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Introduction

Stress urinary incontinence (SUI) is defined as the involuntary loss of urine during physical exertion or effort (1). The prevalence of SUI has been reported to be as high as 49%, but varies depending on the study population and the diagnostic criteria used (2,3). The incidence of this dysfunction becomes more common with age, with ~25% of young women, 44-57% of middle-aged and postmenopausal women, and 75% of elderly women reporting some involuntary urine loss (4). SUI adversely impacts the physical and mental health of patients and also represents a notable medical and economic burden on individuals and healthcare systems (5-7).

Currently, the recommended first-line treatment for SUI is ≥3 months of supervised exercise focused on the pelvic floor muscles (PFMs), known as PFM training (PFMT) (3,6). PFMT involves the patient regularly performing repeated voluntary PFM contractions, with sufficient progression in exercise intensity and duration to produce a training effect on the muscles. Surface electromyography (sEMG) is considered a useful tool for the real-time monitoring of PFM contractions and the assessment of PFM function via the detection of PFM motor unit action potentials (3). The validity of sEMG in the assessment of the bioelectrical activity of PFMs, as well as muscles that act as synergistically with the PFMs, has been demonstrated in numerous studies (8-10). Electrical signals from these muscles reflect the recruitment of motor units during muscle contraction, and a positive association has been observed between muscle strength and the activation of motor units (3). The electrical activity of the PFMs is typically measured using an internal vaginal probe with a rigid two-channel electrode, which is widely used in clinical practice due to its simplicity, speed and ease of operation. However, sEMG cannot determine precisely which specific muscles are being measured, as it captures the cumulative activity from all muscles that contact the electrode plates. Furthermore, the suboptimal probe-muscle interface, traditional two-channel electrode structure, and lack of accurate evaluation methods hinder the accurate measurement of muscle activity under specific conditions, such as when two electrodes are placed on opposite sides of the PFMs (one on the left and the other on the right, or one on the top and the other on the bottom) inside the vagina (11). To address these challenges, the present study evaluated a novel airbag-type stretchable/inflatable electrode array (ASEA) device for sEMG signal acquisition in PFM analysis. As previously reported, the ASEA device can inflate to conform with varying vaginal anatomies, while closely following muscle movement, thereby providing a stable probe-muscle interface with notable biological adaptability (12). The ASEA comprises 24 bipolar electrode pairs, which map to 10 PFM regions and are approximately aligned with the orientation of muscle fibers. These regions include the urethral sphincter (US), vaginal sphincter (VS), external anal sphincter (EAS) and levator ani muscles (LAMs), which consist of the puborectalis (PR), pubococcygeus (PC) and iliococcygeus (IC) muscles. Electrode placement along the muscle fibers improves the accuracy of signal acquisition, and dividing the sEMG signals by region makes it possible to pinpoint the activity of specific muscle regions.

Several key parameters are hypothesized to comprehensively evaluate the regional muscles closely associated with SUI. These parameters, calculated on the basis of regional functional data include: i) Pre-baseline, referred to as the anterior resting potential (ARP); ii) maximum voluntary contraction (MVC), also known as rapid contraction potential; iii) tonic contraction potential (TCP); iv) endurance contraction potential (ECP); and v) post-baseline, referred to as the posterior resting potential (PRP). The aim of the present study was to evaluate the regional characteristics and spatial precision of PFM sEMG signals during rest and contraction using the ASEA device in postmenopausal women with SUI compared with healthy controls.

Materials and methods

Ethics statement

Ethics approval for the present study was obtained from The Ethics Committee of the Women's Hospital, School of Medicine, Zhejiang University (Hangzhou, China; approval nos. IRB-20220008-R and IRB-20240381-R). All participants provided written informed consent to participate in the study, and the ethics committee approved the consent procedure.

Study design

The study was designed as a retrospective data analysis. The data were originally sampled by the same research team during a prior study focusing on the effect of whole-body vibration on PFM activity.

