Investigating endometrial metabolic characteristics in patients with adenomyosis using gas chromatography/mass spectrometry

Background The aetiology and pathogenesis of adenomyosis remain unclear. This study utilised untargeted metabolomics to explore the aberrant amino acid metabolism in adenomyosis. Among the studied metabolites, hydroxyproline is known to promote the proliferation and invasion of tumour cells. Due to detection limitations, hydroxyproline levels were inferred via the expression of prolyl 4-hydroxylase subunit alpha 1 (P4HA1), a crucial enzyme that catalyses the conversion of proline to hydroxyproline.

Endometrial metabolomic analysis was performed using gas chromatography-mass spectrometry (GC-MS) in 15 and 20 patients with and without adenomyosis. The expression levels of P4HA1 mRNA and protein were detected using quantitative PCR (qPCR), western blotting, and immunohistochemistry. In vitro, Cell Counting Kit-8, and 5-Ethynyl-2′-deoxyuridine (EdU) assays were used to investigate the effect of hydroxyproline on the proliferation of human endometrial stromal cells (hESCs). Additionally, a wound-healing assay was conducted to examine the effect of hydroxyproline on hESCs migration.

In total, 22 differentially expressed metabolites were identified in the adenomyosis group compared to the control group. qPCR results demonstrated that P4HA1 mRNA expression levels were significantly higher in the adenomyosis group than in the control group (p < 0.01), which was further validated at the protein level by western blotting and immunohistochemistry. In vitro, functional assays revealed that hydroxyproline promoted hESCs proliferation and migration in a dose-dependent manner. EdU assays showed a significant increase in the number of EdU-positive hESCs in the 5 mM hydroxyproline treatment group than that in the control group (p < 0.01). Additionally, wound healing assays demonstrated enhanced migration of hESCs after treatment with 5 mM hydroxyproline (p = 0.002).

Hydroxyproline levels were significantly elevated in the endometrial tissues of patients with adenomyosis. Furthermore, hydroxyproline promotes the proliferation and migration of hESCs. These findings provide new insights into the pathogenesis of adenomyosis and suggest potential therapeutic strategies. PLAIN LANGUAGE SUMMARY This study utilised untargeted metabolomics to investigate dysregulation of amino acid metabolism in endometrial tissues of patients with adenomyosis. Twenty-two differentially expressed metabolites were identified in the adenomyosis group compared with that in the control group. Hydroxyproline levels were substantially elevated in the adenomyosis group. Further experiments demonstrated that hydroxyproline enhances hESCs migration and proliferation. These findings suggest that aberrant amino acid metabolism plays a critical role in the pathogenesis of adenomyosis and may offer valuable insights into its prevention and treatment.

Adenomyosis is a gynaecological disorder characterised by the invasion of endometrial glands and stroma into the uterine myometrium, leading to compensatory hypertrophy and hyperplasia of uterine smooth muscle cells (Vercellini et al. Citation2006, Cockerham Citation2012). Clinically, this condition presents with symptoms, such as heavy menstrual bleeding (∼40–50%), progressively worsening dysmenorrhoea (25%), abdominal masses, anaemia, and infertility; however, ∼30% of patients remain asymptomatic (Gordts et al. Citation2018). Numerous studies have demonstrated a significant association between adenomyosis and adverse obstetric outcomes, including reduced pregnancy rates, miscarriage, preterm birth, and foetal growth restriction (Rees et al. Citation2023). The aetiology of adenomyosis remains poorly understood. However, researchers have hypothesised that physical and biochemical factors, such as multiple pregnancies, uterine interventions, and chronic endometritis, disrupt the structure of the basal layer, leading to abnormal endometrial cell proliferation and myometrial invasion (Benagiano et al. Citation2006, Khan et al. Citation2022). Several theories have been proposed to explain its pathogenesis: (1) invagination of the endometrial basalis and tissue injury repair, (2) Müllerian duct remnants and adult stem cell differentiation, (3) inflammatory stimulation, and (4) alternative mechanisms involving epithelial-mesenchymal transition, angiogenesis, genetics, and immunology. Although various treatment options are available, hysterectomy remains the only curative approach for adenomyosis. However, its clinical management has several limitations. For instance, pharmacological therapies are primarily suitable for patients with mild symptoms, local surgical interventions often fail to completely remove lesions, and hysterectomy is not a viable option for young patients who desire fertility. Therefore, a deeper understanding of adenomyosis pathogenesis and development of effective preventive and therapeutic strategies are crucial for early diagnosis, timely intervention, and improved patient outcomes. Metabolomics, a powerful tool following genomics, transcriptomics, and proteomics, enables the high-throughput detection and analysis of small-molecule metabolites in tissues, serum, urine, and other biological samples. It has immense potential for biomarker discovery, the elucidation of pathophysiological mechanisms, and the identification of therapeutic targets (Newgard Citation2017, Rinschen et al. Citation2019, Wishart Citation2019). However, few studies have used metabolomics to investigate the pathophysiology of adenomyosis. In 2021, Bourdon et al. used nuclear magnetic resonance (NMR) to identify nine significantly altered metabolites in the serum of patients with adenomyosis compared with healthy controls (Bourdon et al. Citation2021). Similarly, Song et al. employed gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry to analyse uterine myometrial samples, revealing 106 differentially expressed metabolites (Song et al. Citation2022). These findings suggest a strong link between metabolic dysregulation and adenomyosis development and progression. However, research on metabolic alterations in the endometrial tissues of patients with adenomyosis remains limited. This study aims to investigate the metabolic alterations in the endometrial tissues of adenomyosis patients and elucidate their potential role in disease pathogenesis.

