Pilot study on the impact of HIFU treatment on miRNA profiles in vaginal secretions of uterine fibroids and adenomyosis patients

Background High intensity focused ultrasound (HIFU) ablation treatment for uterine fibroids and adenomyosis has been long developed. The aim of this study is to investigate miRNA profile changes in vaginal secretions after HIFU treatment and their clinical relevance.

We prospectively collected vaginal secretions samples from 8 patients (1 with adenomyosis and 7 with fibroids) before and after HIFU treatment. RNA was isolated and miRNA profiles were analyzed using next-generation sequencing (NGS) sequencing.

Our study showed miRNA profile change in vaginal secretion samples after HIFU treatment for uterine fibroids/adenomyosis, with 33 miRNAs upregulated and 6 downregulated overall. In fibroid cases, 31 miRNAs were upregulated and 7 downregulated, while in adenomyosis case, 41 miRNAs were upregulated and 71 downregulated. Four miRNAs (hsa-miR-7977, hsa-miR-155-5p, hsa-miR-191-5p, hsa-miR-223-3p) showed significant differences after HIFU treatment in fibroid cases, except in case 5 with the lowest treatment sonications (425 sonications) and energy input (170000 J). hsa-miR-7977 consistently showed downregulation after HIFU treatment. hsa-miR-155-5p were downregulated in case 4 with lowest treatment efficiency (2439.64 J/cm3), while they were upregulated in other cases. hsa-miR-191-5p and hsa-miR-223-3p were downregulated in cases 4 and 7, with case 7 influenced by high sonication and energy due to multiple fibroids.

HIFU treatment altered miRNA profiles in fibroids/adenomyosis patients. Notably, hsa-miR-7977, hsa-miR-155-5p, hsa-miR-191-5p, and hsa-miR-223-3p showed significant changes in fibroid cases, except in low-energy treatments. hsa-miR-7977 consistently decreased post-treatment, while hsa-miR-155-5p decreased in the least efficient cases. Further research is needed for validation. 1. Introduction Uterine fibroids and adenomyosis are among the most prevalent benign gynecological tumors, affecting approximately 60% and 15% of women, respectively [Citation1]. Both conditions can result in hypermenorrhea, leading to anemia and the formation of bulky masses that can cause compressive effects [Citation2, Citation3]. These effects include abnormal defecation, dysfunctional voiding, abdominal fullness, and even lower back pain. Adenomyosis, a manifestation of endometriosis, may also lead to severe dysmenorrhea during menstruation and potential infertility issues, such as difficulties with embryo implantation [Citation4]. Treatment for uterine fibroids often involves hormonal therapies, including gonadotropin-releasing hormone agonists, ulipristal, and letrozole [Citation5]. Adenomyosis can be treated with medications like dienogest, gestrinone, leuprolide acetate, or the levonorgestrel intrauterine device [Citation6]. Surgical options for both conditions include myomectomy, adenomyomectomy, and hysterectomy, which can be performed through either laparotomic or minimally invasive approaches [Citation7, Citation8]. High-intensity focused ultrasound (HIFU) ablation is another treatment option for reducing the size of fibroids or adenomyosis and alleviating associated symptoms through a noninvasive procedure that has been developed for years [Citation9–11]. By shrinking the tumors and the uterus, HIFU can significantly improve the quality of life for patients suffering from these conditions. MicroRNAs (miRNAs) are a class of non-coding RNA molecules that play critical roles as post-transcriptional regulators of gene expression. By binding to target messenger RNA (mRNA) molecules, miRNAs can either suppress their translation or promote their degradation, thereby modulating gene expression. These small RNA molecules are involved in a wide range of physiological and pathophysiological processes and have emerged as crucial players in the diagnosis and treatment of numerous diseases, including cancer [Citation12]. Aberrant expression of miRNAs has been well-documented in various conditions such as endometriosis and uterine fibroids [Citation13]. For instance, several miRNAs have been identified that target genes implicated in diverse cancer-related pathways [Citation14], such as those governing cell proliferation, extracellular matrix metabolism, and angiogenesis [Citation15]. Recent research has highlighted the presence of distinct miRNA expression profiles in endometriosis including adenomyosis, which significantly influences the expression of associated target mRNAs [Citation16]. These miRNAs are instrumental in regulating key biological processes including epithelial-mesenchymal transition (EMT), angiogenesis, cell proliferation, cell adhesion, and invasion [Citation17, Citation18]. Such processes are critical for cellular functions