Participants

For the purpose of the present study, two groups were established as follows: i) A control group comprising healthy postmenopausal women with no history of pelvic floor dysfunction (n=65); and ii) an SUI group, comprising postmenopausal women with SUI, recruited from the Women's Hospital, School of Medicine, Zhejiang University (n=67). The participants were all female and recruited between August 2022 and September 2023 (Fig. 1 and Table I). The inclusion criteria for the postmenopausal women with SUI were as follows: i) Clinical diagnosis of SUI; ii) a sexual history; and iii) menopausal for ≥1 year. The inclusion criteria for the postmenopausal women without SUI: i) a sexual history; and ii) menopausal for ≥1 year. Since the ASEA device is a vaginal probe, its insertion requires a history of sexual activity. In China, performing vaginal procedures on individuals without prior sexual experience is considered ethically inappropriate; therefore, only individuals with a sexual history were included. The exclusion criteria were the same for both groups and were as follows: i) Acute reproductive organ inflammation; ii) presence of a cardiac pacemaker; iii) malignant tumors; iv) history of pelvic radiotherapy; v) pelvic floor surgery during the previous 6 months; vi) any condition or symptom that could interfere with the implementation of the study or the interpretation of the results; and vii) concurrent participation in another clinical trial. In total, 76 participants were initially screened for inclusion in the control group; however, 11 participants were then excluded due to not meeting the inclusion criteria (3 cases), declining to participate (2 cases), bad comprehension and cooperative degree (3 cases), malignant tumors (1 case) and missing follow-up appointments (2 cases). In total, 84 participants were initially screened for inclusion in the SUI group; however, 17 participants were then excluded due to not meeting the inclusion criteria (6 cases), declining to participate (4 cases), bad comprehension and cooperative degree (3 cases) and missing follow-up appointments (4 cases).

Figure 1 Consolidated Standards of Reporting Trials diagram. SUI, stress urinary incontinence; PFM, pelvic floor muscle; ASEA, airbag-type stretchable/inflatable electrode array; US, urethral sphincter; LAMs, levator ani muscles.

Table I Demographics of participants.

Table I

Demographics of participants.

	Group
Characteristics	SUI (n=67)	Control (n=65)	t-value	Z-value	P-value
Age, years	59.03±5.90	57.38±6.01	1.59	-	0.115
Body mass index, kg/m2	23.68±3.16	23.01±2.46	1.37	-	0.175
Waist-to-hip ratio	0.91±0.60	0.89±0.06	1.85	-	0.067
Years after menopause	7 (4,10)	6 (3,10.5)	-	0.79	0.428
Parity	3 (2,3)	2 (2,4)	-	1.31	0.189
Gravidity	1 (1,2)	1 (1,2)	-	0.60	0.546
Vaginal delivery	1 (1,2)	1 (1,2)	-	0.15	0.878
Cesarean section	0 (0,0)	0 (0,0)	-	1.14	0.253

[i] The control group comprises women with healthy pelvic floor muscles, while the SUI group comprises women with SUI. Data are presented as the mean ± SD or the median (P25, P75) and were analyzed using an unpaired t-test or Wilcoxon rank-sum test, respectively. SUI, stress urinary incontinence.

Procedures and data collection. Vaginal bidigital palpation for the assessment of PFM function

PFM function was assessed by vaginal bidigital palpation following the PERFECT scheme (13,14). During the examination, the participants were instructed how to properly contract their PFMs and asked to perform a maximal contraction. All participants were assigned a PFM testing score of M0-M5 using the modified Oxford grading system: i) M0, no contraction; ii) M1, flicker; iii) M2, weak; iv) M3, moderate; v) M4, good; and vi) M5, strong (14).

ASEA device for the assessment of PFM function. Introduction of the ASEA device

The ASEA is an airbag-type, inflatable vaginal probe designed to maintain tight contact and a stable interface between its electrodes and the PFMs. As previously described, it has 24 bipolar electrode pairs, along with 32 contact pads, which are positioned in alignment with the muscle fibers and cover 10 distinct PFM regions, thereby enabling the acquisition of high-quality sEMG signals (12). The structure of the ASEA and the physical positioning of the contact pads on the muscles are shown in Fig. 2. The ASEA records electrical potential distributions and corresponding frequency information for all major vaginal muscles, including the US, VS and EAS, as well as the PR, PC and IC muscles of the LAM group. Details of the fabrication process, the arrangement of the electrodes (electrical contacts) and clinical reliability assessments of the ASEA have been described previously (15), and have demonstrated that the accuracy and stability of the ASEA are improved compared with those of conventional PFM probes.

