Association Between 3D Transvaginal Ultrasound-Based XI VOCAL Counting Severity and Tissue MMP-9 Levels in Patients with Adenomyosis

A total of 54 patients were included. XI VOCAL Counting severity was positively associated with tissue MMP-9 levels. The correlation was moderate during the proliferative phase (r = 0.680) and stronger during the secretory phase (r = 0.814; p < 0.001).

XI VOCAL Counting severity was positively associated with tissue MMP-9 levels in patients with adenomyosis, with a stronger association observed during the secretory phase. These findings suggest that XI VOCAL Counting may provide a potential imaging-based severity assessment associated with molecular characteristics related to extracellular matrix remodeling. Further prospective multicenter studies are required to validate its reproducibility, clinical utility, and association with clinical outcomes.

adenomyosis, MMP-9, XI vocal counting, 3D transvaginal ultrasound, direct signs

Adenomyosis is a benign uterine disorder characterized by the presence of endometrial glands and stroma within the myometrium, resulting in structural and functional changes of the uterus. This condition is associated with various clinical manifestations, including dysmenorrhea, chronic pelvic pain, abnormal uterine bleeding, and infertility, which may substantially affect women’s quality of life.1,2 The prevalence of adenomyosis varies widely across the globe, ranging from 5–70%, influenced by differences in population, diagnostic methods, and advances in imaging technology.3 Data from Asia are relatively limited, and a Chinese study involving 30,629 women from 2017–2020 reported a low prevalence of adenomyosis at 2.35%, likely influenced by variations in diagnostic access, imaging criteria, and population heterogeneity.4 In the Indonesian population, at Dr. Hasan Sadikin General Hospital in 2016, 70.7% of 116 cases of adenomyosis occurred in women aged over 35 years and 28.4% in the 20–35 age group.5 Although adenomyosis is increasingly recognized as a significant gynecological condition, accurate assessment of disease severity remains challenging due to variations in diagnostic approaches and the lack of standardized quantitative parameters.2,4 Transvaginal ultrasound (TVUS) is widely used for adenomyosis assessment because of its accessibility and ability to identify characteristic morphological features. The Morphological Uterus Sonographic Assessment (MUSA) consensus provides standardized criteria by defining direct and indirect sonographic signs of adenomyosis, including subendometrial lines and buds, hyperechoic islands, and intramyometrial cysts.3,6 However, conventional ultrasound assessment remains primarily focused on morphological characteristics and may not fully reflect the underlying biological activity of adenomyotic lesions.7 The XI VOCAL Counting method has recently been introduced as a three-dimensional transvaginal ultrasound-based approach to quantitatively assess adenomyosis severity by counting transverse uterine sections demonstrating MUSA direct signs. This method may provide a more objective evaluation of lesion distribution compared with conventional subjective assessment.8 However, whether XI VOCAL Counting severity corresponds with molecular characteristics of adenomyotic tissue remains unclear. Matrix metalloproteinase-9 (MMP-9) is an extracellular matrix-degrading enzyme involved in tissue remodeling processes. Increased MMP-9 expression has been reported in adenomyosis tissue, suggesting its potential role as a molecular indicator associated with biological activity and extracellular matrix remodeling in adenomyotic lesions.9,10 Therefore, investigating the relationship between quantitative imaging parameters and molecular markers may provide further insight into the biological relevance of ultrasound-based severity assessment. Previous studies have primarily investigated adenomyosis from two separate perspectives: ultrasound-based studies have focused on identifying and standardizing morphological characteristics using MUSA criteria and severity assessment approaches, whereas molecular studies have explored MMP-9 expression as an indicator related to extracellular matrix remodeling.3,6,8,10 However, the relationship between quantitative ultrasound severity assessment and tissue-level molecular changes remains insufficiently explored. Therefore, this study aimed to investigate the association between 3D transvaginal ultrasound-based XI VOCAL Counting severity and tissue MMP-9 levels in patients with adenomyosis. We hypothesized that higher XI VOCAL Counting severity would be associated with higher tissue MMP-9 levels.