like proliferation, invasion, and angiogenesis [Citation19, Citation20]. In addition to their roles in pathogenesis, miRNAs was reported as promising biomarkers for the detection of endometriosis. Specific miRNAs such as miR-451, the miR-200 family, miR-199, and miR-125 have been published to be associated with this condition [Citation21]. After HIFU treatment for uterine fibroids or adenomyosis, patients often experience an increase in vaginal discharge [Citation22]. Therefore, vaginal discharge serves as an excellent specimen for evaluating the condition of patients post-HIFU treatment and is relatively noninvasive. Previous studies utilizing 16S rRNA gene sequencing to examine vaginal discharge after HIFU have demonstrated significant changes in the diversity of the vaginal microbiota [Citation23]. However, the miRNA profile of vaginal discharge has not been reported in literatures. Therefore, the purpose of this study is to conduct a preliminary analysis of the changes in miRNA profiles in vaginal discharge before and after HIFU treatment and to investigate the correlation between these changes and clinical conditions as well as treatment outcomes. 2. Materials and methods 2.1. Patients recruitments This prospective study recruited patients with uterine fibroids or adenomyosis who decided to undergo HIFU treatment after outpatient consultation at Chang Gung Memorial Hospital of Linkou and Taoyuan Branch. All participants recruited for this study met the following criteria: (1) adult women over 20 years old with sexual experience; (2) patients undergoing HIFU treatment for uterine fibroids and adenomyosis; (3) willingness to sign a written informed consent form. Patients were excluded if they met any of the following conditions: (1) pregnant or breastfeeding women; (2) those with vaginal or urinary tract infections that might affect the sampling results; (3) those with uterine, ovarian, or other cancers; (4) those with acute or chronic inflammatory diseases, autoimmune diseases, or cardiovascular diseases; (5) those with poor renal function or undergoing dialysis. Specifically, to avoid the influence of menstruation and other factors on the components of vaginal discharge, patients who were menstruating at the time of sampling were strictly excluded. In the study, various clinical information of the participating patients, including age, body mass index (BMI), surgical history, chronic disease history, symptoms of uterine fibroids or adenomyosis, and past use of any medications for treating these conditions, was meticulously recorded from the hospital’s electronic medical record system and used as a reference for clinical data analysis. The study was conducted after receiving approval from the local institutional review board (IRB) with the designated IRB number: 202301558B0(2310130019). All patients scheduled for HIFU treatment underwent preoperative evaluations, including clinical symptoms assessment, physical examination, ultrasound, and pelvic MRI focusing on the target fibroids or adenomyosis. Patients were eligible for HIFU treatment if they had no contraindications such as severe abdominal wall scars, extremely retroverted uterus with bowel interference, or malignant uterine sarcomas. Malignant uterine sarcomas were excluded using MRI combined with diffusion-weighted imaging (DWI) and apparent diffusion coefficient (ADC) measurements [Citation24]. MRI also assessed the vascularity of the target tumor to predict HIFU ablation efficacy [Citation25]. 2.2. HIFU treatment HIFU treatment was performed using an ultrasound-guided HIFU machine (JC; Chongqing Haifu Medical Technology, China). According to the manufacturer’s protocol, the treatment area was identified with HIFU ultrasound. Energy was focused within the treatment zone, ensuring a safety margin around the leiomyoma, and ablation energy was applied. Visible coagulation necrosis observed during HIFU indicated effective treatment, prompting additional energy application to extend necrosis throughout the fibroid. Treatment concluded upon ultrasound confirmation of adequate ablation. After HIFU treatment, patients underwent MRI the next day to evaluate the non-perfusion volume (NPV) ratio, assessing immediate ablation response. Adverse events within 30 days post-HIFU were recorded and classified using the Society of Interventional Radiology (SIR) system for minor and major complications [Citation26]. Patients were advised to follow up every 3–6 months in the first year and 6–12 months thereafter, with gynecologic ultrasound included in each visit. 