Uterine-derived exosomes induce the M2 polarization of macrophages via miR210-3p to promote the development of endometriosis

Uterine-derived exosomes induce the M2 polarization of macrophages via miR210-3p to promote the development of endometriosis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Uterine-derived exosomes induce the M2 polarization of macrophages via miR210-3p to promote the development of endometriosis Shengnan Chen, Ying Jiang, Xiaoshan Chai, Zhaoying Chen, Hao Tian, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3030329/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Endometriosis development is associated with peritoneal immune microenvironment abnormality. However, the specific mechanism is uncertain. Investigating peritoneal immune microenvironment regulation mechanisms could introduce novel therapeutic strategies for effective endometriosis treatment. Methods Wound healing assay, Transwell, colony formation were carried out to assess cells invasion and proliferation. Western blotting analysis and ELISA were used to evaluate cells resistance to progesterone. Endometriosis C57BL6 mouse model was conducted to assess the impact of peritoneal inflammatory environment and macrophage miR-210-3p on ectopic lesion implantation and growth. miRNA and proteomics sequencing were carried out to verify the potential mechanisms influencing the development of endometriosis. Results Medium conditioned of peritoneal macrophages from patients with endometriosis, as well as the medium conditioned of macrophages treated with uterine-derived exosomes of endometriosis patients, promoted the proliferation, invasion and progesterone resistance of cells. Interestingly, Uterine-derived exosomes of endometriosis patients increased miR-210-3p expression in peritoneal macrophages. In vivo experiments confirmed that macrophages lentivirally transduced with miR-210-3p inhibitor can significantly decrease the number and volume of endometriotic lesions. Mechanistically, miR-210-3p significantly induced M2 macrophage polarization by inhibiting the expression of ATP5D, and promoted cells migration. Conclusions Uterine-derived exosomes of endometriosis patients upregulated the expression of miR-210-3p in peritoneal macrophages to inhibit ATP5D, driving macrophages polarization towards M2 and promoting the development of endometriosis. Endometriosis exosome macrophage miR-210-3p ATP5D Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Background Endometriosis is a common, oestrogen-dependent, chronic gynaecological disorder associated with pelvic pain and infertility, affecting about 10% of reproductive-aged women[ 1 ]. The development of endometriosis involves hormonal imbalances, immunological, proinflammatory, and proangiogenic processes[ 2 ]. As a result, the precise pathogenic mechanisms of endometriosis are not well-identified. At present, the widely accepted theory is Sampson's theory of retrograde menstruation[ 3 ]. It is not known why only 6–10% of women develop endometriosis despite retrograde menstruation occurring in 76–90% of women[ 4 ]. An attractive hypothesis is that the endometrial stromal cells (hESCs) containing stem cell characteristics or endometrium-derived mesenchymal stem cells (EMSCs)[ 5 – 7 ] may be abnormally shed through the fallopian tube into the peritoneal cavity in affected women, leading to the formation of ectopic implants in endometriosis. There are reports indicating that refluxed ectopic stromal stem cells display diverse features from eutopic stromal stem cells attributable to local microenvironment differences in the abdominal cavity [ 8 , 9 ]. Consequently, some scholars have proposed that the development of endometriosis may involve the interaction between MSCs and peritoneal immune cells. Although the pathogenesis of endometriosis remains elusive, growing evidence has demonstrated that it may be related to an aberrant immune response[ 10 , 11 ]. Peritoneal macrophages play a key role in the establishment and maintenance of endometriosis by regulating the immune and peritoneal environments[ 12 , 13 ]. In endometriosis, abnormal levels of cytokines, interferon, and macrophage colony-stimulating factor in peritoneal fluid lead to functional scavenger receptor deficiency in peritoneal macrophages[ 14 , 15 ]. This decrease in phagocytic activity may contribute to the growth of ectopic endometrial cells. Additionally, based on their activation state and function, macrophages can be classified into 'classically activated' M1 and 'alternatively activated' M2. Bacci et al.[ 16 ] confirmed that patients with endometriosis mainly have peritoneal fluid containing M2-polarized macrophages. Moreover, the focus of endometriosis has a higher concentration of M2 macrophages than surrounding tissues. Injection of M2-polarized macrophages into the peritoneal cavity of mice promotes the formation of large ectopic lesions, while M1-polarized macrophages only produce small lesions, indicating that M2-polarized macrophages can facilitate growth of ectopic lesions[ 17 ]. Our previous research found that[ 18 ] eutopic endometrial cells can alter the immune phenotype of peritoneal macrophages. However, the mechanism by which eutopic endometrial cells regulate the peritoneal immune microenvironment from a distance is unclear as they are located in the uterine cavity. Exosomes[ 19 ] are a circular monolayer structure with a diameter of 40 ~ 100nm, which can carry a variety of proteins, mRNAs, and miRNA. It is a carrier of natural intercellular information, which can remotely regulate the function of receptor cells and participate in the processes of immunity, antigen presentation, cell migration, angiogenesis, and cell growth[ 20 ]. The effect of exosomes on macrophage polarization has been confirmed in various disease states. Zhao et al.[ 21 ] demonstrated that mesenchymal stromal cell-derived exosomes attenuate myocardial ischaemia -reperfusion injury via modification of the polarization of M1 macrophages to M2 macrophages. Xin et al.[ 22 ] constructed a scaffold with mesenchymal stem cell-derived exosomes to promote endometrium regeneration and fertility restoration through macrophage immune regulation. Sun et al.[ 23 ] found that exosomes from endometrial stromal cells of endometriosis patients could induce macrophages into M2 phenotype, decreasing their phagocytic ability. Studies have shown that endometrial exosomes released into the uterine cavity can reflux into the peritoneal cavity and communicate with other cells to influence the implantation of ectopic endometrial lesions[ 24 , 25 ]. Our hypothesis is that uterine-derived exosomes can modulate peritoneal macrophages to create an appropriate environment for the attachment of ectopic endometrial lesions. In this study, we investigated the specific mechanisms by which uterine-derived exosomes regulate peritoneal macrophages to promote the development of endometriosis. Methods Clinical specimens Patients who underwent laparoscopic surgery for ovarian endometriosis were enrolled in the EMs group (n = 30). Patients who underwent surgical treatment for other benign gynecological diseases, such as simple ovarian cysts, uterine incision diverticula, or tubal ligation were enrolled in the control group (n = 30). Samples of peritoneal fluid, uterine fluid, and normal endometrial tissues were collected from all patients. All of the samples were collected at the time of surgery at the Second Xiangya Hospital of Central South University. Informed consent was obtained from each patient, and the study was approved by the Medical Ethics Committee of the Second Xiangya Hospital of Central South University (#2020-065). Endometrial tissue and peritoneal fluid were treated within 6 hours of collection, while the uterine fluid was stored in a freezer at -80℃ after centrifugation. All of the patients had normal ovulation with regular menstrual cycles, and none of the patients had received steroid hormonal medications for at least 3 months. Isolation of primary cells and cell culture Normal endometrial tissues were minced and digested in F12/DMEM containing collagenase I(2.5mg/ml) and deoxyribonuclease I(0.1mg/ml) at 37°C for 40 minutes. The dispersed cells were separated by filtration through a 40µm cell strainer. The cells were plated onto 25cm 2 Falcon tissue culture flasks and maintained in F12/DMEM supplemented with 10% fetal bovine serum (FBS) at 37°C in a humidified atmosphere containing 5% CO2. Endometrial stromal cells (hESCs) were purified by adherent culture, and the cells were used between passages for 3–6. The peritoneal fluid was collected, centrifuged, and recuperated with PBS. Then, it was layered on the same volume of Ficoll and centrifuged for 20 minutes at 2000rpm and at room temperature. The