This study was conducted at the Second Affiliated Hospital of Chongqing Medical University between February and June 2023. Patients in the adenomyosis group were diagnosed based on clinical symptoms, magnetic resonance imaging (MRI) findings, and transvaginal ultrasound, and underwent surgical resection (e.g. hysterectomy) or hysteroscopy. The control group consisted of patients who underwent hysterectomy for uterine fibroids or hysteroscopy for abnormal endometrial echoes with normal endometrial tissues confirmed via histopathological examination. Exclusion criteria included a history of malignant tumours, use of hormone-related medications within the past three months, presence of an intrauterine device, diagnosis of ovarian or other forms of endometriosis, presence of pelvic inflammatory disease, or severe hepatic or renal insufficiency. After surgical removal, endometrial tissues were immediately rinsed with pre-cooled phosphate-buffered saline (PBS), transferred to cryopreservation tubes, and temporarily stored on dry ice. Tissues were then flash-frozen in liquid nitrogen for 15 min and stored at −80 °C for further analyses. In preparation for GC-MS analysis, tissue samples underwent the following pre-treatment procedures. The boiling points of isolated metabolites were reduced using methyl chloroformate, following the protocol established by Smart (Smart et al. Citation2010). Tissue samples weighing 20 mg were transferred into 2 mL Eppendorf tube. Subsequently, 200 μL sodium hydroxide (1 M), 200 μL methanol, 4 μL D4-alanine, and three magnetic beads were added to each tube, and each sample was homogenised using a homogeniser (QIAGEN@, TissueLyser II) to fully cracking, followed by centrifugation (12,000 rpm, 15 min, 4 °C). The supernatant was transferred into a new glass tube, and 34 μL pyridine and 20 μL MCF were added twice and mixed. To separate the derivative metabolites from the reaction mixture, 400 μL of chloroform was added to each tube, followed by the addition of 400 μL of sodium bicarbonate (50 mM). The mixture was vigorously shaken for 10 s and subsequently centrifuged at 2000 rpm for 10 min at 4 °C. The upper aqueous phase was discarded, and sodium sulphate powder was added to the bottom chloroform layer to dry and 200 μL liquid was absorbed to prepare for GC-MS analysis. We conducted metabolomic analysis of the endometrial tissue derivatisation samples using GC-MS (Agilent 7890B GC system equipped with Agilent 5977 A MSD system). The GC used BD-1701 capillary column (30 m × 250 μm id × 0.25 μm, Agilent) to separate non-polar compounds. The GC temperature and MS parameters were set as described by Gao (Gao et al. Citation2022). The samples were infused with helium gas at 290 °C at 1.0 mL/min and the resulting samples were run in a pulseless split-less mode over the GC column. The auxiliary temperature was set at 250 °C, 230 °C for the MS quadrupole, and 50 and 280 °C for the MS source chip and protective chip. The scanning rate was set to 1.562 μ/s, the detection range was 30–550 μm, and the solvent delay time was 5.5 min. Using Automatic Mass Spectrometry Deconvolution and Identification System software, the gas chromatographic peaks were deconvoluted, and the original data were subsequently recognised through a combination of retention time, internal metabolite identification, and peak integration software using the R software package. The relative abundances of metabolites were extracted using the R software, which is based on MassOmics XCMS. Additionally, an internal standard (2,3,3,3-d4-alanine) was used to standardise the peak values of the metabolites to reduce the impact of sample preparation and instrument quality. We extracted total RNA from endometrial tissues using TRIzol reagent (Invitrogen, USA) for quantitative real-time PCR analysis. RNA concentration and purity were determined using a NanoDrop One Ultramicro spectrophotometer (Thermo Scientific, USA). Subsequently, 5 × PrimeScriptTM RT Master Mix (Takara, Japan) was used for reverse transcription in a reverse transcription instrument, then TB Green® Premix Ex TaqTM II (Takara, Japan) was used for quantitative PCR (qPCR) in a C1000TM Thermal Cycler (Bio-RAD, USA). Primer sequences were designed by Tsingke Biotechnology Co., Ltd. (Beijing, China) and are listed in . The 2^−ΔΔCt approach was utilised to determine fold changes in expression, with β-actin expression serving as an internal standard. We isolated total protein from endometrial tissue using radioimmunoprecipitation assay lysis buffer (Beyotime, Beijing, China) for Western blot analysis. After centrifugation at 12,000 rpm, 4 °C, protein concentrations were quantitated utilising a bicinchoninic acid Protein Assay kit (Beyotime, China) according to the manufacturer’s protocol. Lysates were stored at −80 °C. Proteins were subsequently separated via 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (Merck Millipore, Billerica, MA, USA) and transferred to polyvinylidene difluoride membranes. Following a blocking step with 5% bovine serum albumin, membranes were incubated overnight at 4 °C with primary antibody. Following incubation with an HRP-conjugated secondary antibody for 1 h at room temperature, signals were visualised using a BeyoECL Moon Kit (Beyotime) and captured using a ChemiDocTM MP Imaging system (Bio-Rad, Hercules, CA, USA). The intensity of each target protein band was quantitated using Image Lab software (version 6.0, Bio-Rad). We performed immunochemistry on endometrial