2.3. HIFU profile data Based on previous studies, the volume of leiomyomas is calculated using the formula: V = 0.5233 x length x width x depth. The volume reduction rate is determined by subtracting the volume change rate from 1, where the volume change rate is the post-treatment volume divided by the pretreatment volume. Sonication time refers to the duration of HIFU energy emission, while treatment time is the total duration from the start to the end of the procedure. From parameters such as total treatment time, sonication count, total energy input, original volume, and post-treatment MRI-determined NPV ratio, various HIFU profiles can be derived to compare treatment efficacy. These include ‘Energy Efficiency (EEF)’ calculated as energy per volume (J/cm³), ‘Treatment Speed’ as volume per treatment duration (cm³/hr), and ‘Power Rate’ as energy per treatment duration (J/hr). Three indices for NPV generation efficiency involved ‘Treatment Efficiency (TEF)’ as NPV per treatment duration (cm³/min), ‘NPV per Sonication’ as NPV per sonication time (cm³/min), and ‘NPV per Energy’ as NPV per energy (cm³/J). However, it’s important to note that comparisons between multiple and single fibroids are not directly valid. When evaluating total energy, treatment time, and sonications, direct comparisons are feasible, but treatment efficiency should consider representative individual fibroids, such as the largest, to avoid errors due to differing fibroid counts. 2.4. Vaginal secretion samples collection and management As showed, vaginal secretion samples were collected twice in this study: before and after HIFU treatment. Before HIFU, patients received sedation and analgesia for Foley catheter placement, during which vaginal discharge samples were collected using a vaginal swab. For the Post-HIFU sampling, the vaginal secretion samples were collected while the patient was still under sedation and anesthesia, immediately on the operating table, before transferring them to the recovery room for post-anesthesia observation. This approach ensured that patients experienced no discomfort during sample collection. After samples collection, the swabs were placed in 1X PBS (phosphate-buffered saline) for washing and then transferred to the core-laboratory for further managements within 8 h to maintain sample stability. Besides, the entire HIFU treatment process was generally completed within approximately 3 h, allowing for efficient management of sample collection, processing, and storage within the 8-h window, ensuring the accuracy and reliability of our study. After the samples were transferred to our laboratory, total RNA was isolated from three types of body fluids using the miRNeasy Serum/Plasma Kit (Qiagen, Hilden, Germany), following the manufacturer’s instructions. Briefly, QIAzol Lysis Reagent was added to denature protein complexes and RNases, followed by chloroform addition to separate the different phases. The aqueous phase containing RNA was mixed with 100% ethanol and then added to a spin column. After washing the RNA with buffers, it was eluted. The RNA concentration and purity were confirmed by measuring absorbance ratios at 260 nm and 280 nm using a Nanodrop 2000 (Thermo Fisher Scientific, MA, USA). The isolated total RNA was stored at −80 °C for subsequent experiments. 2.5. miRNA detection and analysis by next generation sequencing (NGS) Next-generation sequencing (NGS) involves steps such as library construction, quantification, sequencing, and data analysis. For microRNA sequencing, we used the QIAseq miRNA kit (QIAGEN, Germany, Cat. No. 331502) for library construction, and quantification was performed using Qubit (Thermo Fisher Scientific, USA, Cat. No. Q32851). The sequencing was carried out on the Illumina NextSeq 550 platform (Illumina, CA, USA). The process includes library construction and sequencing, cDNA cleanup, and sequence data analysis. For library construction and sequencing, 3′ adapter ligation was performed using pre-adenylated adapters, followed by 5′ adapter ligation with sequencing primers. cDNA synthesis was conducted using reverse transcription primers containing unique molecular indices (UMIs), which assigned a unique index to each miRNA molecule for individual identification. Following this, cDNA cleanup involved PCR amplification using primers with sample-specific indices. Data quality was checked using the 5200 Fragment Analyzer system (Agilent Technologies, CA, USA) to ensure library sizes ranged from 190 to 220 bp. Quantification was carried out using Qubit (Thermo Fisher Scientific, USA, Cat. No. Q32851), ensuring concentrations exceeded 1 ng/μL for sequencing. The sequencing was then performed on the Illumina NextSeq 550 (Illumina, CA, USA). For sequence data analysis, raw sequence files in Fastq format were obtained. Amplification duplicates were identified and removed using UMI-tools. miRNA reads were counted using SPORTS, with read lengths set between 15 and 55 bp to exclude nonstandard sequences. The analysis was conducted against the human reference genome (GRCh38) and human miRNA sequences from miRBase (v22.1). After obtaining the mapped read counts of miRNAs for each sample, the R package DESeq2 was used to perform normalization between samples and detection of differentially expressed miRNAs between groups. Hierarchical clustering heatmaps were created for miRNA using the R package ‘pheatmap’ to examining correlations between samples. 