresh-frozen female pelves. The location of the pubovaginalis, puboperinealis, puboanalis, puborectalis, and iliococcygeus muscles was identified. Three-dimensional endovaginal ultrasound scans of 22 nulliparous women with normal pelvic floors were obtained, and a three-level evaluation system was constructed. Level 1 contained the muscles as they insert into the perineal body. Level 2 evaluated the insertions into the retropubic area and typically contained the pubovaginalis, puboperinealis, puboanalis, and puborectalis. Level 3 contained muscles that were cephalad to the pubic bone. Two blinded reviewers assessed levator ani subdivision visualization at each level and individual muscle visualization by the origin–insertion points.

RESULTS: There was 98%, 96%, and 92% agreement for levels 1, 2, and 3 muscles, with 95% confidence intervals (CIs) of 0.92–1, 0.95–0.99, and 0.88–0.95, respectively. Kappa values (95% CI) for agreement were calculated for individual muscles as follows: superficial transverse perinei and puborectalis were seen by both raters 100%, puboperinealis, pubovaginalis, and puboanalis 0.645 (0.1–1), and iliococcygeus 0.9 (0.6–1).

CONCLUSION: The location of the pubovaginalis, puboperinealis, puboanalis, puborectalis, and iliococcygeus as seen by three-dimensional endovaginal ultrasonography was confirmed through anatomic dissection of fresh-frozen pelves. Subdivisions of the levator ani muscle were visualized reliably with three-dimensional ultrasonography using a systematic approach.


The location of the pubovaginalis, puboperinealis, puboanalis, puborectalis, and iliococcygeus as seen by three-dimensional endovaginal ultrasonography are confirmed through anatomic dissection of fresh-frozen pelves.

From the 1University of Oklahoma Health Sciences Center and the 2United States Air Force, Department of Obstetrics and Gynecology, Oklahoma City, Oklahoma.

Supported by an equipment loan and a subject recruitment grant from BK Medical (Herlev, Denmark).

Presented at 2008 meeting of the International Urogynecological Association, September 15–17, 2008, Taipei, Taiwan.

Corresponding author: S. Abbas Shobeiri, MD, University of Oklahoma Health Sciences Center, Department of Obstetrics and Gynecology, Associate Professor and Chief, Section of Female Pelvic Medicine & Reconstructive Surgery, 920 Stanton L. Young Blvd. (P.O. Box 26901), WP2410, Oklahoma City, OK 73190; e-mail:

Financial Disclosure The authors did not report any potential conflicts of interest.

Given that we employ concepts such as pelvic floor spasm, levator spasm, and pelvic floor weakness, understanding the basic concepts of pelvic floor musculature is essential to formulating a clinical opinion. The area posterior to the pubic bone is dense with bands of intertwined levator ani muscles that defy the conventional description of the levator ani consisting of the puborectalis, pubococcygeus, and iliococcygeus. The anatomy of distal subdivisions of the levator ani muscle was described further in a recent study.1 Using a nomenclature based on the attachment points of different subdivisions of the levator ani muscles, the muscles posterior to the pubic bone are identified as the pubovaginalis, puboanalis, and puboperinealis as the subdivisions of the pubovisceralis.2 Margulies and colleagues show excellent reliability and reproducibility of visualizing major portions of the levator ani with magnetic resonance imaging (MRI) in nulliparous volunteers.3 Because the puboanalis, pubovaginalis, and puboperinealis are small, they have proven to be hard to visualize in rigid axial, coronal, and sagittal views of the MRI.

The levator ani muscle is arranged as a unique, three-dimensional structure that lends itself to dynamic imaging by ultrasonography. Ultrasound imaging is rapidly evolving to facilitate imaging of the levator ani muscle. Three-dimensional and four-dimensional translabial ultrasound methods for the assessment of puborectalis have been published extensively by centers around the world owing to ease of visualization and availability of equipment. There are no anatomic or correlative studies substantiating the ultrasonic descriptions in these studies. An OVID search from 1950 to March 2009 using the terms “ultrasound,” “pelvic floor,” “levator ani,” “3D,” 3 dimensional,” “vaginal,” and “endovaginal” did not produce any studies that may have used an endovaginal, three-dimensional, pelvic-floor scanning technique as we describe in the current study. The ability to visualize the iliococcygeus, puborectalis, puboperinealis, pubovaginalis, and puboanalis is important because it is hypothesized that dysfunction by any of these muscles would cause respective clinical findings. For example, a detached puboperinealis can be associated with perineal detachment.