cloud-like layer was collected and washed twice with PBS before inoculating the cells in a 6-well plate, with a density of 1 × 10 6 /well. They were cultured in RPMI1640 medium containing 10% FBS for 2 hours to remove non-adherent cells. Only the remaining adherent cells were considered peritoneal fluid macrophages. After culturing for 24h-48h, the supernatant and RNA were collected. Thp1 and RAW264.7 cells were purchased from National Collection of Authenticated Cell Cultures. These cells were cultured in RPMI1640 or DMEM High-Glucose medium supplemented with 10% FBS and 1% penicillin/streptomycin. Surface marker profile and differentiation capacity The characteristics of the stem cells[ 7 ] were identified by flow cytometry. After 3 generations of culture, the hESCs cells were incubated with labeled surface antibodies: PerCP-CyTM5.5 Mouse Anti-Human CD45, APC Mouse Anti-Human CD146, PE Mouse Anti-Human CD140b on ice for 30 minutes. Then, the cells were resuspended in PBS for FCM analysis. hESCs were inoculated in a 6-well plate and cultured in DEME/F12 medium for 24 hours until reaching 70% confluence. Then, an osteogenic differentiation medium was used to induce osteogenesis. The culture medium was changed every 7 days, and alizarin red staining solution was used to detect calcium deposition on the 28th day. Then, an osteogenic differentiation medium was used to induce osteogenesis. The culture medium was changed every 7 days, and alizarin red staining solution was used to detect calcium deposition on the 28th day. Wound healing assay The third generation of hESCs in the logarithmic growth phase were seeded into a 6-well plate with a density of 5 × 10 5 cells/well. When the cell density reached 80 to 90%, a scratch was made in the monolayer using a 100µl pipette tip in the middle of each well. Wound healing within the same scratched line was then observed and photographed at specific time points (0 h, 24 h, or 48 h). Each experiment was performed three times. Transwell 5 × 104 cells were seeded into the upper Transwell chamber using an 8µm pore size filter membrane and culture medium supplemented with 10% FBS. Macrophage conditioned medium was added to the lower chamber at a ratio of 1:1 as a chemoattractant. The cells that had migrated to the lower surface of the filter were fixed in a 4% paraformaldehyde and stained with crystal violet for 30 minutes. The natural air-dried filter membrane was used to count the migrated cells. Three independent experiments were conducted. Colony Formation Assay The third generation hESCs were digested and seeded directly in 6-well plates (3× 10 3 cells/well) for the colony formation assay and cultured in the presence of 10% FBS at 37°C with 5% CO2. Two weeks later, the cells were fixed with 4% paraformaldehyde and stained with crystal violet for 30 minutes. Count the number of colonies with diameter > 0.5mm. Isolation of exosomes from uterine fluid The uterine fluid collected during the operation was thawed at room temperature and centrifuged at 3000 × g for 20min to remove cells. Then, the supernatant is passed through a 0.22µm filter to remove cellular debris. The exosomes were extracted by Exosome Purification kit-exoupur (ECHO BIOTECH, Beijing, China), then concentrated by ultrafilter tube at 30000 rpm for 30min. The isolated exosomes were characterized by transmission electron microscopy, nanoparticle tracking analysis (NTA) and western blot. Treatment of macrophages with uterine fluid-derived exosomes Uterine fluid-derived exosomes were labeled using a PKH67 Fluorescent Cell Linker Kit (Sigma-Aldrich, Saint Louis, MO, USA) to detect the direct transfer of the exosomes into macrophages (Thp1). The purified exosomes were resuspended in PBS and labeled with PKH67. PBS without exosomes was used as control. The internalization of exosomes was conducted by incubating Thp1 cells at 37°C with a final concentration of 100µg/ml. Fluorescent signals were detected after 48h. Cell transfection The miR-210-3p mimics, inhibitor and respective negative controls (scrambled oligos) were obtained from RiboBio. According to the manufacturer's instructions, transfections were performed using Lipofectamine 3000. Transfection of RAW264.7 with miR-210-3p by lentiviral vector miR-210-3p short hairpin RNA lentivirus (miR-210-3p inhibitor) and corresponding negative control lentiviruses (miRNA-NC) were purchased from Genechem Co. Ltd (Shanghai, China). Cells were infected with lentiviruses at 100 MOI (multiplicity of infection) for 6h and replaced with fresh medium for another 48h. The green fluorescent protein signal was detected by a fluorescence microscope and gene transfection efficiency was verified by PCR. Establishment of the endometriosis mouse model. Six-week-old female nonpregnant C57BL6 (20g) were obtained from the Laboratory Animal Research Center of the Second Xiangya Hospital of Central South University. All animal handling and experimental procedures were approved by the Animal Experimental Ethics Committee of the Second Xiangya Hospital of Central South University (#20220059). Donor mice were initially treated with estradiol benzoate (3µg/mouse) and sacrificed after 7 days. The uterus was removed and immediately placed in saline solution. The two uterine horns were excised and split longitudinally with a surgical blade, and the endometrium was carefully separated from muscles. Each endometrium was identically processed and disrupted into segments smaller than 1 mm. On the 0th day of modeling, the fragment was injected into the abdominal cavity of recipient mice. Overall, segments of endometrium from one uterus were placed in 400µl PBS and injected into two recipient mice. The mice were sacrificed by cervical dislocation 28 days after endometrial tissue injection, measure and record the size and volume of the lesions. In the experimental group, mice received intraperitoneal injections of 2mg/kg LPS, every 3 days. (control group, injection of PBS). FCM analysis After euthanizing the animals, peritoneal cells were retrieved by peritoneal lavage with 7ml of ice-cold washing buffer PBS. After shaking the mice, peritoneal cells were collected. CD16/32 was used to block Fc receptors, F4/80 and CD11b were used as pan-macrophage markers, iNOS and CD206 were used to detect M1 and M2 macrophages. The peritoneal macrophages were incubated with antibodies. After washing with PBS, the cells were resuspended in PBS for FCM analysis. RNA extraction and quantitative real-time PCR Total RNA was extracted using TRIzol reagent. RNA was reverse-transcribed into cDNA using the HiScript II Q RT SuperMix for qPCR (+ gDNA wiper) (No. R223-01;Vazyme), followed by qRT-PCR using ChamQ Universal SYBR qPCR Master Mix (No. Q711-02; Vazyme) under the following thermal cycling conditions: initial denaturation at 95°C for 30 s, followed by 40 cycles of 95°C for 5 s and 60°C for 30 s, and lastly 95°C for 15 s, 60°C for 1 min, and 95°C for 15 s. All PCR primers were designed and synthesized by Sangon Biotech (Shanghai, China; Supplemental table S1 ). The threshold cycle method (2 − ΔΔCT) was used to calculate the relative expression levels normalized to GAPDH levels. Western blot analysis The total protein was extracted, and the protein concentration was determined by BCA. The proteins separated via SDS-PAGE and transferred to PVDF membranes, and then blocked in 5% non-fat milk at room temperature for 2h. The membranes were incubated overnight at 4°C with primary antibodies. According to the manufacturer's instructions, HRP conjugated secondary antibodies and ECL kits were used to detect immunoreactive protein. TMT Proteomics Total proteins of Thp1 transfected with miR-210-3p were extracted and their concentrations were quantified by the BCA assay (Thermo Fisher Scientific, Waltham, MA). Peptides were labeled with TMT according to the manufacturer's protocol. For each sample, 2ug of total peptides were separated and analyzed with a nano­UPLC(EASY­nLC1200) coupled to a Q Exactive HFX Orbitrap instrument (Thermo Fisher Scientific) with a nano­electrospray ion source. Data dependent acquisition (DDA) was performed in profile and positive mode with Orbitrap analyzer. Statistical analysis The data are presented as the mean ± SD from at least three independent experiments. Statistical significance was measured using Student’s t test (two-tailed). A value of p < 0.05 indicates that the difference was statistically. All statistical analyses were performed with GraphPad Prism 8.0. Results Abdominal inflammatory microenvironment promotes the development of endometriosis Establishment of a mouse endometriosis model, and simulation of different abdominal environments by injecting LPS and PBS into the peritoneal cavity. Typical endometriosis-like lesions were formed in the mesentery