specimens fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned. Paraffin-embedded sections were heated at 60 °C for 2 h and subsequently underwent xylene dewaxing and gradient alcohol hydration. Slides were submerged in sodium citrate buffer (Servicebio, China) to facilitate antigen retrieval. They were then treated with endogenous peroxidase activity for 10 min before blocking alongside goat serum for 30 min at 37 °C. Subsequently, the sections were incubated overnight at 4 °C with the primary antibody (1:50) against prolyl 4-hydroxylase subunit alpha 1 (P4HA1) (proteintech, Wuhan, China). The following day, after rewarming and washing steps, the slides were incubated with the secondary antibody and subsequently underwent staining with 3,3′-diaminobenzidine (DAB). Haematoxylin was used as a counterstain to visualise nuclei. Using the ImageJ software, the expression levels of P4HA1 were evaluated based on an assessment of staining intensity and the extent of the positive staining area. We cultured human endometrial stromal cells (hESCs) obtained from ProCell (Wuhan, China) in Dulbecco’s modified Eagle medium (DMEM) (Gibco, United States) supplemented with 10% foetal bovine serum (FBS; PAN Biotech, United States) and 1% penicillin-streptomycin. To maintain ideal growth conditions, cells were cultured at 37 °C with 5% CO2. The culture medium was changed every 2–3 days. We assessed cell viability using the cell counting kit-8 (CCK-8) assay. The hESCs were digested with trypsin (Mengbio, China), resuspended in a single-cell suspension in complete DMEM, and seeded at a density of 3 × 103 cells/well in a 96-well plate. After overnight incubation at 37 °C in a 5% CO2 incubator, the hESCs were pre-treated with various concentrations of hydroxyproline (Meilunbio, China) for 24 h in completed DMEM medium following starvation in serum-free medium for 12 h. Cell viability was assessed using a CCK-8 kit (Beyotime, China). One hundred microlitres of DMEM supplemented with 10% CCK-8 reagent for 1 h, and the optical density (OD) was measured at a wavelength of 450 nm (Bio-Rad, USA). The experiments were performed independently three times for each group. We examined DNA synthesis using the 5-Ethynyl-2′-Deoxyuridine (EdU) assay. hESCs were resuspended in DMEM complete medium and were plated in 96-well at a density of 1 × 103 cells/well in a 37 °C incubator with 5% CO2. Hydroxyproline (5 mM) was added for 24 h after starvation in a serum-free medium for 12 h. According to the EdU kit (product number: C0075L, Beyotime, China), EdU was added to each well and incubated for 2 h, then each well was fixed with 4% paraformaldehyde (Beyotime, China) and the membrane was broken through immunostaining (Beyotime, China). The click reaction solution was added and incubated for 20 min in the dark. Nuclei were stained with Hoechst 33342, observed, and photographed under a microscope (BIO-RAD, The United States). We evaluated the migratory capacity of hESCs using the wound-healing assay. Briefly, 5 × 105 cells were plated in six-well plates at a confluence of 80–90%. Subsequently, 10 μL sterile plastic pipette tips were used to scratch cells. After gentle washing with PBS, the cells were incubated in DMEM containing different concentrations of hydroxyproline (0.5, 1, and 5 mM) without FBS. At 0 and 24 h, the wounds were observed under an optical microscope (OLYMPUS, Japan). Finally, the percentage of scratch healing in each group was calculated. All statistical analyses were performed in accordance with methodological standards. The original GSE185392 dataset was downloaded from the Gene Expression Omnibus (GEO) database. Genes and their Log2 fold change (FC) data were obtained using GEO2R analysis. The downloaded data were sorted according to log2 FC. Eighty-four genes from ten metabolic pathways were selected from the Kyoto Encyclopaedia of Genes and Genomes (KEGG) database, and enrichment analysis was conducted using Gene Set Enrichment Analysis (GSEA) software. SPSS was used for statistical analyses. The Shapiro-Wilk test was used to evaluate whether the constant factors were normally distributed. If the data displayed normal distribution, independent t-tests were used. The Mann-Whitney U test was used when data did not fit a normal distribution. Data with normal distributions are presented as means ± standard deviations, and data with non-normal distributions are expressed as medians (25th and 75th percentiles). In multivariate statistical analyses, Partial Least Squares Discrimination Analysis (PLS-DA) and Variable Importance Projection (VIP) values were calculated using the open data platform MetaboAnalyst5.0 (Liu et al. Citation2023). During univariate statistical analyses, the statistical significance of the difference between the two groups for each metabolite was established by calculating the p-value, and the false discovery rate (FDR) was determined using the Student’s t-test. Specifically, only double-tailed p < 0.05 and FDR < 0.1 were considered statistically significant. All procedures performed in this study involving human participants were reviewed and approved by the Ethics Committee of the Second Affiliated Hospital of Chongqing Medical University and followed the 1964 Helsinki Declaration and its later amendments or comparable ethical standards. All participants provided written informed consent to participate in this study. The study protocol was approved by the Ethics Committee of Chongqing Medical University (No. 13, 30 January 2024).