Figure 2 Structure and configuration of the ASEA probe. (A) Distribution of the pelvic muscles, numbered in a clockwise manner. (B) Details of the double-layer electrode unit and (C) distribution of main pelvic muscles in a 2D format. The locations of the contact pads of the ASEA (black dots) are distributed according to the major muscle distributions and muscle fiber directions, and each muscle is shown in a different color to allow for easy distinction. (D) Shape and assembly of the ASEA, which was composed of two layers. The inner layer had an electrode sheet, and the outer layer had a perforated diaphragm. ‘Pack’ refers to wrapping the electrode sheet with the perforated diaphragm, in which only the contact electrode holes were exposed. Macro internal structure of ASEA (left), and the probe before (middle) and after inflation (right). ASEA, airbag-type stretchable/inflatable electrode array.

Use of the ASEA device for the assessment of PFM function. To ensure consistency, all assessments were performed by a single therapist, experienced in pelvic floor rehabilitation. The patients were first asked to adopt a supine position with slightly bent knees and a neutral hip position, and then the ASEA probe was inserted into the vagina and the patients were instructed to activate their PFMs, which was controlled with electromyographic biofeedback. The PFM activity was measured using the novel ASEA probe (patent no. ZL 202210927100.3; China). Patients were first instructed to squeeze their PFMs freely to acclimate to the test probe, while the therapist monitored the abdomen, perineum, hip and gluteal muscles to ensure isolated activation of the PFMs. Subsequently, the ASEA probe was carefully inserted into the vagina and its proper placement confirmed before any measurements were recorded.

The protocol of all measurements of PFM activity consisted of the assessment of the following elements: i) Pre-baseline, also known as the ARP, where a 10-sec measurement of PFM activity at rest was taken before the functional tests; ii) MVC, also termed ‘flick contractions’, consisting of a 10-sec measurement while participants performed short, quick contractions of the PFMs; iii) TCP, comprising 5 repetitions of 10-sec contractions, during which participants attempted to contract and hold the PFMs for 10 sec; iv) ECP, where participants attempt to hold a PFM contraction for 60 sec; and v) post-baseline, also known as the PRP, consisting of a 10-sec measurement of the PFM at rest after the functional tests.

Statistical analysis

Statistical analysis was performed using SPSS 26.0 (IBM Corporation). The distributions of baseline characteristics in the SUI and control groups were compared using unpaired t-tests or Wilcoxon rank-sum tests. To determine the optimal cut-off level for sEMG activation of the PFMs for use in the diagnosis of SUI, receiver operating characteristic (ROC) curve analysis was performed and the area under the curve (AUC), 95% confidence intervals (CIs), sensitivity and specificity were calculated. P<0.05 was considered to indicate a statistically significant result.

Results

Demographic and clinical characteristics

In total, 132 postmenopausal women were included in the present study, with 67 participants in the SUI group and 65 in the control group. In the SUI group, the median age was 58 years and the age range was 48-74 years, while in the control group, the median age was 57 years and the age range was 46-78 years. The demographic and clinical characteristics of the participants are presented in Table I. No statistically significant difference was identified between the two groups for age, BMI, waist-to-hip ratio, parity, gravidity and mode of delivery.

PFM function assessment by vaginal bidigital palpation

All patients in each group were assigned an initial PFM testing score of M0-M5 according to the modified Oxford grading system (Table II). Notably, the scores in the SUI group were significantly lower compared with those in the control group.

Table II Pelvic floor muscle functional assessment by vaginal bidigital palpation.

Table II

Pelvic floor muscle functional assessment by vaginal bidigital palpation.

	Group
Type of muscle fiber	SUI (n=67)	Control (n=65)	Z-value	P-value
Type I	1 (1,2)	2 (1,3)	4.48	<0.001
Type II	1 (1,2)	2 (1.5,3)	4.53	<0.001

[i] The control group comprises women with healthy pelvic floor muscles, and the SUI group comprises women with SUI. Scores were determined using the modified Oxford grading scale (M0-M5), with the ‘M’ prefix omitted from the scores in the table for simplicity. Ranked data were analyzed using the Wilcoxon rank-sum test. SUI, stress urinary incontinence.