2.6. miRNA target interaction and functional analysis The miRNAs with significantly differential expression were subjected to further microRNA target interaction (MTI) analysis using miRTarBase, one of the largest databases containing experimentally validated miRNA-target interactions, was utilized for subsequent target gene analysis in our study. These MTIs were then used for f predict their potential regulatory functions by performing enrichment analysis with clusterProfiler and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. Through this analytical approach, we identified potential biological functions and signaling pathways regulated by miRNAs with significant expression differences between samples collected before and after HIFU treatment. The detection platform allowed us to delineate the functional roles and regulatory mechanisms of these differentially expressed miRNAs. 3. Results 3.1. Enrolled patients in our study From January 2024, we collected data on 8 patients undergoing HIFU ablation treatment for uterine fibroids (7 patients) and adenomyosis (1 patient). These patients were monitored for changes over a 6-month period post-treatment. In accordance with the research protocol, vaginal secretion samples were collected pre- and post-operatively for analysis. The patient characteristics are detailed in . The ages of the patients ranged from 31 to 49 years, with BMI between 18.97 and 25.2. Among the patients with fibroids, 4 had multiple fibroids with individual counts of 3, 4, 5, and 6, while the remaining 3 patients had single fibroids. The preoperative volumes of the single fibroids were 157.35, 316.41, and 277.76 cm³, with maximum diameters of 9.7, 10.2, and 7.7 cm, respectively. For patients with multiple fibroids, the largest fibroid was selected for representation, with volumes of 184.21, 76.55, 199.09, and 240.94 cm³ and maximum diameters of 7.4, 6.0, 9.1, and 10.9 cm, respectively. The adenomyosis had a size of 96.91 cm³ with a maximum diameter of 6.9 cm. The total treatment time for fibroids ranged from a minimum of 52 min for case 5, involving 425 sonications and an energy output of 170,000 Joules, to a maximum of 193 min, with 1,465 sonications and an energy output of 585,550 Joules. The NPV ratio for fibroid ablation exceeded 95% in all cases. The adenomyosis treatment (case 3) lasted 60 min, with 460 sonications, an energy output of 183,550 Joules, and an NPV ratio of 84%. 3.2. HIFU treatment profile To accurately evaluate the treatment efficiency of HIFU, we selected the largest fibroid from patients with multiple fibroids (cases 1, 4, 6, and 7) as a representative, with the results summarized in . The energy efficiency, defined as the energy required per cm³ volume, varied among cases. Case 4 required the highest energy at 2439.64 J/cm³, while cases 5 and 7 required the least at 537.27 J/cm³ and 542.88 J/cm³ respectively. The treatment speed was fastest in case 5, achieving 365.09 cm³/hr, and slowest in case 4, with 64.69 cm³/hr. When examining the power rate, which indicates energy consumption per hour, case 2 had the highest at 202,289.06 J/hr, whereas case 6 had the lowest at 130,951.46 J/hr. Treatment efficiency, in terms of Non-Perfused Volume (NPV) produced per minute, was highest in case 5 at 8.23 cm³/min and lowest in case 4 at 1.31 cm³/min. Additionally, NPV efficiency per joule of energy and per sonication was highest in case 5, at 0.00252 cm³/J and 1.01 cm³/sonication, respectively. Conversely, case 4 had the lowest efficiency at 0.00049 cm³/J and 0.19 cm³/sonication. Regarding the reduction rate of fibroid size post-treatment, case 2 exhibited the fastest reduction at 71.37% and 77.20% at 3 and 6 months post-treatment, respectively. In contrast, cases 4 and 5 showed slower reduction rates of 36.32% and 23.93% at 3 months post-treatment. Other patients who returned for follow-up demonstrated reduction rates of 40–60% at 3 months and 60–80% at 6 months. 