Study Design and Participants This analytical observational study used a cross-sectional design to investigate the association between three-dimensional transvaginal ultrasound (3D-TVUS)-based XI VOCAL Counting severity and tissue MMP-9 levels in patients with adenomyosis. The study was conducted at Dr. Hasan Sadikin General Hospital and Bandung Kiwari District General Hospital, Bandung, Indonesia, from February to April 2026. The study protocol was approved by the Health Research Ethics Committee of Dr. Hasan Sadikin General Hospital, Bandung (No. DP.04.03/D.XIV.6.5/51/2026). Written informed consent was obtained from all participants before enrollment. The study population consisted of women with adenomyosis who underwent surgical management at the study centers. Adenomyosis diagnosis was initially established through TVUS evaluation based on the MUSA criteria and subsequently confirmed through histopathological examination of surgical specimens. All participants underwent preoperative 3D-TVUS examination for XI VOCAL Counting assessment, and adenomyotic tissue samples obtained during surgery were analyzed for tissue MMP-9 levels. Eligibility Criteria Participants were included if they were women aged 20–49 years with adenomyosis confirmed by histopathological examination after surgery. Participants were required to have available preoperative 3D-TVUS volume datasets for XI VOCAL Counting assessment and adequate adenomyotic tissue samples for MMP-9 analysis. Patients were excluded if they had uterine malignancy, uterine fibroids, concomitant endometriosis, active infection, abnormal uterine bleeding, irregular menstrual cycles, or were in the menstrual phase during ultrasound examination. Patients who had received hormonal therapy within the previous three months, including gonadotropin-releasing hormone agonists, progestin-only contraceptives, or combined estrogen-progesterone therapy, were also excluded. Patients receiving anti-inflammatory medication, including corticosteroids or non-steroidal anti-inflammatory drugs, within the previous three months were excluded. Patients with immunological, endocrine, or metabolic disorders were also excluded. Participants with normal body mass index (BMI) according to World Health Organization criteria were selected to minimize potential confounding effects of obesity-related inflammation and altered estrogen metabolism on MMP-9 expression.11 Sample Size Estimation The sample size was calculated based on two analytical objectives: comparison of tissue MMP-9 levels among XI VOCAL Counting severity groups and correlation analysis between XI VOCAL Counting severity and tissue MMP-9 levels. For comparison analysis, the sample size calculation was based on previous findings by Inagaki et al using an alpha error of 0.05, statistical power of 80%, pooled standard deviation of 2.85, and expected mean difference of 4.7.12 For correlation analysis, the sample size calculation was based on a minimum expected correlation coefficient of 0.5, alpha error of 0.05, and statistical power of 80% based on previous findings by Li et al.13 The minimum calculated sample size was 34 participants. To compensate for possible incomplete data and participant exclusion, the final sample consisted of 54 participants. Participants were subsequently categorized into mild, moderate, and severe adenomyosis groups according to XI VOCAL Counting severity classification. Data Collection and Measurements Baseline Characteristics The participants completed a questionnaire concerning sociodemographic variables, including age, body mass index (BMI), parity history, curettage history, reported complaints (infertility and pain), endometrial phase, and type of surgical procedure. Body Mass Index (BMI), calculated by dividing body weight in kilograms by the square of height in meters, is a commonly utilized statistic for assessing an individual’s nutritional condition. The BMI is classified into four categories: underweight (below 18.5 kg/m2), normal weight (18.5–24.9 kg/m2), overweight (25.0–29.9 kg/m2), and obese (30.0 kg/m2 or more).11 The endometrial phase was assessed in participants with confirmed adenomyosis based on clinical data and TVUS findings, specifically the proliferative and secretory phases with regular menstrual cycles. The definition of a regular menstrual cycle based on the criteria of The International Federation of Gynecology and Obstetrics (FIGO) is a frequency of 24 to 38 days, a duration of ≤ 8 days, regularity with a variation of ≤7-9 days, and a menstrual volume that does not disrupt the quality of life of women.14 Three-Dimensional Transvaginal Ultrasound and XI VOCAL Counting Assessment Preoperative 3D-TVUS examinations were performed using Voluson E10 ultrasound equipment at Dr. Hasan Sadikin General Hospital and Voluson E8 ultrasound equipment at Bandung Kiwari District General Hospital. The ultrasound examination consisted of conventional two-dimensional assessment and three-dimensional volume acquisition using Tomographic Ultrasound Imaging (TUI) and Volume Contrast Imaging (VCI) modes. The XI VOCAL Counting method was performed using the acquired three-dimensional uterine volume dataset. Adenomyosis severity was assessed by counting the number of transverse uterine sections demonstrating MUSA direct signs. A total of 20 transverse sections were evaluated. The assessed MUSA direct signs included subendometrial lines and buds, hyperechoic islands, and intramyometrial cysts. XI VOCAL Counting severity was classified according to the number of sections demonstrating at least one direct sign: mild adenomyosis: 0–6 affected sections; moderate adenomyosis: 7–14 affected sections; and severe adenomyosis: 15–20 affected sections.8 Before data collection, examiner standardization and calibration procedures were performed to ensure consistency in XI VOCAL Counting assessment. The ultrasound volume datasets were evaluated independently from molecular analysis results. The reliability of XI VOCAL Counting assessment demonstrated excellent interobserver agreement, with an intraclass correlation coefficient (ICC) of 0.982 (95% confidence interval: 0.958–0.995) and Cronbach’s alpha coefficient of 0.983. Tissue Sampling and Histopathological Confirmation Adenomyotic tissue samples were obtained during surgical procedures. Tissue sampling was performed based on intraoperative identification of adenomyotic lesions and subsequently confirmed by histopathological examination. Histopathological confirmation of adenomyosis was based on the presence of ectopic endometrial glands and stroma within the myometrium. Tissue Preparation and MMP-9 Measurement Tissue samples measuring approximately 1 cm3 were prepared for molecular analysis. The samples were washed using cold phosphate-buffered saline (PBS) with pH 7.4 and homogenized using PBS at a ratio of 1:9 with a Magnaliser homogenizer. The homogenized tissue samples were centrifuged at 1000 rpm for 5 minutes, and the supernatant was collected for MMP-9 analysis. Tissue samples were stored according to the study protocol. Samples were stored at 4°C for short-term storage up to five days, at −20°C for storage up to one month, and at −80°C for longer storage up to six months. Aliquoting procedures were performed to minimize repeated freeze-thaw cycles. Tissue MMP-9 levels were measured using the Human MMP-9 Enzyme-Linked Immunosorbent Assay (ELISA) method at the Molecular Genetics Laboratory, Faculty of Medicine, Padjadjaran University-Dr. Hasan Sadikin General Hospital. The ELISA procedure was performed according to the manufacturer’s protocol. The analysis involved standard preparation, sample incubation, washing procedures, antibody incubation, enzymatic reaction, substrate incubation, and optical density measurement at 450 nm. Statistical Analysis Statistical analysis was performed using IBM SPSS Statistics version 26.0 for Windows. Data processing included data editing, coding, entry, and cleaning before statistical analysis. Descriptive analysis was performed to summarize participant characteristics. Normally distributed continuous variables were presented as mean ± standard deviation, while non-normally distributed continuous variables were presented as median and range. Categorical variables were presented as frequencies and percentages. The distribution of numerical variables was assessed using the Shapiro–Wilk test, and homogeneity of variance was evaluated using the Levene test. Differences in tissue MMP-9 levels among mild, moderate, and severe adenomyosis groups based on XI VOCAL Counting severity classification were analyzed using one-way analysis of variance (ANOVA) for normally distributed data or the Kruskal–Wallis test for non-normally distributed data. Associations between categorical variables and XI VOCAL Counting severity categories were evaluated using the Chi-square test or Fisher’s exact test when appropriate. The correlation between XI VOCAL Counting severity score and tissue MMP-9 levels was analyzed using the Spearman rank correlation test, considering the ordinal characteristics of XI VOCAL Counting severity categories and the numerical characteristics of MMP-9 levels. The strength and direction of correlation were interpreted based on the correlation coefficient (r). Statistical significance was determined at a p-value ≤0.05.