The current study was designed to 1) use three-dimensional endovaginal ultrasound imaging to better visualize distal levator ani subdivisions, 2) authenticate and identify the muscles as seen on three-dimensional endovaginal ultrasound scans in cadaveric dissections, and 3) validate our findings in live, nulliparous women.

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All phases of the current study were approved by the institutional review board at our institution. All participants signed appropriate research consent forms. Because this study investigated a new technology for visualization of pelvic floor musculature, all the authors were involved in all aspects of design and implementation of the study to ensure that we developed a sound methodology for identification of the target muscles. There were three phases in this study.

Phase I was designed to identify pelvic floor muscles as seen on three-dimensional endovaginal ultrasonography performed in fresh cadavers. Fresh-frozen pelves were prepared from unperfused/unfixed specimens. Immediately after the pelves were defrosted for 36 hours and brought to room temperature in an external bath, three-dimensional endovaginal ultrasonography was performed by S.A.S. and E.L. BK Medical (Wilmington, MA), with a 2050 probe with three-dimensional imaging technology, was used for acquisition of images. Scanning was performed from 6 cm cephalad to the hymen to 3 cm caudad to the hymenal ring. Two overlapping scans were performed. Echogenic structures suspected of being the superficial transverse perinei, pubovaginalis, puboperinealis, puboanalis, puborectalis, and iliococcygeus were tagged with biopsy needles (MPM Medical, Elmwood Park, NJ) and marked with 1 cm3 of indigo carmine dye for localization. Additionally, any other unknown structures and possible defects were tagged in the same manner (Fig. 1). The findings were cataloged and images captured. After each pelvis was scanned ultrasonographically, the findings were recorded digitally and the pelves were dissected by S.A.S. while the other authors participated to locate each of the numbered needles in all the cadaveric specimens (Fig. 2).





Phase II was the development of a scoring system for visualization of the pelvic floor muscles in nulliparous individuals. The principal investigator (S.A.S.) performed the three-dimensional scans in 50 volunteers. Initially, a sample of 28 scans of women with varied pelvic support and etiology was evaluated to gain experience with the morphological appearance of levator ani subdivisions using previously published anatomic information.2,3 Volunteers were recruited by campus e-mails and flyers. The nulliparous patients were provided with $100 recruitment incentives. The ultrasound imaging was performed. A length of 6 cm was scanned in 60 seconds with scans every 0.25 mm for a cumulative 240 scans from which a three-dimensionally rendered cube was calculated. Each three-dimensional cube was digitally catalogued for future analysis. The ultrasound cubes were reviewed to develop a method of classification based on presence or absence of muscles at different levels.

The investigators discussed the morphology of the subdivisions and arrived at a consensus on the characteristic features. The characteristic features of each of the five separate levator subdivisions were determined for each scan plane by the following two criteria: 1) a clear and visible separation between adjacent structures or 2) differing origin–insertion points of the muscle.

All of the investigators agreed on a three-level system before commencing the evaluation. Level 1 contained the muscles that insert into the perineal body, namely the superficial transverse perinei, puboperinealis and puboanalis. The superficial transverse perinei served as the reference point. Level 2 contained the attachment of the pubovaginalis, puboperinealis, puboanalis, puborectalis, and iliococcygeus to the pubic bone. Level 3 contained subdivisions visible cephalad to the inferior pubic ramus, namely the pubococcygeus and iliococcygeus, which winged out toward the ischial spine. The visualization of the pubococcygeus was debatable, and, because this structure was not visualized reliably during pelvic-floor dissection, it was not included (Fig. 3). Two investigators, (S.A.S. and E.L.) individually reviewed each of the 22 scans from the nulliparous group. The levator ani subdivisions in these scans were examined at levels 1, 2, and 3 according to the two criteria noted above. An exemplary scan of a 25-year-old nulliparous woman was selected for image clarity based on a described classification method.2 Next, a three-dimensional PowerPoint (Microsoft, Redmond, WA) library was created, describing what structures needed to be identified at each level (Figs. 4 and 5). The creation of a PowerPoint reference was useful for displaying the three-dimensional ultrasound characteristic features of the levator ani muscle subdivisions.







