The Application of Shear Wave Elastography to Determine the Elasticity of the Levator Ani Muscle and Vaginal Tissue in Patients With Pelvic Organ Prolapse

The changes of the extracellular matrix of the connective tissue have significantly contributed to the incidence of pelvic organ prolapse (POP). It seems reasonable that sonoelastography could be a useful tool to evaluate the elasticity of pelvic floor tissue in patients with POP and compare it to those without POP. The main aim of this pilot study was to determine if there are differences in the elasticity of the levator ani muscle (LAM) and vaginal tissue between patients with and without POP.

involved such as the musculature, ligaments and other supporting tissue of the pelvic floor.Numerous risk factors have been described for the appearance of POP, mainly associated with damage of pelvic floor muscles and connective tissue, which are directly caused by pregnancy and vaginal delivery. 2,3Amongst them, the changes of the extracellular matrix of the connective tissue have significantly contributed to the appearance of POP. 4,5The most common types of collagen in the pelvic tissue are type I and III. 6Type I collagen as a high resistance to traction, while type III provides more flexibility.Patients with POP have been described to possess changes in the proportion of collagen type and reticulation. 7,8In fact, a recent meta-analysis showed that patients with POP have a lower expression of type I collagen than patients without POP, 9 without significant differences after stratification in terms of biopsy site (utero-sacral ligaments, cardinal ligaments, and vaginal tissue). 92][13][14][15] In fact, patients with POP more often have defects in the levator ani and generate less vaginal closure force during a maximal contraction. 12t seems reasonable that, likewise, an increased elasticity of the LAM might facilitate the appearance of POP.
][18][19][20][21] Tissue elasticity changes throughout pathological processes such as trauma, inflammation and tumors.Furthermore, the stiffness of any new formation associated with a higher risk for the appearance of dysplasias in the tissue studied. 227][18][19][20][21] The pressure on the tissue may be made by either a light mechanic compression (strain elastography, SE), or ultrasound waves (shear-wave sonoelastography, SWE).3][34] It seems reasonable that sonoelastography should be an useful tool to evaluate the elasticity of pelvic floor tissue in patients with POP and compare it to those without POP, allowing in vivo knowledge of the characteristics of the tissues of patients affected by POP.Thus, the main aim of this pilot study was to determine if there are differences in the elasticity of the LAM and vaginal tissue between patients with and without POP.

Study Population
A pilot observational and prospective study was conducted, including 60 patients (30 with POP and 30 without POP), who were consecutively recruited (first 30 patients with POP and first 30 patients without POP) in the Gynecology Unit at the Valme University Hospital (Seville, Spain) between September 1, 2022 and December 31, 2022.

Subjects
Patients were consecutively recruited, including 30 patients with POP and 30 patients without pelvic floor dysfunctions.The patients were recruited from the general gynecology clinic by a single specialist gynecologist who performed the clinical examination of each of them.All patients with a prior history of pelvic floor corrective surgery, hysterectomy or pelvic radiotherapy were excluded from the study.

Clinical Examination
All patients were submitted to a physical examination after a directed medical history, to evaluate the type (anterior vaginal wall prolapse, uterine prolapse, rectocele, or enterocele) and stage of prolapse of each compartment, according to the pelvic organ prolapse quantification system of the international continence society (ICS-POP-Q).Stage 0: No prolapse is demonstrated.Stage I: Most distal portion of the prolapse is more than 1 cm above the level of the hymen.Stage II: The most distal portion of the prolapse is situated between 1 cm above the hymen and 1 cm below the hymen.Stage III: The most distal portion of the prolapse is more than 1 cm beyond the plane of the hymen but everted at least 2 cm less than the total vaginal length.Stage IV: Complete eversion or eversion at least within 2 cm of the total length of the lower genital tract is demonstrated. 1