or peritoneum 28 days after implantation (Fig. 1 a, b). The results showed that the volume of lesions in LPS group was significantly larger than that in PBS group, and the number of lesions showed the same trend (Fig. 1 c). We conducted immunocytochemical staining for Ki-67 (proliferation marker) and vimentin (mesenchymal marker) followed by fluorescence microscopy, and then observed that abdominal inflammation significantly promoted the proliferation of endometrial stromal cells (Fig. 1 d). At the same time, the peritoneal fluid of mice was collected for FCM analysis, it was found that the number of macrophages in LPS group was more than that in PBS group, and M2 macrophages were dominant in both groups (Fig. 1 e, f). Peritoneal macrophages promote the proliferation and migration of hESCs Primary isolated hESCs expressed high levels of mesenchymal stem cell surface markers CD140b and CD146, and have the potential for osteogenic and adipogenic differentiation (Supplemental Fig. 1a, b, c). Peritoneal fluid macrophages conditioned medium from patients with endometriosis (EMs) and patients without endometriosis (Control) was used to interfere with hESCs. As shown in Fig. 2 a, b, the results of wound healing assays and transwell migration were performed to assess the influence of macrophages on the migration of hESCs. As expected, macrophages from EMs remarkably enhanced the migration capacity of hESCs. In addition, Western blot analysis (Fig. 2 c, d) showed that compared to the Control group, macrophages from EMs enhanced the expression of MMP9, MMP2 and N-Cadherin in hESCs. Subsequently, FCM analysis and colony formation revealed that macrophages from EMs promoted the proliferative capacity of hESCs (Fig. 2 e, f). To assess the influence of macrophages on progesterone resistance of hESCs, we induced decidualization in vitro. Then we examined the expression of estrogen (ER) and progesterone receptors (PR) in hESCs (Fig. 2 g). Western blot analysis revealed that peritoneal macrophages in patients with endometriosis reduced the expression of PR and epithelial marker E-cadherin and enhanced the expression of mesenchymal marker N-Cadherin, which means that the differentiation ability of hESCs into epithelial cells and decidualization was impaired. Consistent with the findings, ELISA result showed (Fig. 2 h) macrophages from EMs decreased the expression of the decidualization markers IGFBP-1 in hESCs. The effect of macrophages treated with exosomes on hESCs EMs-exosomes (EMs-exo) from uterine fluid of patients with endometriosis and Control-exosomes (Control-exo) from uterine fluid of patients without endometriosis showed similar characteristics of 100nm diameter, biconcave morphological features (Fig. 4 a, b), and positive staining for exosomal makers TSG101, HSP70 and CD63(Fig. 4 c). To elucidate the role of exosomes in the macrophages, we used the Thp1 cell line as model of macrophages in vitro. The exosomes were labeled with PKH67 and incubated with the macrophages. Uterine fluid-derived exosomes with green fluorescence were observed in the macrophages (Fig. 4 d). The results show that we successfully extracted uterine-derived exosomes and that macrophages can phagocytose uterine-derived exosomes. To investigate the function of macrophages treated with uterine-derived exosomes in hESCs. Thp1 were incubated with EMs-exo and Control-exo for 48 hours, conditioned medium was collected and co-cultured with hESCs. Wound healing assays and transwell assays revealed that EMs-exo group enhanced the migration of hESCs (Fig. 3 a, b). Similarly, western blot analysis showed that EMs-exo group increased the protein level of MMP9, MMP2 and N-Cadherin in hESCs (Fig. 3 c, d). Moreover, FCM analysis and colony formation indicated that compared to the Control-exo group, EMs-exo group promoted the proliferative capacity of hESCs (Fig. 3 e, f). Furthermore, co-culturing EMs-exo conditioned medium with hESCs resulted in a downregulation of PR and E-cadherin expression and an upregulation of N-cadherin in hESCs (Fig. 3 g). Similarly, ELISA results showed that the secretion of IGFBP1 in hESCs was significantly decreased in the EMs-exo group compared to the control group (Fig. 3 h). These results suggested that macrophages co-incubated with EMs-exo can promote the proliferation and migration of hESCs, resulting in progesterone resistance and impairs decidualization. miR-210-3p is overexpressed in macrophages treated with EMs-exo To assess endometriosis-induced changes in the expression of exosomal miRNAs, we conducted sequencing of small RNAs from endometriosis and non-endometriosis uterine-derived exosomes and eutopic endometrium, 9 differentially expressed miRNAs were identified in exosomes, while 22 differentially expressed miRNAs were identified in endometrium. The miRanda and TargetScan databases were used to predict the target genes of the differentially expressed miRNAs. Functional enrichment analysis showed that the target genes of exosomal miRNA were mainly involved in the positive regulation of macrophage differentiation, cytokine activity, ATP binding and glycogen metabolic process (Supplemental Fig. 2a, b). miR-210-3p expression was significantly upregulated in uterine-derived exosomes and eutopic endometrium from patients with endometriosis (Fig. 4 e, f), and qRT-PCR results also confirmed that it was markedly elevated in EMs-exo and eutopic endometrium[ 26 ]. Therefore, we selected this miRNA for further analyses. Notably, as shown in Fig. 4 g, miR-210-3p levels were significantly higher in peritoneal fluid macrophages from endometriosis, compared with control group. Similarly, qRT-PCR results also confirmed that miR-210-3p expression of Thp1 was obviously upregulated after EMs-exo treatment for 48h (Fig. 4 h). Based on the above results, we speculated that the uterine-derived exosomes of patients with endometriosis may promote the development of endometriosis by upregulating the expression of miR210-3p in macrophages. miR-210-3p promotes the proliferation and migration of hESCs in vitro To clarify the effect of miR-210-3p on endometriosis, we first silenced miR-210-3p by transfecting miR-210-3p inhibitor in Thp1. Next, transwell migration and wound healing assays were performed to assess whether silenced miR-210-3p could reverse the effect of peritoneal macrophages on the migration of hESCs. As shown in Fig. 5 a, b, c, d, the low-expression of miR-210-3p inhibited the migration capacity in hESCs. Subsequently, the results of colony formation and FCM assays revealed that the low-expression of miR-210-3p in macrophages inhibited the proliferative capacity of hESCs (Fig. 5 e, f). Western blot analysis revealed that downregulating miR-210-3p enhanced the expression of epithelial marker E-cadherin and reduced the expression of mesenchymal marker N-cadherin (Fig. 5 g). The results showed that the ability of mesenchymal stem cells to differentiate into epithelial cells was enhanced and the decidualization ability was improved after the inhibition of macrophage miR-210-3p, and the decidualization marker IGFBP1 showed the same trend (Fig. 5 h). These results suggested that inhibiting the expression of miR-210-3p in macrophages can inhibit the migration and proliferation of hESCs, and improve the progesterone resistance of hESCs. miR-210-3p promotes the development of endometriosis in vivo Macrophage-depleting liposomes containing clodronate were used to deplete mouse peritoneal macrophages, then the miR-210-3p of the mouse macrophage cell line RAW264.7 was knocked out using a lentivirus and intraperitoneally injected (Fig. 6 a). At sacrifice, 28 days after intraperitoneal injection of syngeneic endometrial tissue, lesions had developed in all mice (Fig. 6 b, c). Endometriotic lesions with low-expression of miR-210-3p in mouse macrophages were significantly bigger, which was reflected by their total volume and number (Fig. 6 d). Immunofluorescent staining of Ki-67 and vimentin were performed that downregulation of miR-210-3p in macrophages inhibited the proliferation capacity of endometrial stromal cells (Fig. 6 e). These results implied that inhibiting miR-210-3p in macrophages effectively protected mice from endometriosis, and show that miR-210-3p may be a valuable biomarker for non-invasive diagnosis and treatment of endometriosis. miR-210-3p induces M2 macrophage polarization by ATP5D To identify the molecular mechanism of miR-210-3p in macrophages, we transfected miR-210-3p mimics and inhibitors, and then conducted proteomic sequencing. According to the results, we identified a total of 360 differentially expressed proteins between miR-210-3p overexpression and the control group, and 37 differentially expressed proteins between miR-210-3p underexpression and the control group.