3.3. miRNA profile changes before and after HIFU treatment We examined the differential expression of miRNA in vaginal secretion samples collected from 8 patients before and after HIFU treatment. To eliminate the influence of low-expression and non-differential miRNAs on the clustering analysis, all samples were filtered to include miRNA with Normalized read counts > = 5 for Coefficient of Variation (CV) calculation. The CV, a standardized measure of data variability, is defined as the ratio of the standard deviation to the mean. After calculating the CV, we selected the top 100 miRNAs with the highest CV values for hierarchical clustering analysis, which is visualized as a heatmap in . Within the CV top 100 miRNA profile, no significant differences were observed before and after treatment. Subsequently, we performed Principal Components Analysis (PCA) on miRNAs with Normalized read counts > = 5 across all samples to observe the relationships between samples, as shown in . PCA is a dimensionality reduction technique that transforms high-dimensional data into a lower-dimensional space while retaining the most variance information. PCA is commonly used to visualize data and observe the relationships between samples. The PCA revealed that case 2’s 2 A (pretreatment) and 2 C (post-treatment) samples were outliers. Figure S1 furtherly supported the results by calculating the frequency of outlier sample occurrence in each miRNA based on group (A as pretreatment and C as post-treatment) showing that case 2’s 2 A and 2 C samples frequently appeared as miRNA outliers. Consequently, case 2 was excluded from further analysis in our study. 3.4. miRNA changes and functional analysis in patients before and after HIFU treatment We identified differentially expressed miRNAs in patients with fibroids and adenomyosis before and after treatment, selecting those with |Log2 Fold Change| ≥ 1 and P-value < 0.05, as shown in . The histogram plot revealed a intermediate-enriched normal distribution of miRNA fold change count. The volcano plot, used to observe the relationship between miRNA fold changes and P-values, showed a trend extending toward the upper left and upper right quadrants. Our selection focused on these two regions (P-value < 0.05, log2 fold change ≥ 1 or ≤ −1), revealing 33 upregulated miRNAs and 6 downregulated miRNAs. showed the functional analysis of differentially expressed miRNAs before and after treatment, highlighting fibroid-related functions and overall functional annotations. The overall functional annotations predominantly included ‘miRNAs in cancer’, ‘proteoglycans in cancer’, and ‘hepatitis B’. The fibroid-related functions were associated with pathways such as ‘FoxO signaling pathway’, ‘PI3K-Akt signaling pathway’, ‘MAPK signaling pathway’, ‘EGFR tyrosine kinase inhibitor resistance’, and ‘TGF-beta signaling pathway’. 3.5. miRNA changes and functional analysis in fibroid patients before and after HIFU treatment presented the miRNA analysis focusing solely on fibroid cases, revealing 31 upregulated miRNAs and 7 downregulated miRNAs. Tables S2 and 4 displayed the functional analysis of differentially expressed miRNAs in fibroid cases, highlighting both overall functional annotations and fibroid-related functions. The overall functions included ‘MicroRNAs in cancer’, ‘Hepatitis B’, and ‘Proteoglycans in cancer’. The fibroid-related functions involved the ‘FoxO signaling pathway’, ‘MAPK signaling pathway’, ‘PI3K-Akt signaling pathway’, and ‘EGFR tyrosine kinase inhibitor resistance’. 3.6. miRNA changes and functional analysis in adenomyosis patients before and after HIFU treatment demonstrated the miRNA analysis for case 3, the only patient with adenomyosis, showing 41 upregulated and 71 downregulated miRNAs before and after treatment. Tables S3 and 5 showed the functional analysis of these differentially expressed miRNAs, encompassing both overall functional annotations and adenomyosis-related functions. The overall functional annotations were similar to previous findings, primarily including ‘microRNAs in cancer’. The adenomyosis-related functions involved the ‘MAPK signaling pathway’, ‘PI3K-Akt signaling pathway’, ‘FoxO signaling pathway’, and ‘EGFR tyrosine kinase inhibitor resistance’. 3.7. Significantly differential miRNAs in all fibroid cases and the related functional analysis listed four miRNAs that appeared in the differential expression analysis across multiple cases. Notably, case 2 was excluded as an outlier, and case 5 showed fewer differential genes between pre- and post-treatment samples, thus these four miRNAs did not appear in case 5. Among these miRNAs, hsa-miR-7977 consistently showed downregulation after treatment compared to before treatment. Hsa-miR-155-5p was downregulated in case 4. The remaining miRNAs were generally upregulated. Specifically, hsa-miR-191-5p and hsa-miR-223-3p exhibited downregulation in cases 4 and 7 but were upregulated in other cases. provide the functional analysis of these four miRNAs, including both fibroid-related and overall functional annotations. The overall functional annotations primarily involve pathways such as ‘AGE-RAGE signaling pathway in diabetic complications’, ‘Human T-cell leukemia virus 1 infection’, and ‘Epstein-Barr virus infection’. In contrast, the fibroid-related functional