Comparison of sEMG results between the SUI and control groups

Multi-channel sEMG signals were successfully acquired from all participants. Differences in the sEMG activity of the PFMs between the SUI and control groups were determined using unpaired t-tests (Table III). The PFM activity measurements included ARP, MVC, TCP, ECP and PRP. In the resting state, the pre-baseline and post-baseline assessments of PFM activity, namely the ARP and PRP, exhibited no statistically significant differences between the two groups, except the ARP US and PR pre-baselines muscles. The ARP of US in SUI was significantly lower compared with the control, while the ARP of PR was significantly higher compared with the control. However, the activation amplitudes of the US, PR and PC muscles in the SUI group during flick, tonic and endurance contractions were significantly reduced compared with those in the control group (Fig. S1).

Table III Surface electromyography results of the urethral sphincter and levator ani muscles in postmenopausal women.

Table III

Surface electromyography results of the urethral sphincter and levator ani muscles in postmenopausal women.

				Levator ani muscles
	Urethral sphincter			Puborectalis			Pubococcygeus			Iliococcygeus
Parameter	SUI, µV	Control, µV	P-value	SUI, µV	Control, µV	P-value	SUI, µV	Control, µV	P-value	SUI, µV	Control, µV	P-value
Anterior resting potential	9.12±6.37	16.33±31.60	0.014	52.92±122.67	10.44±7.30	0.017	11.48±9.37	13.86±15.36	0.447	19.61±50.47	14.24±17.32	0.518
Maximum voluntary contraction	43.4±43.11	142.88±495.68	0.031	45.64±48.39	127.19±388.94	0.014	41.76±41.91	58.14±73.70	0.017	59.22±80.31	64.5±81.11	0.345
Tonic contraction potential	24.74±29.00	47.51±98.40	0.038	25.75±30.54	48.51±92.66	0.009	22.36±17.68	39.64±70.73	0.021	30.61±40.85	33.5±52.39	0.657
Endurance contraction potential	23.64±24.19	51.17±116.60	0.017	23.28±17.65	52.92±122.67	0.002	23.22±16.26	36.86±60.42	0.041	29.75±23.12	29.51±19.68	0.636
Posterior resting potential	12.43±20.68	19.97±65.12	0.464	11.56±8.17	14±13.71	0.514	11.01±6.73	12.99±13.64	0.904	14.23±12.35	13.37±12.87	0.224

[i] Data are presented as the mean ± SD and were analyzed using an unpaired t-test. SUI, stress urinary incontinence.

Predictive capability of the sEMG

ROC curves were constructed separately for each sEMG parameter (Fig. 3) to evaluate their predictive performance for SUI. The optimum predicted cut-offs (µV) for the US were: MVC, 48.55 (AUC, 0.61; 95% CI, 0.51-0.71; sensitivity, 76.1%; specificity, 46.2%); TCP, 25.79 (AUC, 0.61; 95% CI, 0.51-0.70; sensitivity, 77.6%; specificity, 44.6%); and ECP, 23.02 (AUC, 0.62; 95% CI, 0.53-0.72; sensitivity, 70.1%; specificity, 60.0%). For the PR, the optimum predicted cut-offs (µV) were: MVC, 34.48 (AUC, 0.62; 95% CI, 0.53-0.72; sensitivity, 55.2%; specificity, 67.7%); TCP, 26.96 (AUC, 0.63; 95% CI, 0.54-0.73; sensitivity, 74.6%; specificity, 50.8%); and ECP, 26.20 (AUC, 0.65; 95% CI, 0.56-0.75; sensitivity, 70.1%; specificity, 60.0%). Finally, for the PC, the optimum predicted cut-offs (µV) were: MVC, 35.05 (AUC, 0.62; 95% CI, 0.52-0.72; sensitivity, 64.2%; specificity, 67.7%); TCP, 17.36 (AUC, 0.62; 95% CI, 0.52-0.71; sensitivity, 50.7%; specificity, 73.8%); and ECP, 34.68 (AUC, 0.60; 95% CI, 0.51-0.70; sensitivity, 85.1%; specificity, 33.8%). Notably, no significant difference in sEMG results for the IC muscles was observed between the SUI and control groups.

Figure 3 ROC curves for sEMG activity of pelvic floor muscles in postmenopausal women with SUI compared with those without SUI. ROC curves for sEMG activity of the (A) urethral sphincter, (B) puborectalis, (C) pubococcygeus and (D) iliococcygeus muscles. ROC, receiver operating characteristic; sEMG, surface electromyography; SUI, stress urinary incontinence; MVC, maximum voluntary contraction; TCP, tonic contraction potential; ECP, endurance contraction potential.