We conducted a clinical analysis of adenomyosis by enrolling 35 individuals, comprising 15 patients diagnosed with adenomyosis and 20 individuals without adenomyosis (who served as the control group). The flow chart of this study is presented in . The clinical demographics of the participants are presented in . The two groups exhibited comparable profiles in terms of age, body mass index, parity, and interval between the last menstruation and surgery. The adenomyosis group demonstrated a slightly higher number/proportion of multigravida women than the control group [4 (3, 6) vs. 3 (1, 3.75), p = 0.001]. We performed PLS-DA on GC-MS data to compare endometrial metabolic profiles between adenomyosis and control groups. As shown in , a clear differential clustering was observed between the adenomyosis and control groups, reflecting distinct metabolic profiles. The PLS-DA score plot showed significant separation between the two groups, with good model reliability and predictive power (R2Y = 0.808, Q2 = 0.650) (). A permutation test (n = 1000) was conducted to validate the PLS-DA model. The results (p < 0.05) confirmed the robustness of the model, indicating no overfitting (). The volcano plot () illustrates the 170 metabolites that differed between the adenomyosis and control groups. Based on the PLS-DA model, to find substantial variations within metabolites among the two categories, factors with VIP values >1.0 were selected using the criteria of p < 0.05 and FDR < 0.1. A total of 22 distinct metabolites were identified as being significantly different, as shown in the volcano plot () and heatmap (). Specifically, the levels of amino acids, including proline, N-acetyl-L-leucine, N-acetyl-L-alanine, and N-acetyl-aspartate, were lower in the adenomyosis group than in the control group. Conversely, the concentrations of citric acid, hydroxyproline, N-acetyl-L-lysine, 2-hydroxyphenylacetic acid, and 3-methyl-2-oxopentanoic acid were higher in the adenomyosis group. Levels of fatty acids, such as 10-pentadecenoic acid and docosahexaenoic acid (DHA) were also higher in the adenomyosis group. Additionally, some alkanes were altered. Statistically significant differential metabolites were imported into MetaboAnalyst 5.0 for enrichment analysis to identify potential metabolic pathways associated with adenomyosis compared to the control group. KEGG bubble plot analysis () indicated that differential metabolites were mainly enriched in pathways related to aspartate metabolism, arginine and proline metabolism, tyrosine metabolism, tryptophan metabolism, and the citrate cycle. To further analyse the metabolic pathways in adenomyosis, the transcriptomic data of endometrial tissues during the window of implantation from patients with and without adenomyosis were extracted from the NCBI database (GSE185392). A total of 84 metabolic pathways were identified from the 10 KEGG analyses. GSEA revealed () that the gene set related to proline metabolism was upregulated in the adenomyosis category compared to the control category, indicating metabolic dysregulation of proline in the endometrial tissues of adenomyosis. Our qPCR results revealed disrupted proline metabolism in the endometrial tissues of adenomyosis patients, as evidenced by significantly higher P4HA1 mRNA expression levels in the adenomyosis group compared to controls (p < 0.01; ). Protein expression levels of P4HA1 were assessed by western blotting and immunohistochemistry (). The results demonstrated that P4HA1 expression was notably higher in the endometrial tissues of patients with adenomyosis than in controls. Immunohistochemical analysis revealed that P4HA1 was predominantly expressed in the cytoplasm of hESCs. Through in vitro experiments using the CCK-8 assay, we found that hydroxyproline promotes the proliferation of hESCs in a dose-dependent manner (). To further validate this effect, we assessed DNA synthesis using the EdU assay (). The results revealed a significant increase in EdU-positive hESCs following treatment with 5 mM hydroxyproline compared to the control group (p < 0.01). To examine the effect of hydroxyproline on hESCs migration, a wound-healing assay was conducted after treatment with 0, 0.5, 1, or 5 mM hydroxyproline for 24 h. As shown in , 5 mM hydroxyproline significantly promoted hESCs migration (p = 0.002).