A total of 54 patients with adenomyosis were included in this study and categorized according to XI VOCAL Counting severity into mild (n=19), moderate (n=12), and severe (n=23) groups. Baseline characteristics are presented in Table 1. Significant differences among severity groups were observed in age, parity, history of curettage, infertility complaints, pain complaints, endometrial phase distribution, and MUSA direct signs, including subendometrial lines and buds, hyperechoic islands, and intramyometrial cysts (p<0.05). | Table 1 Baseline Characteristics of Patients According to XI VOCAL Counting Severity | The comparison of tissue MMP-9 levels according to XI VOCAL Counting severity stratified by endometrial phase is presented in Table 2. Tissue MMP-9 levels increased significantly across increasing severity categories in both phases. During the proliferative phase, mean tissue MMP-9 levels were 31.6 ± 5.3 ng/mL in the mild group, 43.3 ± 1.7 ng/mL in the moderate group, and 65.2 ± 18.4 ng/mL in the severe group (p=0.002). During the secretory phase, mean tissue MMP-9 levels were 37.3 ± 1.1 ng/mL, 56.1 ± 6.6 ng/mL, and 112.7 ± 45.2 ng/mL in the mild, moderate, and severe groups, respectively (p<0.001). | Table 2 Tissue MMP-9 Levels According to XI VOCAL Counting Severity Stratified by Endometrial Phase | The correlation analysis between XI VOCAL Counting score and tissue MMP-9 levels is summarized in Table 3 and visualized in Figure 1. A significant positive correlation was identified between XI VOCAL Counting score and tissue MMP-9 levels in both endometrial phases. In the proliferative phase (n=22), XI VOCAL Counting score showed a moderate positive correlation with tissue MMP-9 levels (Spearman r=0.680; p=0.001). In the secretory phase (n=32), a stronger positive correlation was observed (Spearman r=0.814; p<0.001). The scatter plots demonstrated a consistent upward distribution pattern between XI VOCAL Counting score and tissue MMP-9 levels across both phases, with a stronger association observed during the secretory phase (Figure 1). | Table 3 Correlation Between XI VOCAL Counting Severity and Tissue MMP-9 Levels According to Endometrial Phase |