(Fig. 7 a, b). Subsequently, functional analysis of the differential proteins was performed. It was found that inhibition of miR-210-3p mainly affected ATP metabolic pathways, such as oxidative phosphorylation pathway, ATP metabolism and ATP synthase activity (Supplemental Fig. 3b, d). KEGG showed that miR-210-3p overexpression mainly affected immunoinflammatory pathways (Supplemental Fig. 3a, c), such as IL-17 signaling pathway, TNF signaling pathway and Nod-like receptor signaling pathway. We screened out differential proteins with consistent expression trends in the two groups (Fig. 7 c), and found that the expression of ATP5F1D, FAU and MST1L was inhibited in macrophages when miR-210-3p was overexpressed, while the three proteins were up-regulated in macrophages when miR-210-3p was underexpressed. The qRT-PCR and WB results confirmed that ATP5F1D (ATP5D) expression was consistent with the proteomic results (Fig. 7 d, e). The expression of ATP5D was significantly reduced in abdominal macrophages of patients with endometriosis as compared to non-endometriosis patients (Fig. 7 f). Therefore, we hypothesize that the excessive expression of miR-210-3p in macrophages inhibits the expression of ATP5D, affecting the function of macrophages. By knocking down ATP5D expression in macrophages, the expressions of CD206, CD163 and IL10 in macrophages were significantly increased, while the expressions of iNOS and CCR7 were significantly decreased (Fig. 7 g), suggesting that inhibiting ATP5D expression drive the M2 polarization of macrophages. Subsequently, the supernatant of macrophages with low-expression of ATP5D was co-cultured with hESCs for 48 hours, wound healing assays and transwell indicated that macrophages with decreased expression of ATP5D remarkably enhanced the migration capacity of hESCs (Fig. 7 h, i). Discussion Endometriosis is a common benign gynecological disease; however, its biological behavior regarding distant metastasis and invasion is similar to that of malignant tumors[ 27 ]. The pathogenesis of endometriosis is still unclear[ 1 , 2 , 10 ], despite a large body of evidence suggests that, in addition to hormonal and intrinsic abnormalities of the endometrium, the immune system plays a key role in its development[ 28 , 29 ]. Immune dysfunction is believed to play a role in both the initiation and progression of endometriosis by creating a microenvironment that encourages the ectopic survival and implantation of endometrial cells[ 30 , 31 ]. Our study has demonstrated that the inflammatory abdominal microenvironment is conducive to the implantation and growth of ectopic endometrial cells, and that peritoneal macrophages are primarily M2 macrophages. Furthermore, we found that peritoneal macrophages from endometriosis patients can stimulate the migration, invasion, and proliferation of hESCs while also inducing their progesterone resistance. This evidence suggests that the changes of macrophages establish an "immune tolerance" microenvironment locally in the abdominal cavity, providing a suitable "soil" for the implantation and growth of ectopic lesions[ 32 – 34 ]. Consequently, there is a new research focus on regulating the abdominal immune microenvironment, reprogramming macrophage epigenetic, and disrupting the "peaceful coexistence" between ectopic endometrial tissue and the local microenvironment. Exosomes act as natural carriers of intercellular information and play a crucial role in immune regulation, particularly in remote regulation[ 19 ]. Our results demonstrate that uterine-derived exosomes from endometriosis patients, when administered to peritoneal macrophages, stimulate the proliferation and migration of hESCs while also inducing their resistance to progesterone. Our earlier study revealed that miR-210-3p expression was upregulated in both uterine-derived exosomes and eutopic endometrial tissue from women with endometriosis[ 26 ]. Our present study shows that miR-210-3p expression is upregulated in peritoneal macrophages from women with endometriosis. Therefore, we hypothesize that exosomes from the uterine cavity may cause the ectopic implantation and growth of endometrial cells by elevating miR-210-3p expression in peritoneal macrophages. In the present study, we show that upregulation of macrophage miR-210-3p inhibits ATP5D expression, thereby enhancing the invasion and migration of hESCs. Moreover, downregulating macrophage ATP5D expression induces M2 polarization, which may be a mechanism by which uterine-derived exosomes promote endometriosis development. ATP5D is a subunit of mitochondrial ATP synthase that plays an important role in catalyzing the synthesis of ATP in the mitochondria[ 35 ]. Decreased mitochondrial ATP generation typically increases AMP concentration and shifts metabolism from synthetic to catabolic[ 36 ]. The metabolic state of macrophages not only reflects the intrinsic stability of the local microenvironment but also affects the local microenvironment by altering their own function[ 37 – 40 ]. Therefore, exosomes derived from the uterine cavity may affect the function of macrophages by disrupting their metabolism through the alteration of ATP synthase in peritoneal macrophages. Similar to the "tumor niche" hypothesis[ 41 ], the immune microenvironment of the abdomen ("soil") undergoes changes before endometrial cells ("seeds") reach the peritoneal cavity, promoting the escape of the "seeds" from immune surveillance and aiding in their settlement and growth. Uterine-derived exosomes may function as a mode of information transfer. In fact, endometriotic exosomes may retrogradely enter the pelvic area and adjust local cell functions, shaping a suitable "soil" for ectopic endometrial growth, which might be key molecules in the pathogenesis of endometriosis[ 42 – 44 ]. Our data suggest that uterine-derived exosomes inhibit macrophage ATP5D expression, driving macrophages towards M2 polarization, establishing a suitable soil of local immune inhibition and promoting ectopic endometrial settlement. Additionally, study found that exosomes induce macrophage polarization to create a favorable microenvironment for angiogenesis, regulating the development of endometriosis[ 45 , 46 ]. Our study demonstrates that uterine-derived exosomes can regulate the expression of miR-210-3p in peritoneal macrophages to induce the formation of an immunosuppressive microenvironment. These findings provide new insights into the role of uterine-derived exosomes in mediating the establishment of an immunosuppressive peritoneal microenvironment and show that miR-210-3p may be a valuable biomarker, and that inhibiting the expression of miR-210-3p in peritoneal macrophages may become a novel immunotherapeutic strategy for endometriosis. However, our study has some limitations. Although the isolated extracellular vesicles from the uterus cavity may reflect the initial state of the cavity, it is hard to identify a specific source of exosomes. Because of species variation, we were unable to directly investigate the impacts of exosomes that are derived from the uterus of endometriosis patients on the implantation and growth of ectopic lesions and peritoneal macrophages in a mouse model. It is crucial to investigate the particular regulatory mechanism of uterine-derived exosomes on the function of macrophages, to gain a more profound understanding of the role they plays in the growth and metastasis of hESCs. Conclusion In summary, we presented evidence of the mediation of growth and migration of hESCs by exosomes derived from the uterine cavity in endometriosis patients. We discovered that uterine-derived exosomes upregulate the expression of miR-210-3p in peritoneal macrophages to inhibit ATP5D, thereby promoting M2 polarization and reshaping the peritoneal immune microenvironment, which leads to the development of endometriosis. Abbreviations EMs Endometriosis hESCs Human Endometrial Stromal Cells EMSCs Endometrium-derived Mesenchymal Stem Cells ATP5D ATP synthase, H + transporting, mitochondrial F1 complex, δ subunit PBS Phosphate buffer saline LPS Lipopolysaccharide PMA Phorbol-12-myristate-13-acetate WB Western blot analysis PR Progesterone Receptor ER Estrogen Receptor E-Ca Epithelial Cadherin N-Ca Neural Cadherin EMs-exo exosomes from uterine fluid of patients with endometriosis Control-exo exosomes from uterine fluid of patients without endometriosis NTA Nanoparticle Tracking Analysis TEM Transmission Electron Microscope Thp1 Human myeloid leukemia mononuclear cells MMP Matrix Metalloproteinase IGFBP1 Insulin-like Growth Factor Binding Protein 1 mimics NC Mimics negative control Inhibitor NC Inhibitor negative control siRNA Small interfering RNA GO Gene Ontology KEGG Kyoto Encyclopedia of Genes Declarations Ethics approval and consent to participate This study was approved by the Medical Ethics Committee of the Second Xiangya Hospital of Central South University (approval number:2020-065). Informed consent was obtained from each patient. All animal handling and experimental procedures were approved by the Animal Experimental Ethics Committee of the Second Xiangya Hospital of Central South University (approval number:20220059). Consent for publication Not applicable Availability of data and materials All data produced or analyzed in this study are contained in this published article and its supplementary information files. All data used in the current study are available from the corresponding author on reasonable request. Competing interests The authors declare that they have no competing interests in this work. Funding This study was supported by a grant from the National Natural Science Foundation of China (#81873826). Authors' contributions SN.C and XQ.W conceived and designed the experiments; SN.C, Y.J and XQ.W prepared the manuscript; SN.C, Y.J and XS.C performed the experiments; ZY.C, H.T, M.L, TY.Z, WW.SG collected the samples; SN.C and XQ.W analyzed the data. All authors read and approved the final manuscript. Acknowledgements None References Zondervan KT, Becker CM, Missmer SA. Endometriosis. Longo DL, editor. N Engl J Med [Internet]. 