Phase III was estimating interrater reliability. The scans were captured by the principal investigator (S.A.S.). For each participant, two raters were asked whether muscle subdivisions were “visible” or “not visible” at levels 1, 2, and 3. The visibility was scored by these two observers, who were blinded to the identity of the volunteers and the scores of the other observer. Interrater reliability was calculated by taking the number of agreements and dividing by the number of observations in the total number of scans.

If the observer wanted to confirm the identity of the structure, they simply manipulated the planes on the three-dimensional cube to trace the entire muscle subdivision to look at the origination–insertion points (Fig. 5). Cohen’s kappa index and 95% confidence intervals (CIs) were assessed for visualization of each individual muscle as well. Kappa values of 0.8–1.0 were considered excellent agreement, 0.6–0.8 good, 0.4–0.6 moderate, 0.2–0.4 fair, and less than 0.2 poor.

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Phase I. The pelves were selected randomly. The specimens were obtained under research agreement from our institution’s anatomy program between August 2006 and March 2007. Of the five dissected pelves, three were posthysterectomy. The average age for the pelves was 54 years (range 52–59). In the ultrasound imaging and the correlative dissections, the superficial transverse perinei was the first muscle visualized. Immediately cephalad to it was the puboperinealis insertion into the perineal body. In the dissections, the puboanalis was located deep and lateral to the puboperinealis and had a wide base inserting itself into the anorectal fibers. Puboanalis fibers intermixed with lateral supportive fibers of the rectum to form the posterior arcus,4,5 which in turn fused with laterally located fibers of the iliococcygeus. The pubovaginalis was a short band 3 cm cephalad to the ischiopubic rami, causing an indentation in the anterior vaginal epithelium. Puborectalis insertion was lateral, wrapping itself around the rectum 3 cm cephalad to the anus. By ultrasonography, the puboperinealis had mixed echogenicity and was located immediately cephalad to the superficial transverse perinei. The puboanalis was identified as a triangular hypoechoic area lateral to the puboperinealis. The pubovaginalis was identified as dense muscular bands at the level of the midurethra in cadavers and as hypoechoic areas causing heart-shaped angulation of the anterior vaginal mucosa. All of these structures and the iliococcygeus were identified accurately by needle identification during three-dimensional endovaginal ultrasonography and authenticated by gross dissection.

Phase II. The 22 nulliparous volunteers had a mean age of 24 years (range 23–30) and a mean body mass index of 24 (range 20–34) (body mass index is calculated as weight (kg)/[height (m)]2); 21 were white, and one was Asian. None had any prior surgeries and none suffered from any acute or chronic illnesses. The three-dimensional endovaginal ultrasound scans were reviewed by two observers using the scoring system outlined in the Methods section.

Phase III. There was 98%, 96%, and 92% agreement for levels 1, 2, and 3 muscles, with 95% CIs of 0.92–1, 0.95–0.99, and 0.88–0.95, respectively. Kappa values (95% CI) for agreement were calculated for individual muscles as follows: the superficial transverse perinei and puborectalis were seen by both raters 100%, puboperinealis, pubovaginalis, and puboanalis 0.645 (0.1–1), and iliococcygeus 0.9 (0.6–1).