Ultrasound Assessment
7][18][19][20][21] Ultrasound assessment was performed using a Toshiba Aplio i700 ultrasound scanner (Canon Medical systems, Japan) with a transvaginal probe with the following machine settings for sonoelastography: elastogram speed map between 6.5 cm/s (red) and 0.5 cm/s (blue) meters/second with blue being indicative of softer tissues; 11C3, diffT7, 0.4 fps, G: 100, DR: 75, SW 6, 10.0 k, SF: 1.The probe transvaginal prove was introduced without any pressure, and was placed to obtain a midsagittal plane of the different study areas: vagina at the level of middle third of the urethra; vagina at the level of the trigone; vaginal in the anterior and posterior fornix; vagina at the level of middle third of the anorectal canal (Figures 1 and 2).Afterwards, we obtained an axial plane of the anal canal at the level of the LAM, and then the probe was lightly tilted to the right side of the patient, capturing the posterior third of the LAM (Figures 1 and 2).First, we performed an anatomical ultrasonography evaluation in B mode.Then, the sonoelastography region of interest is centered on the study area and the shear wave activated to obtain an elastogram speed map with an adequate propagation, which is confirmed with the visualization of parallel lines in the study area in the wavefront propagation map.The elastogram map should stay stable for at least 3 seconds before obtaining the elasticity measurements.Measurements were made in each study area by means of a 2-mm circular study window to obtain the mean, standard deviation and median of the elasticity, in kilopascals (kPa).That way we obtained quantitative measurements of the anatomical study areas of the study.

Statistical Analysis
Numeric variables were summarized by means, median, and standard deviation, while percentages were used for qualitative variables.The Shapiro-Wilk test was used to evaluate the normality of the data.The Student's t-test or the non-parametric Mann-Whitney test was used according to the normality of the data.For the comparison between more than two study groups, the following tests were used: ANOVA test for parametric data with equal variances (Levene test); ANOVA test with Welch correction for parametric data with different variances; and Kruskal-Wallis test for non-parametric data.Finally, we performed multiple linear regression models predictive of elastography variables.The hypotheses of linearity, normality, independence and equality of variances were verified.Based on a previously published study, 34 we calculated the sample size needed to detect a difference of 20 kPa in the elasticity of the LAM between the groups with and without POP, considering a common standard deviation of 25 kPa, an 5% alpha error and a 10% beta error (Power 90%).Thus, we needed at least 26 patients per study group.

Ethical Approval
The study was conducted in accordance with the Declaration of Helsinki (as revised in 2013).The study (0554-N-21) was approved by the local Ethics and Research Committees on April 13, 2021.
All patients gave their written informed consent before starting the study.

Results
A total of 60 patients completed the study: 30 patients with POP and 30 without POP.In the POP group,  Sonoelastographic parameters are shown in Table 2.We can observe that patients with POP had values suggesting higher elasticity in all anatomical study areas, with statistically significant differences in the anterior fornix (13.6 vs 11.2 kPa; P: .012).However, when controlling age and parity with a multiple regression (Table 2) we can see that other areas also had statistically significant differences, such as the posterior fornix (P: .034)and LAM (P: .014).
Likewise, we performed the multiple linear correlation coefficient adjusting for age, menopausal stage, parity and POP (Table 3), observing statistically significant values in the vagina at the level of the middle third of the urethra (r: .531;P: .003), in the vagina in the anterior fornix (r: .563;P < .001), in the vagina at the level of the middle third of the anorectal canal (r: .420;P: .049)and in the posterior third of the levator ani muscle (r: .501;P: .008).Table 4 shows the