2020 [cited 2023 May 31];382:1244–56. Available from: https://pubmed.ncbi.nlm.nih.gov/32212520/ Giudice LC, Kao LC. Endometriosis. Lancet [Internet]. 2004 [cited 2023 May 31];364:1789–99. Available from: https://pubmed.ncbi.nlm.nih.gov/15541453/ Halme J, HAMMOND M, … JH-O&, 1984 undefined. Retrograde menstruation in healthy women and in patients with endometriosis. journals.lww.com [Internet]. [cited 2023 May 31]; Available from: https://journals.lww.com/greenjournal/Abstract/1984/08000/Retrograde_Menstruation_in_Healthy_Women_and_in.1.aspx Kjerulff KH, Erickson BA, Langenberg PW. Chronic gynecological conditions reported by US women: findings from the National Health Interview Survey, 1984 to 1992. Am J Public Health [Internet]. 1996 [cited 2023 May 31];86:195–9. Available from: https://pubmed.ncbi.nlm.nih.gov/8633735/ Zhang Z, Suo L, Chen Y, Zhu L, International GW-SC, 2019 undefined. Endometriotic peritoneal fluid promotes myofibroblast differentiation of endometrial mesenchymal stem cells. hindawi.com [Internet]. [cited 2023 May 31]; Available from: https://www.hindawi.com/journals/sci/2019/6183796/ Khatun M, Sorjamaa A, Kangasniemi M, Sutinen M, Salo T, Liakka A, et al. Niche matters: The comparison between bone marrow stem cells and endometrial stem cells and stromal fibroblasts reveal distinct migration and cytokine profiles in response to inflammatory stimulus. PLoS One [Internet]. 2017 [cited 2023 May 31];12. Available from: https://pubmed.ncbi.nlm.nih.gov/28419140/ Gargett CE, Schwab KE, Deane JA. Endometrial stem/progenitor cells: the first 10 years. Hum Reprod Update [Internet]. 2016 [cited 2023 May 31];22:137–63. Available from: https://pubmed.ncbi.nlm.nih.gov/26552890/ Kao AP, Wang KH, Chang CC, Lee JN, Long CY, Chen HS, et al. Comparative study of human eutopic and ectopic endometrial mesenchymal stem cells and the development of an in vivo endometriotic invasion model. Fertil Steril [Internet]. 2011 [cited 2023 May 31];95. Available from: https://pubmed.ncbi.nlm.nih.gov/21047634/ Nair ARKG, Pandit H, Warty N, Madan T. Endometriotic mesenchymal stem cells exhibit a distinct immune phenotype. Int Immunol [Internet]. 2015 [cited 2023 May 31];27:195–204. Available from: https://pubmed.ncbi.nlm.nih.gov/25416515/ Burney RO, Giudice LC. Pathogenesis and pathophysiology of endometriosis. Fertil Steril [Internet]. 2012 [cited 2023 May 31];98:511–9. Available from: https://pubmed.ncbi.nlm.nih.gov/22819144/ Ma S, Xie N, Li W, Yuan B, Shi Y, Wang Y. Immunobiology of mesenchymal stem cells. Cell Death Differ [Internet]. 2014 [cited 2023 May 31];21:216–25. Available from: https://pubmed.ncbi.nlm.nih.gov/24185619/ Vallvé-Juanico J, Houshdaran S, Giudice LC. The endometrial immune environment of women with endometriosis. Hum Reprod Update [Internet]. 2019 [cited 2023 May 31];25:565–92. Available from: https://pubmed.ncbi.nlm.nih.gov/31424502/ Ramírez-Pavez TN, Martínez-Esparza M, Ruiz-Alcaraz AJ, Marín-Sánchez P, Machado-Linde F, García-Peñarrubia P. The Role of Peritoneal Macrophages in Endometriosis. Int J Mol Sci [Internet]. 2021 [cited 2023 May 31];22. Available from: https://pubmed.ncbi.nlm.nih.gov/34639133/ Berbic M, Schulke L, Markham R, Tokushige N, Russell P, Fraser IS. Macrophage expression in endometrium of women with and without endometriosis. Hum Reprod [Internet]. 2009 [cited 2023 May 31];24:325–32. Available from: https://pubmed.ncbi.nlm.nih.gov/19049988/ Weinberg J, Haney A, Xu F, Ramakrishnan S. Peritoneal fluid and plasma levels of human macrophage colony-stimulating factor in relation to peritoneal fluid macrophage content. 1991 [cited 2023 May 31]; Available from: https://ashpublications.org/blood/article-abstract/78/2/513/168804 Bacci M, Capobianco A, Monno A, Cottone L, Di Puppo F, Camisa B, et al. Macrophages are alternatively activated in patients with endometriosis and required for growth and vascularization of lesions in a mouse model of disease. Am J Pathol [Internet]. 2009 [cited 2023 May 31];175:547–56. Available from: https://pubmed.ncbi.nlm.nih.gov/19574425/ Jeljeli M, Riccio LGC, Chouzenoux S, Moresi F, Toullec L, Doridot L, et al. Macrophage Immune Memory Controls Endometriosis in Mice and Humans. Cell Rep [Internet]. 2020 [cited 2023 May 31];33. Available from: https://pubmed.ncbi.nlm.nih.gov/33147452/ Xie Q, He H, Wu YH, Zou LJ, She XL, Xia XM, et al. Eutopic endometrium from patients with endometriosis modulates the expression of CD36 and SIRP-α in peritoneal macrophages. J Obstet Gynaecol Res [Internet]. 2019 [cited 2023 May 31];45:1045–57. Available from: https://pubmed.ncbi.nlm.nih.gov/30843336/ Kalluri R, LeBleu VS. The biology , function , and biomedical applications of exosomes. Science [Internet]. 2020 [cited 2023 May 31];367. Available from: https://pubmed.ncbi.nlm.nih.gov/32029601/ Whiteside TL. Exosome and mesenchymal stem cell cross-talk in the tumor microenvironment. Semin Immunol [Internet]. 2018 [cited 2023 May 31];35:69–79. Available from: https://pubmed.ncbi.nlm.nih.gov/29289420/ Zhao J, Li X, Hu J, Chen F, Qiao S, Sun X, et al. Mesenchymal stromal cell-derived exosomes attenuate myocardial ischaemia-reperfusion injury through miR-182-regulated macrophage polarization. Cardiovasc Res [Internet]. 2019 [cited 2023 May 31];115:1205–16. Available from: https://pubmed.ncbi.nlm.nih.gov/30753344/ Xin L, Lin X, Zhou F, Li C, Wang X, Yu H, et al. A scaffold laden with mesenchymal stem cell-derived exosomes for promoting endometrium regeneration and fertility restoration through macrophage immunomodulation. Acta Biomater [Internet]. 2020 [cited 2023 May 31];113:252–66. Available from: https://pubmed.ncbi.nlm.nih.gov/32574858/ Sun H, Li D, Yuan M, Li Q, Zhen Q, Li N, et al. Macrophages alternatively activated by endometriosis-exosomes contribute to the development of lesions in mice. Mol Hum Reprod [Internet]. 2019 [cited 2023 May 31];25:5–16. Available from: https://pubmed.ncbi.nlm.nih.gov/30428082/ Campoy I, Lanau L, Altadill T, Sequeiros T, Cabrera S, Cubo-Abert M, et al. Exosome-like vesicles in uterine aspirates: a comparison of ultracentrifugation-based isolation protocols. J Transl Med [Internet]. 2016 [cited 2023 May 31];14. Available from: https://pubmed.ncbi.nlm.nih.gov/27317346/ Harp D, Driss A, Mehrabi S, Chowdhury I, Xu W, Liu D, et al. Exosomes derived from endometriotic stromal cells have enhanced angiogenic effects in vitro. Cell Tissue Res [Internet]. 2016 [cited 2023 May 31];365:187–96. Available from: https://pubmed.ncbi.nlm.nih.gov/26841879/ Jiang Y, Chai X, Chen S, Chen Z, Tian H, Liu M, et al. Exosomes from the Uterine Cavity Mediate Immune Dysregulation via Inhibiting the JNK Signal Pathway in Endometriosis. Biomedicines [Internet]. 2022 [cited 2023 May 31];10. Available from: /pmc/articles/PMC9775046/ Giudice LC, Kao LC. Endometriosis. Lancet [Internet]. 2004 [cited 2023 May 31];364:1789–99. Available from: https://pubmed.ncbi.nlm.nih.gov/15541453/ Symons LK, Miller JE, Kay VR, Marks RM, Liblik K, Koti M, et al. The Immunopathophysiology of Endometriosis. Trends Mol Med [Internet]. 2018 [cited 2023 May 31];24:748–62. Available from: https://pubmed.ncbi.nlm.nih.gov/30054239/ Agostinis C, Balduit A, Mangogna A, Zito G, Romano F, Ricci G, et al. Immunological Basis of the Endometriosis: The Complement System as a Potential Therapeutic Target. Front Immunol [Internet]. 2021 [cited 2023 May 31];11. Available from: https://pubmed.ncbi.nlm.nih.gov/33505394/ Saunders PTK, Horne AW. Endometriosis: Etiology, pathobiology, and therapeutic prospects. Cell [Internet]. 