We conducted untargeted metabolomic analysis on endometrial tissue samples from patients with adenomyosis, complemented by bioinformatic investigations, to elucidate local amino acid metabolism. Our analysis identified 22 significantly altered metabolites, among which hydroxyproline was markedly upregulated in the adenomyosis group compared to the control group. Due to the technical limitations of direct detection methods, hydroxyproline levels can be indirectly assessed by measuring P4HA1 expression, a key enzyme responsible for catalysing the conversion of proline to hydroxyproline. Elevated P4HA1 expression has been demonstrated to correlate with increased hydroxylation activity (Nwogu et al. Citation2001, Shi et al. Citation2020). Previous studies have emphasised the critical role of enhanced proliferation and migration of hESCs in adenomyosis progression (Yang et al. Citation2007, Huang et al. Citation2021). We demonstrated that hydroxyproline promotes hESCs proliferation and migration in a dose-dependent manner. These findings indicate dysregulated proline metabolism in the endometrial tissues of adenomyosis patients, characterised by increased catalytic activity of prolyl hydroxylases and elevated local hydroxyproline levels. This metabolic dysregulation provides compelling evidence for further investigation into the mechanisms underlying adenomyosis pathogenesis. Dutta et al. found that proline levels were significantly decreased in the endometrial tissue of patients with endometriosis (Dutta et al. Citation2018). In this study, we also found that the proline content of the endometrial tissue in patients with adenomyosis was notably lower than that in the control group. GSEA of the transcriptome revealed significant upregulation of arginine-proline metabolism in adenomyosis during the receptive phase of the endometrium. Proline synthesis involves the convertion of glutamate to Δ1-pyrroline-5-carboxylate (P5C) by the enzyme ALDH18A1, followed by the generation of proline by P5C reductase; while degradation involves the generation of P5C catalysed by PRODH (Liu et al. Citation2012). Another pathway of proline metabolism involves the generation of hydroxyproline by P4HA1 (Duan et al. Citation2018). Our results showed that the mRNA and protein levels of P4HA1 were remarkably elevated in the adenomyosis group than that in the control group. According to previous studies, P4HA1 promotes the development, invasion, and metastasis of tumours (Gilkes et al. Citation2013, Duan et al. Citation2018). However, whether P4HA1 is associated with adenomyosis development remains unexplored and warrants further investigation. We found that compared with the control group, patients with adenomyosis had higher levels of hydroxyproline in their endometrial tissue samples. Previous studies have reported that hydroxyproline facilitates hepatocellular carcinoma cell proliferation and invasion by inducing HIF1α expression under hypoxic conditions (Tang et al. Citation2018). Additionally, hydroxyproline promotes pancreatic cancer cell proliferation and invasion by downregulating the EGLN/HIF1α pathway (Chiba et al. Citation2020). In this study, treatment of endometrial stromal cells with different concentrations of exogenous hydroxyproline increased the proliferation and migration vitality of endometrial stromal cells. We hypothesise that the consumption of proline and subsequent accumulation of hydroxyproline in the endometrial tissue could have significant implications in the onset and progression of adenomyosis. Moreover, compared to the control