This study demonstrated that higher XI VOCAL Counting severity scores were associated with increased tissue MMP-9 levels in patients with adenomyosis. The association was observed in both endometrial phases, with a moderate correlation during the proliferative phase (r=0.680) and a stronger correlation during the secretory phase (r=0.814). These findings suggest that XI VOCAL Counting may provide an objective ultrasound-based assessment reflecting molecular characteristics associated with adenomyosis severity. Previous studies have mainly evaluated adenomyosis severity through imaging characteristics or molecular alterations separately. The MUSA consensus has standardized the evaluation of direct ultrasound signs, including subendometrial lines and buds, hyperechoic islands, and intramyometrial cysts, while molecular studies have demonstrated increased expression of extracellular matrix-related markers such as MMP-9 in adenomyotic tissue.6,10 Our findings extend previous evidence by demonstrating an association between a quantitative ultrasound severity approach and tissue-level molecular changes. The progressive increase in MMP-9 levels across XI VOCAL severity categories suggests that greater sonographic involvement may correspond with increased extracellular matrix remodeling activity in adenomyotic tissue. MMP-9 has been reported to contribute to extracellular matrix degradation and tissue remodeling processes; therefore, increased MMP-9 expression may represent one biological characteristic associated with more extensive adenomyotic lesions.10 However, because this study used a cross-sectional design, the observed relationship should be interpreted as an association rather than evidence of causality or disease progression. The stronger correlation observed during the secretory phase may indicate that endometrial phase influences the relationship between ultrasound findings and tissue MMP-9 expression. Previous studies have suggested that hormonal alterations, including progesterone resistance, may influence inflammatory responses and extracellular matrix regulation in adenomyosis.15 Nevertheless, hormonal pathways and molecular regulators were not directly measured in this study; therefore, these mechanisms should be considered possible explanations rather than demonstrated mechanisms. The findings also support the potential clinical relevance of XI VOCAL Counting as an imaging-based severity assessment. Conventional ultrasound evaluation relies mainly on morphological interpretation, whereas XI VOCAL Counting provides a semi-quantitative approach by measuring the number of uterine sections containing MUSA direct signs. This quantitative approach may improve objective assessment of adenomyosis severity, although further validation is required before implementation as a standardized clinical marker.8 The main strength of this study is the integration of quantitative 3D transvaginal ultrasound assessment using XI VOCAL Counting with tissue-level MMP-9 evaluation, providing an imaging–molecular correlation approach for adenomyosis severity assessment. The phase-specific analysis according to the endometrial cycle also provides additional information regarding the variation of this association. Several limitations should be acknowledged. The cross-sectional design limits causal interpretation between XI VOCAL severity and MMP-9 expression. In addition, the study population consisted of surgical patients from referral hospitals, which may limit generalizability. The absence of a non-adenomyosis control group, MRI comparison, and external reproducibility assessment also limits further clinical validation of XI VOCAL Counting. Additionally, the unequal distribution of endometrial phases among severity categories may have influenced the observed association between XI VOCAL Counting severity and MMP-9 levels. Future multicenter prospective studies are needed to validate XI VOCAL Counting, establish standardized severity thresholds, and evaluate its association with clinical outcomes. Integration with MRI parameters and additional molecular markers may further clarify the biological and clinical relevance of this imaging-based assessment.

In this study of patients with adenomyosis, XI VOCAL Counting severity was positively associated with tissue MMP-9 levels. Higher XI VOCAL severity scores corresponded with increased MMP-9 expression, with stronger correlations observed during the secretory phase. These findings suggest that XI VOCAL Counting may provide a potential imaging-based severity assessment reflecting molecular characteristics related to extracellular matrix remodeling. Further prospective, multicenter studies with standardized thresholds and control of potential confounding factors are required to validate its reproducibility, clinical utility, and association with disease outcomes. Ethics Approval and Consent to Participate This study complies with the Declaration of Helsinki and was approved by the Ethics Committee of Dr. Hasan Sadikin General Hospital, Bandung with number of DP.04.03/D.XIV.6.5/51/2026. Informed consent was obtained from all individual participants included in the study. Author Contributions All authors made a significant contribution to the work reported, whether that is in the conception, study design, execution, acquisition of data, analysis and interpretation, or in all these areas; took part in drafting, revising or critically reviewing the article; gave final approval of the version to be published; have agreed on the journal to which the article has been submitted; and agree to be accountable for all aspects of the work. Funding There is no funding to report. Disclosure The author(s) report no conflicts of interest in this work.

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