2021 [cited 2023 May 31];184:2807–24. Available from: https://pubmed.ncbi.nlm.nih.gov/34048704/ Wang Y, Nicholes K, Shih IM. The Origin and Pathogenesis of Endometriosis. Annu Rev Pathol [Internet]. 2020 [cited 2023 May 31];15:71–95. Available from: https://pubmed.ncbi.nlm.nih.gov/31479615/ Capobianco A, Rovere-Querini P. Endometriosis, a disease of the macrophage. Front Immunol [Internet]. 2013 [cited 2023 May 31];4. Available from: https://pubmed.ncbi.nlm.nih.gov/23372570/ Wu J, Xie H, Yao S, Liang Y. Macrophage and nerve interaction in endometriosis. J Neuroinflammation [Internet]. 2017 [cited 2023 May 31];14. Available from: https://pubmed.ncbi.nlm.nih.gov/28288663/ Klemmt PA, Starzinski-Powitz A. Molecular and Cellular Pathogenesis of Endometriosis. Curr Womens Health Rev [Internet]. 2018 [cited 2023 May 31];14:1–11. Available from: https://pubmed.ncbi.nlm.nih.gov/29861704/ Oláhová M, Yoon WH, Thompson K, Jangam S, Fernandez L, Davidson JM, et al. Biallelic Mutations in ATP5F1D, which Encodes a Subunit of ATP Synthase, Cause a Metabolic Disorder. Am J Hum Genet [Internet]. 2018 [cited 2023 May 31];102:494–504. Available from: https://pubmed.ncbi.nlm.nih.gov/29478781/ Weinberg SE, Sena LA, Chandel NS. Mitochondria in the regulation of innate and adaptive immunity. Immunity [Internet]. 2015 [cited 2023 May 31];42:406–17. Available from: https://pubmed.ncbi.nlm.nih.gov/25786173/ He L, Jhong JH, Chen Q, Huang KY, Strittmatter K, Kreuzer J, et al. Global characterization of macrophage polarization mechanisms and identification of M2-type polarization inhibitors. Cell Rep [Internet]. 2021 [cited 2023 May 31];37. Available from: https://pubmed.ncbi.nlm.nih.gov/34731634/ Verdeguer F, Aouadi M. Macrophage heterogeneity and energy metabolism. Exp Cell Res. 2017;360:35–40. Yan J, Horng T. Lipid Metabolism in Regulation of Macrophage Functions. Trends Cell Biol [Internet]. 2020 [cited 2023 May 31];30:979–89. Available from: https://pubmed.ncbi.nlm.nih.gov/33036870/ Van den Bossche J, O’Neill LA, Menon D. Macrophage Immunometabolism: Where Are We (Going)? Trends Immunol [Internet]. 2017 [cited 2023 May 31];38:395–406. Available from: https://pubmed.ncbi.nlm.nih.gov/28396078/ Liu Y, Cao X. Characteristics and Significance of the Pre-metastatic Niche. Cancer Cell [Internet]. 2016 [cited 2023 May 31];30:668–81. Available from: https://pubmed.ncbi.nlm.nih.gov/27846389/ Horne AW, Saunders PTK. SnapShot: Endometriosis. Cell [Internet]. 2019 [cited 2023 May 31];179:1677-1677.e1. Available from: https://pubmed.ncbi.nlm.nih.gov/31951524/ Sun H, Li D, Yuan M, Li Q, Li N, Wang G. Eutopic stromal cells of endometriosis promote neuroangiogenesis via exosome pathway†. Biol Reprod [Internet]. 2019 [cited 2023 May 31];100:649–59. Available from: https://pubmed.ncbi.nlm.nih.gov/30295741/ Qiu JJ, Lin XJ, Zheng TT, Tang XY, Zhang Y, Hua KQ. The Exosomal Long Noncoding RNA aHIF is Upregulated in Serum From Patients With Endometriosis and Promotes Angiogenesis in Endometriosis. Reprod Sci [Internet]. 2019 [cited 2023 May 31];26:1590–602. Available from: https://pubmed.ncbi.nlm.nih.gov/30808247/ Sun H, Li D, Yuan M, Li Q, Zhen Q, Li N, et al. Macrophages alternatively activated by endometriosis-exosomes contribute to the development of lesions in mice. Mol Hum Reprod [Internet]. 2019 [cited 2023 May 31];25:5–16. Available from: https://pubmed.ncbi.nlm.nih.gov/30428082/ Li Y, Lyu P, Ze Y, Li P, Zeng X, Shi Y, et al. Exosomes derived from plasma: promising immunomodulatory agents for promoting angiogenesis to treat radiation-induced vascular dysfunction. PeerJ [Internet]. 2021 [cited 2023 May 31];9. Available from: https://pubmed.ncbi.nlm.nih.gov/33859878/ Additional Declarations No competing interests reported. Supplementary Files SupplementalFig1.tif SupplementalFig2.tif SupplementalFig3.tif SupplementaryMaterial..docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3030329","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":207721861,"identity":"d2b3d2e7-d041-4962-a471-484abe0d252b","order_by":0,"name":"Shengnan Chen","email":"","orcid":"","institution":"The Second Xiangya Hospital of Central South University","correspondingAuthor":false,"prefix":"","firstName":"Shengnan","middleName":"","lastName":"Chen","suffix":""},{"id":207721862,"identity":"96d51cb7-7d08-4e62-99bf-b516527b882a","order_by":1,"name":"Ying Jiang","email":"","orcid":"","institution":"The Second Xiangya Hospital of Central South University","correspondingAuthor":false,"prefix":"","firstName":"Ying","middleName":"","lastName":"Jiang","suffix":""},{"id":207721863,"identity":"a18c8068-6672-46db-8ebc-60e9890170b8","order_by":2,"name":"Xiaoshan Chai","email":"","orcid":"","institution":"The Second Xiangya Hospital of Central South University","correspondingAuthor":false,"prefix":"","firstName":"Xiaoshan","middleName":"","lastName":"Chai","suffix":""},{"id":207721864,"identity":"87fa8f76-42e0-40d5-94d1-876aa785af37","order_by":3,"name":"Zhaoying Chen","email":"","orcid":"","institution":"The Second Xiangya Hospital of Central South University","correspondingAuthor":false,"prefix":"","firstName":"Zhaoying","middleName":"","lastName":"Chen","suffix":""},{"id":207721865,"identity":"bca4f66d-8ab4-454b-9653-28ea8892d39f","order_by":4,"name":"Hao Tian","email":"","orcid":"","institution":"The Second Xiangya Hospital of Central South University","correspondingAuthor":false,"prefix":"","firstName":"Hao","middleName":"","lastName":"Tian","suffix":""},{"id":207721866,"identity":"15c9176a-56c7-403a-af13-16085f1ccfe2","order_by":5,"name":"Min Liu","email":"","orcid":"","institution":"The Second Xiangya Hospital of Central South University","correspondingAuthor":false,"prefix":"","firstName":"Min","middleName":"","lastName":"Liu","suffix":""},{"id":207721867,"identity":"94db6575-5d46-42e7-beff-c1ccd1e5557e","order_by":6,"name":"Tianyu Zhu","email":"","orcid":"","institution":"The Second Xiangya Hospital of Central South University","correspondingAuthor":false,"prefix":"","firstName":"Tianyu","middleName":"","lastName":"Zhu","suffix":""},{"id":207721868,"identity":"f1d71bd5-7339-48c9-8525-d6d4cb081d81","order_by":7,"name":"Wanwan ShangGuan","email":"","orcid":"","institution":"The Second Xiangya Hospital of Central South University","correspondingAuthor":false,"prefix":"","firstName":"Wanwan","middleName":"","lastName":"ShangGuan","suffix":""},{"id":207721869,"identity":"47236908-a3b5-498f-8295-e1ac947171d3","order_by":8,"name":"Xianqing Wu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA/0lEQVRIiWNgGAWjYDACZhBhwMDAxt/Y+OADRMyAOC38EocPG84gSgsMSDakpUnzEKPF4DjzswdvCu7YbThwxkDapqYusYG9eZsEQ80d3IY3s5kbzjF4lrzhcI+Bcc6xw4kNPMfKJBiOPcOphZ+ZwUyax+BwsgHQluTchgOJDRI5ZhKMDYdxamFjZv8G1ZJjcNiyAegw+Tf4tfAz84BtsQN6P7GZsYEZaAsPfi2SzTxlknMMDieAApmx59hh4zaetGKLhGO4tRicP75N4s2fw/bAqGz/8aOmTraf/fDGGx9qcGsBA2B0JDbAfQciEvBrAGuxJ6RmFIyCUTAKRjAAAAnMU6l7ZmOJAAAAAElFTkSuQmCC","orcid":"","institution":"The Second Xiangya Hospital of Central South University","correspondingAuthor":true,"prefix":"","firstName":"Xianqing","middleName":"","lastName":"Wu","suffix":""}],"badges":[],"createdAt":"2023-06-06 15:59:19","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3030329/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3030329/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":38298344,"identity":"861edf14-335e-4f15-8d6c-b5613e38eb6d","added_by":"auto","created_at":"2023-06-09 15:49:57","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1891309,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAbdominal inflammatory microenvironment promotes the development of endometriosis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(a) Representative endometriosis-like lesions in the indicated treatment groups. (b)HE staining of ectopic endometrial tissues. (c)Total volume and number of lesions (n=7), the volume is the sum of the volumes of all lesions per mouse. (d)Immunofluorescent staining of Ki-67 (red) and vimentin (green) in representative lesions. (e-f) Peritoneal macrophages were identified by flow cytometry in the indicated treatment groups. Data are presented as mean ± SD. *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001, ****P \u0026lt; 0.0001. S = stroma, E = epithelium, G = gland.