Discussion

To the best of our knowledge, the present study is the first to objectively quantify regional differences in PFM function between postmenopausal women with and without SUI using both vaginal digital palpation and an intravaginal physiology-based airbag-type electrode array probe. Firstly, vaginal digital palpation was used to evaluate PFMs according to the modified Oxford grading system, revealing that the scores of the SUI group were lower than those of the control group. Although vaginal digital palpation has certain limitations, such as subjectivity, poor reliability and low sensitivity for the detection of small changes in pressure, it is still recommended by the International Urogynecological Association and International Continence Society for the evaluation of PFMs (1). In the present study, vaginal digital palpation clearly identified that the PFM strength of postmenopausal women with SUI was lower compared with that of postmenopausal women without this condition. Furthermore, previous studies by Ignácio et al (16) and Yang et al (17) endorse the routine clinical use of digital palpation examination for PFM evaluation. The findings of the present study also support vaginal digital palpation as a useful and simple strategy for the evaluation of PFM function.

Several studies have reported the value and application of sEMG in the evaluation of PFMs in cases of pelvic floor dysfunction (18-20). However, conventional sEMG signals reflect the overall activity of the PFMs, and do not provide region-specific data. In the present study, a novel ASEA probe was used to collect sEMG signals from different PFM regions based on anatomical positioning, including the US, VS, EAS and LAMs. The majority of existing vaginal probes can record only 1-4 channels of sEMG signals using custom configurations (21,22), and their rigid structure cannot adapt to anatomical variability of the vaginal structure, leading to reduced accuracy. In addition, differences in impedance, muscle depth and muscle fiber orientation hinder valid comparisons between groups without signal normalization. By contrast, the inflatable probe used in present study is able to adapt to diverse vaginal anatomies and align with specific muscle regions, which is not possible using existing technologies. Therefore, the present study describes a novel evaluation method for the accurate examination of PFMs.

Regarding specific regional PFM abnormalities and insufficiencies, significant differences in muscle strength were observed between the two groups based on sEMG results, particularly during flick, tonic and endurance contractions. In the resting state, as assessed by the pre- and post-baseline measurements, most PFMs exhibited no significant differences between the SUI and control groups, except the US muscles in the SUI group was lower, while the PR muscles in the SUI group was higher compared with the control. However, a statistically significant reduction in the activity of the US, PR and PC muscles was observed during flick, tonic and endurance contractions in the SUI group compared with the control group.

The pathogenesis of SUI is associated with anatomical abnormalities involving the urethra, urinary bladder and urogenital diaphragm. Insufficiency of the US and vesicourethral ligament, as well as weakening of the muscle-fascial structures of the whole pelvic floor, impairs normal urinary continence. Falah-Hassani et al (21) highlighted the potential role of routine clinical EMG examination in the evaluation of US insufficiency. The ASEA device used in the present study provides tight contact and a stable interface between the electrode units and PFMs, enabling the collection of high-quality sEMG signals from specific muscle regions. Notably, the mean sEMG values in the US of the SUI group were significantly lower compared with those in the control group. Although US insufficiency has attracted increasing attention (21,23) its functional assessment remains challenging. The present study describes a promising method for the evaluation of US function and supports the involvement of US insufficiency in the pathogenesis of SUI.

DeLancey (24) described the ‘hammock’-like structure formed by the continuity of the intrapelvic fascia, vaginal wall and LAMs, which helps to maintain urethral stability and urethral closure pressure during increases in abdominal pressure. The LAMs are considered a functional unit that supports the pelvic organs by lifting them in the transverse plane and compresses the urethra against the anterior vagina in the mid-sagittal plane to aid in urethral closure. Damage or dysfunction of the LAMs is a recognized contributor to SUI (23). Various approaches are available for the evaluation of LAM structure and function (21,25,26), and intravaginal dynamometry has been proposed to be the most direct approach for measuring the force-generating capacity of the LAMs (21). However, existing intravaginal devices only record the combined force of all PFMs, which may not precisely reflect the activity of individual PFMs. By contrast, the ASEA device used in the present study provides separate signals from the PR, PC and IC muscles.

Notably, the sEMG activity of the LAMs, specifically the PR and PC muscles, in the SUI group was lower compared with that in the control group. The present study demonstrates that LAM insufficiency is associated with the pathogenesis of SUI, and suggests that the PR and PC muscles, located near the center of the PFMs, play a key role in SUI pathogenesis. This suggests that dysfunction in specific LAM regions, rather than impairment of the whole muscle group, contributes to the pathogenesis of SUI.