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Our study substantiates that three-dimensional endovaginal ultrasonography can be used for the evaluation of the pelvic floor muscles. In the current study, we demonstrated that three-dimensional endovaginal ultrasonography can visualize subdivisions of the levator ani muscle reliably. Our scientific knowledge about injuries to the pelvic floor muscles has increased dramatically by the use of techniques such as electromyography, MRI, and transperineal three-dimensional ultrasonography.3,6 Magnetic resonance imaging has shown that, during a vaginal delivery, the levator ani muscles can be injured as a result of mechanical stretch.3 The puborectalis is implicated as the muscle that sustains the most stretch.7 There is a lack of knowledge about the puboperinealis, puboanalis, and pubovaginalis muscles and the injuries they may sustain during vaginal delivery.8 Pinpointing where the specific injury occurs will help in development of better treatment options. The ease with which three-dimensional endovaginal ultrasonography can be performed in a clinical setting provides an opportunity to investigate levator ani injury in large populations.

The current study has several weaknesses. The phase 1 pelves were from older women with unknown obstetrical histories, three of whom were posthysterectomy. The inability to visualize the pubococcygeus portion of the pubovisceralis may have been avoided in a study of younger pelves. In total, 10 hemipelves were dissected. The sample size of dissected pelves (five) and the nulliparous volunteers scanned (22) was not as large as we would have liked owing to funding limitation. Lastly, one of the ultrasound observers was also the principal investigator, which may predispose to bias. However, this aspect was minimized by making the ultrasound images unidentifiable in respect to the volunteer’s personal information.

The endovaginal three-dimensional ultrasound technique allowed us to document the complex anatomy of the levator ani muscle in graphic detail and in a permanent, objective way. The prospect that this could be used to study dynamic changes is exciting. We used an extensive, methodical, anatomical approach to authenticate our technique, which has not been the case for transperineal ultrasonography. Validation of three-dimensional ultrasonography findings will enable us to use ultrasonography as a tool to study and diagnose anatomic changes in the pelvic floor muscles of living women. Three-dimensional endovaginal ultrasonography may be a less expensive and more comfortable option than MRI for the evaluation of distal pelvic floor muscles, which are poorly visualized by MRI.3 Magnetic resonance imaging scan software has not yet been refined to the point of allowing us to visualize fiber bundles in their entirety if they are not in axial, coronal, or sagittal views, as is the case with subdivisions of the levator ani muscle. Because the levator ani has a complicated three-dimensional geometry, MRI has limitations because these muscles defy any one orthogonal scan plane to distinguish the subdivisions.3

In living women, we have visualized differing origin–insertion points of five separate subdivisions of the levator ani muscle by endovaginal three-dimensional ultrasonography through a systematic approach. Unlike MRI, which requires time-consuming reconstruction of the levator ani muscle to document its distal components, endovaginal ultrasonography creates an instant three-dimensional image of the levator ani muscle. Additionally, unlike MRI, the three-dimensional ultrasound cube can be manipulated instantly to visualize the origin and insertion points, creating clear images for the trained ultrasonographer. The ease of use is important because it would take the imaging modality out of the hands of a few experts and put it into the hands of practicing physicians who can screen patients for pelvic floor defects. This technology is already available to general practitioners for endoanal ultrasound imaging. The procedure takes less than 5 minutes to perform and can be taught proficiently.

Knowing exactly which muscle is damaged is not inconsequential in clinical practice. The levator ani anatomy and the function of the pelvic floor governing micturition, defecation, and intercourse are inseparable. Many of the functions have just recently been understood by describing the subdivisions of the levator ani muscle. Attachments are important because the muscles exert their action by contraction. For example, a patient with defecatory dysfunction due to a detached puboperinealis will not benefit from a posterior repair. Also, reattachment of the puboperinealis does not address defecatory dysfunction due to loss of the anorectal angle from a damaged puborectalis.

In this study of women with normal support, we found that two examiners consistently could identify the visibility of the levator ani subdivisions puboperinealis, puboanalis, and pubovaginalis, which are not easily visible by MRI and have yet to be described in any other ultrasound study. Future research will focus on investigating whether the presence or absence of the puboperinealis, puboanalis, and pubovaginalis subdivisions of the levator ani can be identified in women with disease.9,10

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Appearance of the Levator Ani Muscle Subdivisions in Endovaginal Three-Dimensional Ultrasonography
Timor-Tritsch, IE
Obstetrics & Gynecology, 114(5): 1145.
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© 2009 by The American College of Obstetricians and Gynecologists.