analysis highlights pathways such as ‘FoxO signaling pathway’, ‘PI3K-Akt signaling pathway’, ‘MAPK signaling pathway’, ‘JAK-STAT signaling pathway’, and ‘TGF-beta signaling pathway’. Regarding the differential expression of miRNAs in adenomyosis and fibroids before and after HIFU treatment, we identified the overlap of differentially expressed miRNAs between these two groups, as illustrated in Figure S2. Three miRNAs, including hsa-miR-126-3p, hsa-miR-20b-5p, and hsa-miR-345-5p, were found to be upregulated in both fibroids and adenomyosis post-HIFU treatment. In contrast, only hsa-miR-7977 was consistently downregulated in both conditions. Table S5 presents the functional analysis of these three upregulated miRNAs, which are commonly associated with pathways such as ‘MicroRNAs in cancer’, ‘FoxO signaling pathway’, ‘Prostate cancer’, ‘Pancreatic cancer’, and ‘Chronic myeloid leukemia’. 4. Discussion This study investigated whether the use of HIFU in treating uterine fibroids and adenomyosis induces changes in the miRNA profile within the uterine environment, using vaginal secretions as the primary study samples. We found that HIFU treatment indeed alters the miRNA profile in the vaginal secretions of patients. Overall, 33 miRNAs were upregulated and 6 were downregulated post-treatment. Specifically, in fibroid cases, 31 miRNAs were upregulated and 7 were downregulated, whereas in adenomyosis case, 41 miRNAs were upregulated and 71 were downregulated. Additionally, when compared with the clinical HIFU treatment profiles, four miRNAs (hsa-miR-7977, hsa-miR-155-5p, hsa-miR-191-5p, and hsa-miR-223-3p) showed significant differences before and after treatment in fibroid cases. However, in case 5, which had the shortest treatment duration and the least energy input (52 min and 425 sonications), these four miRNAs did not show any differences. In other fibroid cases, hsa-miR-7977 consistently showed downregulation. The other three miRNAs (hsa-miR-155-5p, hsa-miR-191-5p, and hsa-miR-223-3p) were downregulated in case 4, which had the highest energy consumption per cm³, the smallest treatment volume per hour, and the least NPV generated per minute, per energy (J), or per sonication, indicating poor treatment efficiency. These three miRNAs were upregulated in all other cases except case 7. In case 7, hsa-miR-155-5p was also upregulated, but hsa-miR-191-5p and hsa-miR-223-3p were downregulated, similar to case 4. Case 7 involved multiple fibroids and had the longest total treatment time among all patients, yet its treatment efficiency for the largest representative fibroid was the second fastest, following case 5. Therefore, the miRNA changes in such cases may differ from those in other cases with similarly fast treatment efficiency, such as cases 1, 6, and 8. HIFU delivers high-intensity acoustic energy exceeding 100 W/cm2, which can be focused on the target tumor area, providing localized heat above 60 °C. This heat, along with accompanying cavitation and mechanical physical effects, induces tissue ablation [Citation27, Citation28]. Post-ablation, uterine fibroids may undergo coagulative necrosis and subsequently trigger cell apoptosis [Citation29, Citation30]. The necrotic fibroid cells may then be phagocytosed or digested by immune or inflammatory cells, such as macrophages, leading to the shrinkage of the fibroids [Citation31–33]. HIFU has been applied in the noninvasive treatment of uterine fibroids for many years, demonstrating significant advantages in improving the quality of life [Citation34]. A prospective ‘IDEAL’ study conducted by Chen et al. in 2017 also revealed that HIFU ablation improves the quality of life more rapidly and reduces morbidity compared to surgery [Citation35]. In our previous studies, the average reduction rates of uterine fibroids post-treatment were observed to be 54.5% at 3 months, 60.8% at 6 months, and 68.6% at 12 months [Citation36]. HIFU may also alter the body’s immune response. In 2013, Wang et al. observed that patients undergoing myomectomy showed decreased CD4/CD8 T cell ratios and serum IL-2 levels [Citation37]. The hyperthermic reaction induced by HIFU energy can stimulate dendritic cells and macrophages to secrete IL-12 and TNF-α, respectively [Citation38]. To investigate whether HIFU alters the uterine environment, we can examine vaginal secretion samples, but it was rarely discussed before. Previous research using 16S rRNA gene sequencing found that post-HIFU, the microbial α-diversity in vaginal secretions decreased, some pathogenic bacteria were reduced, but Proteobacteria increased [Citation23]. miRNAs are also involved in the pathogenesis of fibroids, as demonstrated in previous studies. Compared to the myometrium, fibroid cells exhibit significant upregulation of miRNAs such as let-7s, miR-21, miR-23b, miR-27a, and