\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-3030329/v1/e1a2cfd19fbf23c0ae3a0204.png"},{"id":38296760,"identity":"367ce2bf-61f0-42c3-a2aa-b55846d4fe63","added_by":"auto","created_at":"2023-06-09 15:41:57","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1361342,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePeritoneal macrophages promote the proliferation and migration of hESCs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(a) Wound healing assay results showing the differences in migration capacities in hESCs(left), statistical analysis of the wound healing assay results (right). (b) Representative transwell results comparing the effects of macrophages on cell migration between the Control and EMs groups(left), statistical analysis of the transwell assay results(right). (c, d) Protein expression of MMP9, MMP2 and N-Cadherin in hESCs, treatment without E2 and P4, as determined by Western blotting. GAPDH was used as a loading control. (e) CD140b+CD146+ cell count by flow cytometry (left); statistical analysis of flow results (right). (f) Representative images of the colony formation assay in the indicated cells (left); statistical analysis of the colony formation results(right). (g)Western blotting analysis of the expression of PR, N-cadherin and E-cadherin in hESCs after induced decidualization in vitro. (h)Expression of IGFBP1 in supernatant of hESCs after induced decidualization in vitro. *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001, ****P \u0026lt; 0.0001. EMs = endometriosis, Con = Control.\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-3030329/v1/59661f9e8c8a7bfb3b665489.png"},{"id":38298347,"identity":"aefdc2f9-209d-4a2d-b0e9-5dc82f482bd0","added_by":"auto","created_at":"2023-06-09 15:49:57","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1471921,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe effect of macrophages treated with exosomes on hESCs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(a, b, c, d) The invasion and migration of hESCs after treatment with macrophage-conditioned medium by wound healing assay (a), transwell (b) and protein expression of MMP9, MMP2 and N-Cadherin detected by Western blotting (c, d) in the indicated treatment groups. (e, f) hESCs proliferative after treatment with macrophage-conditioned medium by flow cytometry(e) and colony formation assay(f) in the indicated treatment groups. （g, h）hESCs progesterone resistance after treatment with macrophage-conditioned medium by the levels of IGFBP1 detected by enzyme-linked immunosorbent assay kit (h), and protein expression of PR , E-cadherin N-cadherin detected by Western blotting (g). *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001, ****P \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-3030329/v1/641c3f62fa45f4a77782139f.png"},{"id":38299497,"identity":"8dda47a2-8bce-4e17-a433-50964242f32b","added_by":"auto","created_at":"2023-06-09 15:57:57","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":768159,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003emiR-210-3p is overexpressed in macrophages treated with EMs-exo\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(a)Transmission electron microscopy image of the morphology of exosomes in uterine fluid (scale bar = 0.5μm). (b)Nanoparticle tracking analysis result on the size distribution of uterine fluid-derived exosomes. (c)Western blot analysis of exosome positive markers CD63, HSP70 and TSG101. (d)Representative images of PKH67-labelled exosomes co-cultured with Thp1\u003cstrong\u003e. \u003c/strong\u003e(e,f) The Volcano plot of the differentially expressed miRNAs in uterine-derived exosomes and eutopic endometrium from the EMs compared with the control. (g) qRT-PCR assay of miR-210-3p expression in peritoneal fluid macrophages of patients with endometriosis (EMs, n=4) and patients without endometriosis (Control, n=4). (h) qRT-PCR assay of miR-210-3p expression in Thp1 co-cultured with EMs-exo and Control-exo. *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001, ****P \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-3030329/v1/3a27509bd40504878e1a0d6d.png"},{"id":38299498,"identity":"53100180-dda5-4337-aa58-c9d663c89710","added_by":"auto","created_at":"2023-06-09 15:57:57","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1457119,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003emiR-210-3p promotes\u003c/strong\u003e \u003cstrong\u003ethe proliferation and migration of hESCs in vitro\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(a, b, c, d) Wound healing assay (a), Transwell (b) and expression of MMP9, MMP2 and N-Cadherin (c, d) comparing the effects of macophages on cell migration between the miR-inhibitor NC and miR-210-3p inhibitor groups. (e, f) Flow cytometry (e) and colony formation assay(f) results showing the differences in proliferation capacities in the indicated group. (g, h) Effect of miR-210-3p expression in macrophages on decidualization of hESCs as determined by the expression of PR, E-cadherin and N-cadherin (g) and the levels of IGFBP1 (h).*P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001, ****P \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-3030329/v1/6e75ca17b3bd4dc3abbfbfe9.png"},{"id":38296763,"identity":"a9d44048-fbac-4521-a864-705d0861975e","added_by":"auto","created_at":"2023-06-09 15:41:57","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1686397,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003emiR-210-3p promotes\u003c/strong\u003e \u003cstrong\u003ethe proliferation and migration of hESCs in vivo\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(a) Schematic of experimental timeline and procedures. 24-48 hours before modeling, 150μl of liposomes were injected into the mouse peritoneal cavity to deplete the original macrophages, followed by a maintenance dose of 100μl biweekly. RAW264.7 cells, 1 × 106 per transfer, were transferred every three days. (b, c) Representative images of ectopic endometriotic lesions in the indicated treatment groups (b) and the HE staining of ectopic endometrial tissues (c). (d)Total volume and number of lesions in the indicated groups (n = 6). (e) Immunofluorescent staining of Ki-67 (red) and vimentin (green) in representative lesions. *P \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"fig6.png","url":"https://assets-eu.researchsquare.com/files/rs-3030329/v1/c533c5d0c8b8dc65ddd9b3d5.png"},{"id":38298348,"identity":"d68d00de-1fc4-4af8-ac2b-d5e93fdeaba0","added_by":"auto","created_at":"2023-06-09 15:49:57","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1005047,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003emiR-210-3p induces M2 macrophage polarization by ATP5D\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(a, b) The Volcano plot of the differentially expressed proteins between the indicated group. (c) The Venn diagram shows proteins in the indicated groups with corresponding expression trends. (d) qRT-PCR analyses to verify the expression of differential proteins with corresponding expression trends. (e) Protein expression of ATP5D in the indicated cells as determined by Western blotting. (f) Western blot analysis of ATP5D in peritoneal macrophages from patients with endometriosis and non-endometriosis patients. (n=10). (g)qRT-PCR analysis of M1/M2 markers in the indicated infected cells. (h, i) hESCs invasion and migration after treatment with macrophage-conditioned medium by transwell (H) and wound healing assay (I) in the indicated treatment groups.*P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001, ****P \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"fig7.png","url":"https://assets-eu.researchsquare.com/files/rs-3030329/v1/e859f82f3998a487011070b6.png"},{"id":38320525,"identity":"922e2b6f-ee27-4803-ae96-15fe3442f50a","added_by":"auto","created_at":"2023-06-10 04:29:35","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4838684,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3030329/v1/aa8f27b0-c703-45ad-b2ec-bb790ea5ee37.pdf"},{"id":38296770,"identity":"e736ce5b-c84c-4e90-9d73-60a1f3c596f1","added_by":"auto","created_at":"2023-06-09 15:41:57","extension":"tif","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":3582128,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementalFig1.tif","url":"https://assets-eu.researchsquare.com/files/rs-3030329/v1/b7937a55706eee0272a6f30c.tif"},{"id":38296769,"identity":"3e3f2a17-808f-4c17-8ea8-f5e12b413607","added_by":"auto","created_at":"2023-06-09 15:41:57","extension":"tif","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":1355388,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementalFig2.tif","url":"https://assets-eu.researchsquare.com/files/rs-3030329/v1/8fe57f2b13028efc96dfa7ed.tif"},{"id":38296765,"identity":"c6885e1e-bae0-46df-bffc-32d8cbe24c07","added_by":"auto","created_at":"2023-06-09 15:41:57","extension":"tif","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":1800820,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementalFig3.tif","url":"https://assets-eu.researchsquare.com/files/rs-3030329/v1/d509b97d9c64eeb64a8e3645.tif"},{"id":38299496,"identity":"be623bac-214b-4a27-b24f-e41345c848ad","added_by":"auto","created_at":"2023-06-09 15:57:57","extension":"docx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":16585,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryMaterial..docx","url":"https://assets-eu.researchsquare.com/files/rs-3030329/v1/8cae70d95cde365bc95673ed.