group, adenomyosis patients had higher levels of citrate in their endometrial tissues. Iizuka et al. detected high levels of citrate in the uterine tissue of a mouse model of adenomyosis using NMR spectroscopy (Iizuka et al.Citation1996). Citrate is essential for insulin production, cancer, and inflammation (Iacobazzi et al. Citation2014). This is a crucial step in the tricarboxylic acid (TCA) cycle and acts as a substrate for cellular energy metabolism. The elevation in citrate levels suggests a disruption of the TCA cycle. Furthermore, our results revealed that the adenomyosis group had higher lactate levels than the control group (without statistical significance). Lactate, which is the final product of anaerobic glycolysis, is a major contributor to hypoxia. Our results suggest that the endometrial tissues of patients with adenomyosis may be in a hypoxic environment, which could impair endometrial receptivity and affect reproductive potential. In addition to citrate metabolic abnormalities, there may be energy metabolism abnormalities in the endometrial tissues of patients with adenomyosis. However, there is little information on the role of citrate in adenomyosis, and how it affects endometrial stromal cells remains unclear. Therefore, further studies are needed. These findings have important clinical implications. Understanding metabolic disruption and its effects on cellular behaviour in adenomyosis may pave the way for novel diagnostic and therapeutic strategies. Additionally, this study had some limitations, including the use of 15 adenomyosis and 20 non-adenomyosis control samples for metabolomic analysis. Although this sample size was feasible and consistent with those of previous studies in the field, it remains relatively small. Future studies with larger sample sizes should provide greater statistical power and improve the generalisability of the findings. This study primarily explored the effects of hydroxyproline on the proliferation and migration of human endometrial stromal cells at the functional level. However, the specific molecular mechanisms underlying these metabolic changes were not elucidated and require further investigation in subsequent studies.

In conclusion, we identified specific metabolites, including proline, hydroxyproline, and citrate, in the endometrial tissues of patients with adenomyosis. Aberrant metabolism of these amino acids may affect energy metabolism, inflammatory responses, protein synthesis, and hydrolysis. Future research on the function of these amino acids in adenomyosis may have a significant impact on its prevention and treatment.

The authors would like to thank all the teachers in the Department of Gynecology and Obstetrics of The Second Affiliated Hospital of Chongqing Medical University, for their technical assistance. Lina Hu and Fan He contributed to the study design. Clinical specimens were collected by Ju Ren. GC-MS metabolomics and data processing were performed by Ju Ren, Jingcong Dai, and Li Wang. Cell experiments were performed by Ju Ren and Jiashuo Liu. The first draft of the manuscript was written by Ju Ren and Jiashuo Liu. Fan He and Lina Hu revised the manuscript. All authors read and approved the final manuscript. Disclosure statement No potential conflict of interest was reported by the authors. Data availability statement The entirety of the data that was produced or subjected to analysis throughout this study has been comprehensively incorporated within this published article. Additional information Funding

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