miR-30a, and downregulation of miRNAs such as miR-29b, miR-32, miR-144, miR-197, and miR-212 [Citation39]. Other literature also highlights that, compared to the myometrium, fibroids show upregulation of miR-181a-5p, miR-127-3p, miR-28-3p, miR-30b-5p, and let-7c-5p. These miRNAs affect cell proliferation, extracellular matrix metabolism, and angiogenesis [Citation15]. Furthermore, studies have found that miR-363, miR-490, miR-137, miR-217, and miR-4792 exhibit significant abnormal expression in fibroids compared to normal myometrium [Citation40]. In our current study, we observed that hsa-miR-7977 was consistently downregulated in patients with fibroids following HIFU treatment. Previous studies have not reported any association between hsa-miR-7977 and fibroids. However, hsa-miR-7977 has been identified as a diagnostic biomarker for epithelial ovarian cancer in plasma exosomes and as a novel biomarker for lung adenocarcinoma in serum exosomes [Citation41, Citation42]. Moreover, miR-7977 may be involved in the failure of normal hematopoiesis in myeloid neoplasms [Citation43]. Overexpression of miR-7977 in CD4+ T cells has been associated with multiplex autoimmunity in patients with Addison’s disease [Citation44]. Additionally, mesenchymal stromal cell line HTS-5 transduced with miR-7977 showed elevated saturation density and enhanced cell cycle entry [Citation45]. However, for fibroids, further research is needed to elucidate their specific associations. If HIFU treatment reduces their expression, it may suggest that the mechanisms promoting fibroid formation are inhibited by HIFU. From our study, differences in the expression of hsa-miR-155-5p, hsa-miR-191-5p, and hsa-miR-223-3p were observed in fibroid patients before and after HIFU treatment. Specifically, hsa-miR-155-5p was downregulated in the least efficient case 4, while it was upregulated in other fibroid cases. Previous studies have shown that hsa-miR-155 acts as a tumor suppressor and anti-apoptotic mediator and is associated with the formation of fibroids [Citation46]. It is considered a proto-oncogene and is significantly increased in patients with large B cell lymphoma and Hodgkin lymphoma [Citation47]. Additionally, research has indicated a higher presence of macrophages in fibroids, and exposure to pro-inflammatory cytokines (such as TNF-α) leads to the overexpression of miR-155 [Citation48]. Hsa-miR-155 regulates autophagic activity and is involved in hypoxia-induced autophagy [Citation49]. Its loss results in reduced accumulation of M1 macrophages and improved obesity-related metabolic profiles [Citation50]. In ovarian cancer, miRNA 155 is classified as a tumor suppressor miRNA that regulates the expression of certain oncogenic proteins. Administration of miR-155-5p increases the sensitivity of ovarian cancer cells to cisplatin, and its downregulation reduces the ability to suppress oncogenic protein production, leading to a worse cancer prognosis [Citation51]. There is no literature on the role of hsa-miR-155 in fibroids treated with HIFU. If it functions as a tumor suppressor, this could explain the reduction of miR-155-5p in case 4 and its increase in more efficient cases, although further research is needed for validation. Two other miRNAs, hsa-miR-191-5p and hsa-miR-223-3p, were found to be downregulated in case 4, which had low HIFU efficiency, and also downregulated in case 7, which had high HIFU efficiency. However, case 7 involved the highest total sonication and energy levels. Whether these factors influence the expression of these miRNAs requires further research. miR-191 has been found to be overexpressed in endometriotic cyst tissues and is a key regulator of the proliferative and invasive characteristics of endometriotic cells [Citation52]. Previous studies have frequently used miR-191 as a reference gene for normalization or as a control gene [Citation53, Citation54]. miR-191 may serve as a diagnostic biomarker to distinguish Crohn’s disease from ulcerative colitis [Citation55]. Similarly, miR-191 has been shown to be a potential biomarker for pulmonary hypertension, enabling early detection and indicating disease severity [Citation56]. In serum samples from patients with ovarian clear cell carcinoma, miR-191-5p levels were significantly higher compared to healthy controls, suggesting its potential as a biomarker for ovarian cancer [Citation57]. There is no relevant literature on miR-223-3p in fibroids. miR-223-3p can mediate intercellular communication among blood cells, immune cells, and vascular cells via high-density lipoproteins, platelet-derived exosomes, or macrophage-derived microparticles, playing a role in anti-atherosclerosis [Citation58]. miR-223-3p exhibits opposite functions in different types of cancers. It may reduce the invasiveness of breast cancer