In the present study, ROC curves were constructed for certain sEMG test results to evaluate the predictive ability of each variable for SUI. Optimum cut-off points were determined based on MVC, TCP and ECP measurements of the US and LAMs, and the corresponding AUC, 95% CI, sensitivity and specificity were assessed. In a previous study by Ptaszkowski et al (18), ‘quick flicks’, ‘contractions’ and ‘static hold’ were used to assess PFM activity in women with SUI. Sensitivities ranging from 60 to 70% were obtained, which are similar to those in the present study. However, the specificities were reported to range from 90 to 94%, which were notably higher than those in the present study. It must be noted the PFM activities in the previous study were based on the mean functional activity of all PFMs, while the present study collected signals from specific PFMs, including the US, PR and PC. These findings suggest that measuring sEMG activity in targeted PFMs may serve as a valuable tool in the diagnosis of SUI.

Based on the aforementioned analyses, the present study suggests a point-to-point evaluation and targeted treatment approach for the US and LAM regions to improve muscle function. Coordinated training may be used to increase the functional association between the US, LAMs and other muscles. The approach used in the present study may be more accurate than traditional PFM evaluation and treatment methods. However, additional clinical research is required to verify its effectiveness.

Several factors are associated with an increased risk of female SUI, including pregnancy, multiple vaginal deliveries, menopause, obesity and pelvic surgeries such as hysterectomy (27,28). Although vaginal delivery is considered an independent high-risk factor for PFM injury, the present study did not find a significant association. However, a previous study suggests that the number of vaginal deliveries is a risk factor for SUI, with one vaginal delivery being associated with a lower risk of SUI than multiple deliveries (29). Furthermore, a long-term study reported no association of stress or urgency urinary incontinence with the mode of delivery in women aged ≥50 years (30). The present study may not have found a significant association of vaginal delivery with SUI due to the low number of multiparous women among the participants. However, a variety of factors may be involved, and further verification with a larger and more diverse population, including both postpartum and menopausal women, is necessary.

To conclude, in postmenopausal women with SUI, the novel ASEA device revealed that the functional activities of specific US and LAMs, particularly the PR and PC, were lower than those in postmenopausal women without pelvic floor dysfunction. This suggests that US defects and dysfunctional LAMs play an important role in the pathogenesis of SUI in postmenopausal women. In addition, the ability to assess the US and LAMs separately may support the development of a reliable and optimized treatment strategy for the precise rehabilitation of PFMs.

Supplementary Material

Comparison of surface electromyography results for PFMs between postmenopausal women with or without SUI. Comparison of (A) MVC, (B) TCP and (C) ECP in the US, PR, PC and IC muscles. PFMs, pelvic floor muscles; SUI, stress urinary incontinence; MVC, maximum voluntary contraction; TCP, tonic contraction potential; ECP, endurance contraction potential; US, urethral sphincter; PR, puborectalis; PC, pubococcygeus; IC, Iliococcygeus; sEMG, surface electromyography. *P<0.05 as indicated.

Acknowledgements

Not applicable.

Funding

Funding: This study was funded by the 4+X Clinical Research Project of the Women's Hospital, School of Medicine, Zhejiang University (No. ZDFY2021-4X102) and the Major Health and Health Issues in Zhejiang Province Technology Plan Project of National Health Commission Scientific Research Fund (grant no. WKJ-ZJ-2507).

Availability of data and materials

The data generated in the present study may be requested from the corresponding author.

Authors' contributions

ZZ was responsible for conceptualization, software, visualization and writing the original draft of the manuscript. QC and SH contributed to methodology and validation. ZZ, QC and SH performed the formal analysis and reviewed and edited the manuscript. WL performed the investigation. SW was responsible for data curation and collecting electrical signals. ZX conceived and designed the study, analyzed and interpretated the data, reviewed and edited the final manuscript, and supervised the research. ZZ and ZX confirm the authenticity of all the raw data. All authors read and approved the final version of the manuscript.

Ethics approval and consent to participate

The study was conducted in accordance with the Declaration of Helsinki and approved by the Ethics Committee of the Women's Hospital, School of Medicine, Zhejiang University (approval nos. IRB-20220008-R and IRB-20240381-R). All participants provided written informed consent for participation.

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

References