Discussion
Patients with POP have a higher elasticity of vaginal tissue and LAM than patients without POP, as measured by sonoelastography.These sonoelastographic differences observed in patients with POP have been previously described in a study on the LAM. 34In fact, it has been established that, in patients with POP, LAM elasticity is significantly higher in patients with POP at rest (27.9 vs 31 kPa; P < .001),and lower during Valsalva maneuver (57.3 kPa vs 53.1 kPa; P < .05),describing lower elastic changes between rest and Valsalva maneuver in patients with POP and patients without POP, given the lower passive stretching capacity of LAM in patients with POP. 34his increased elasticity of the LAM in patients with POP could influence the presence of ballooning, being this hiatal ballooning is independent risk factors for symptoms and signs of prolapse.. 35 Now, our study shows by sonoelastography that patients with POP also present a higher degree of elasticity of vaginal tissues than patients without POP, as in a previous work applying vaginal tactile imaging. 36For correct sonoelastographic assessment, it is important to control the pressure exerted by the vaginal probe on the tissue to avoid erroneous measurements.To avoid this possible effect, we applied an elastogram speed map with an adequate propagation, confirming it with the visualization of parallel lines in the study area in the auxiliary wave front map (Figure 2).Nevertheless, the integrity of the LAM is not the only mechanism behind POP, as it origin is also influenced by the quality of the ligament support 37 and collagen type. 7,8A meta-analysis showed that patients with POP have a lower expression of type I collagen than patients without POP, 9 without significant differences after stratification in terms of biopsy site (utero-sacral ligaments, cardinal ligaments, and vaginal tissue). 9This could support our general results, as we observed a higher elasticity of vaginal tissue in patients with POP.Although POP is influenced by age, the biomechanical evaluation of vaginal tissue showed little difference between pre and postmenopausal patients. 38However, there are different in vaginal cells between premenopausal patients with and without POP. 392][43] Based on these premises, we adjusted our results controlling the age, menopausal stage and parity of patients, observed statistically significant differences in elasticity in the vagina at the level of the middle third of the urethra (r: .531;P: .003), in the anterior fornix (r: .563;P < .001),at the level of the middle third of the anorectal canal (r: .420;P: .049)and in the posterior third of the levator ani muscle (r: .501;P: .008).This increased elasticity in these specific areas might be explained by the type of POP that was included in the study as the majority were prolapse of the anterior and posterior compartment (30% anterior vaginal wall prolapse, 50% rectocele).We might presume that the high elasticity in these anatomical areas might enable the appearance of prolapse in these compartments.The main strength of this study was its novel design.To date, there are no other studies applying sonoelastography to evaluate vaginal elasticity in patients with POP.Currently, the lack of knowledge behind this aspect is quite relevant and, although we may not yet establish definite conclusions, these results pave the way for future studies in this field, where elasticity cutoff points are determined to optimize the type of treatment in each patient.The design of this work was based on the previously published study 34 in which we needed to recruit a small sample per study group.This might have facilitated the observed differences in the clinical characteristics of both study groups included, which might pose a limitation.However, we tried to reduce its impact on our results by controlling the age, parity, and menopausal stage when comparing study groups.However, with the data presented we cannot determine sonoelastographic values that allow us to make clinical decisions about our patients.Another limitation of this study is the small number of cases with uterine prolapse, which were all stage II.Furthermore, the measurement area could vary from patient to patient depending on the anatomical characteristics of each woman's vagina.In future studies that should include more severe prolapse in this compartment might show sonoelastographic differences between study groups, as well as determine the interand intraobserver variability of said technique.

Conclusions
In conclusion, we observed that it is possible to evaluate the elasticity of the LAM and vaginal tissue using sonoelastography, with a higher elasticity in patients with POP than in those without POP.

Figure 1 .
Figure 1.Anatomical study areas in the midsagittal plane [vagina at the level of middle third of the urethra (A); vagina at the level of the trigone (B); vagina in the anterior fornix (C); vagina in the posterior fornix (D); vagina at the level of middle third of the anorectal canal (E)] and axial plane [posterior third of the LAM (F)].

Figure 2 .
Figure 2. Sonoelastographic captures of the anatomical study areas in the midsagittal plane [vagina at the level of middle third of the urethra (A); vagina at the level of the trigone (B); vagina in the anterior fornix (C); vagina in the posterior fornix (D); vagina at the level of middle third of the anorectal canal (E)] and axial plane [posterior third of the LAM (F)].

Table 1 .
General and Clinical Characteristics of the Patient Included

Table 3 .
Sonoelastographic Parameters-Square Multiple Correlation Coefficient Adjusting by Age, Menopausal Stage, Parity, and POP

Table 4 .
Sonoelastographic parameters-Multiple Linear Regression Adjusting by Age, Menopausal Stage, Parity, and POP a Variables without predictive capacity.García-Mejido et al-Elasticity of the Vaginal Tissue in Patients With POP