cells but enhance the biological behavior of prostate cancer cells [Citation59, Citation60]. In ovarian cancer tissues and cell lines, miR-223-3p is overexpressed, and using an inhibitor to downregulate miR-223-3p can suppress ovarian cancer cell proliferation, migration, and invasion [Citation61]. The roles of both miR-191-5p and miR-223-3p in fibroids remain unclear, and their response to HIFU requires further investigation to draw definitive conclusions. In our study, it was evident that HIFU treatment of fibroids and adenomyosis potentially altered the uterine environment. Changes in the miRNA profile were observed in vaginal secretions post-HIFU treatment. In fibroid patients, four miRNAs (hsa-miR-7977, hsa-miR-155-5p, hsa-miR-191-5p, hsa-miR-223-3p) showed significant changes. However, in case 5, which had the shortest treatment duration and the least energy input, no differences in these four miRNAs were observed. This suggested that the expression of these miRNAs might require sufficient HIFU energy exposure to manifest. In fibroid patients, hsa-miR-7977 generally showed downregulation post-HIFU treatment, potentially indicating that hsa-miR-7977 could serve as a marker for evaluating the mechanism of HIFU treatment for fibroids. A similar downregulation pattern was also observed in adenomyosis case. hsa-miR-155-5p was downregulated only in case 4, while it was upregulated in other cases. Case 4 had the least energy efficiency among the fibroid cases and also exhibited a poorer reduction rate three months post-treatment. This suggested that changes in hsa-miR-155-5p could be a potential indicator for evaluating the efficacy of HIFU treatment in the future. hsa-miR-191-5p and hsa-miR-223-3p were downregulated in both case 4 and case 7. Case 4 was characterized by low energy efficiency, while case 7 had high energy efficiency but involved multiple fibroids with larger volumes, resulting in more total sonication, energy, and time compared to other fibroid cases. Thus, these two miRNAs might also represent indicators of energy efficiency, although their expression could be influenced by excessive treatment duration and energy input. The clinical significance of these findings still required further examination and validation through more cases and samples. To the best of our knowledge, this is the first study to investigate the changes in miRNA profiles in vaginal secretions before and after HIFU treatment for uterine fibroids. This is a preliminary study, and the limited number of cases is one of our limitations. Despite the small sample size, we observed differences in miRNA expression, which encourages us to continue collecting more patient data for broader research and validation. Additionally, since this is a preliminary study, there were discrepancies in the number of cases for adenomyosis patients, single fibroids, and multiple fibroids, making statistical comparisons challenging. In this study, although there was only one case of adenomyosis, it exhibited a greater number of differentially expressed miRNAs compared to fibroids. This may be related to the fact that adenomyosis is considered one of the manifestations of endometriosis, a condition known to involve inflammatory cellular responses [Citation62]. This could explain the more pronounced miRNA differential expression following HIFU treatment. Given the absence of previous reports on this topic, further studies are warranted to explore this finding in more detail. Balancing these case numbers will be necessary for future studies. Moreover, our research focused solely on miRNAs, which is another limitation. This study opens the door for more in-depth research on HIFU treatment for fibroids and adenomyosis, suggesting that future studies could consider combining other biomarkers, such as cytokines, for a more comprehensive analysis. Authors’ contributions Conceptualization, W.-C.C., T.-C.C., and C.-M.C.; methodology, W.-C.C., L.P., and C.-M.C.; software, W.-C.C. and J.-J.H.; validation, W.-C.C., L.P., and C.-M.C.; formal analysis, W.-C.C. and C.-M.C.; investigation, W.-C.C., T.-C.C., and C.-M.C.; resources, M.-H.C., J.-J.H., and C.-M.C.; data curation, W.-C.C. and C.-M.C.; writing—original draft preparation, W.-C.C.; writing—review and editing, W.-C.C., T.-C.C., and C.-M.C.; visualization, W.-C.C. and C.-M.C.; supervision, T.-C.C. and C.-M.C.; project administration, W.-C.C. and C.-M.C.; funding acquisition, W.-C.C. and C.-M.C. Supplemental material Supplemental Material Download Zip (316.1 KB)Supplemental MaterialDisclosure statement No potential competing interest was reported by the authors. Data availability statement Data will be made available upon reasonable request to the corresponding author. Additional information Funding

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