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Uterine-derived exosomes induce the M2 polarization of macrophages via miR210-3p to promote the development of endometriosis","fulltext":[{"header":"Background","content":"\u003cp\u003eEndometriosis is a common, oestrogen-dependent, chronic gynaecological disorder associated with pelvic pain and infertility, affecting about 10% of reproductive-aged women[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. The development of endometriosis involves hormonal imbalances, immunological, proinflammatory, and proangiogenic processes[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. As a result, the precise pathogenic mechanisms of endometriosis are not well-identified.\u003c/p\u003e \u003cp\u003eAt present, the widely accepted theory is Sampson's theory of retrograde menstruation[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. It is not known why only 6\u0026ndash;10% of women develop endometriosis despite retrograde menstruation occurring in 76\u0026ndash;90% of women[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. An attractive hypothesis is that the endometrial stromal cells (hESCs) containing stem cell characteristics or endometrium-derived mesenchymal stem cells (EMSCs)[\u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e] may be abnormally shed through the fallopian tube into the peritoneal cavity in affected women, leading to the formation of ectopic implants in endometriosis. There are reports indicating that refluxed ectopic stromal stem cells display diverse features from eutopic stromal stem cells attributable to local microenvironment differences in the abdominal cavity [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Consequently, some scholars have proposed that the development of endometriosis may involve the interaction between MSCs and peritoneal immune cells.\u003c/p\u003e \u003cp\u003eAlthough the pathogenesis of endometriosis remains elusive, growing evidence has demonstrated that it may be related to an aberrant immune response[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Peritoneal macrophages play a key role in the establishment and maintenance of endometriosis by regulating the immune and peritoneal environments[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. In endometriosis, abnormal levels of cytokines, interferon, and macrophage colony-stimulating factor in peritoneal fluid lead to functional scavenger receptor deficiency in peritoneal macrophages[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. This decrease in phagocytic activity may contribute to the growth of ectopic endometrial cells. Additionally, based on their activation state and function, macrophages can be classified into 'classically activated' M1 and 'alternatively activated' M2. Bacci et al.[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e] confirmed that patients with endometriosis mainly have peritoneal fluid containing M2-polarized macrophages. Moreover, the focus of endometriosis has a higher concentration of M2 macrophages than surrounding tissues. Injection of M2-polarized macrophages into the peritoneal cavity of mice promotes the formation of large ectopic lesions, while M1-polarized macrophages only produce small lesions, indicating that M2-polarized macrophages can facilitate growth of ectopic lesions[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Our previous research found that[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e] eutopic endometrial cells can alter the immune phenotype of peritoneal macrophages. However, the mechanism by which eutopic endometrial cells regulate the peritoneal immune microenvironment from a distance is unclear as they are located in the uterine cavity.\u003c/p\u003e \u003cp\u003eExosomes[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e] are a circular monolayer structure with a diameter of 40\u0026thinsp;~\u0026thinsp;100nm, which can carry a variety of proteins, mRNAs, and miRNA. It is a carrier of natural intercellular information, which can remotely regulate the function of receptor cells and participate in the processes of immunity, antigen presentation, cell migration, angiogenesis, and cell growth[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. The effect of exosomes on macrophage polarization has been confirmed in various disease states. Zhao et al.[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e] demonstrated that mesenchymal stromal cell-derived exosomes attenuate myocardial ischaemia -reperfusion injury via modification of the polarization of M1 macrophages to M2 macrophages. Xin et al.[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e] constructed a scaffold with mesenchymal stem cell-derived exosomes to promote endometrium regeneration and fertility restoration through macrophage immune regulation. Sun et al.[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e] found that exosomes from endometrial stromal cells of endometriosis patients could induce macrophages into M2 phenotype, decreasing their phagocytic ability.\u003c/p\u003e \u003cp\u003eStudies have shown that endometrial exosomes released into the uterine cavity can reflux into the peritoneal cavity and communicate with other cells to influence the implantation of ectopic endometrial lesions[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Our hypothesis is that uterine-derived exosomes can modulate peritoneal macrophages to create an appropriate environment for the attachment of ectopic endometrial lesions. In this study, we investigated the specific mechanisms by which uterine-derived exosomes regulate peritoneal macrophages to promote the development of endometriosis.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eClinical specimens\u003c/h2\u003e \u003cp\u003ePatients who underwent laparoscopic surgery for ovarian endometriosis were enrolled in the EMs group (n\u0026thinsp;=\u0026thinsp;30). Patients who underwent surgical treatment for other benign gynecological diseases, such as simple ovarian cysts, uterine incision diverticula, or tubal ligation were enrolled in the control group (n\u0026thinsp;=\u0026thinsp;30). Samples of peritoneal fluid, uterine fluid, and normal endometrial tissues were collected from all patients. All of the samples were collected at the time of surgery at the Second Xiangya Hospital of Central South University. Informed consent was obtained from each patient, and the study was approved by the Medical Ethics Committee of the Second Xiangya Hospital of Central South University (#2020-065). Endometrial tissue and peritoneal fluid were treated within 6 hours of collection, while the uterine fluid was stored in a freezer at -80℃ after centrifugation. All of the patients had normal ovulation with regular menstrual cycles, and none of the patients had received steroid hormonal medications for at least 3 months.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eIsolation of primary cells and cell culture\u003c/h2\u003e \u003cp\u003eNormal endometrial tissues were minced and digested in F12/DMEM containing collagenase I(2.5mg/ml) and deoxyribonuclease I(0.1mg/ml) at 37\u0026deg;C for 40 minutes. The dispersed cells were separated by filtration through a 40\u0026micro;m cell strainer. The cells were plated onto 25cm\u003csup\u003e2\u003c/sup\u003e Falcon tissue culture flasks and maintained in F12/DMEM supplemented with 10% fetal bovine serum (FBS) at 37\u0026deg;C in a humidified atmosphere containing 5% CO2. Endometrial stromal cells (hESCs) were purified by adherent culture, and the cells were used between passages for 3\u0026ndash;6.\u003c/p\u003e \u003cp\u003eThe peritoneal fluid was collected, centrifuged, and recuperated with PBS. Then, it was layered on the same volume of Ficoll and centrifuged for 20 minutes at 2000rpm and at room temperature. The cloud-like layer was collected and washed twice with PBS before inoculating the cells in a 6-well plate, with a density of 1 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e/well. They were cultured in RPMI1640 medium containing 10% FBS for 2 hours to remove non-adherent cells. Only the remaining adherent cells were considered peritoneal fluid macrophages. After culturing for 24h-48h, the supernatant and RNA were collected.\u003c/p\u003e \u003cp\u003eThp1 and RAW264.7 cells were purchased from National Collection of Authenticated Cell Cultures. These cells were cultured in RPMI1640 or DMEM High-Glucose medium supplemented with 10% FBS and 1% penicillin/streptomycin.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eSurface marker profile and differentiation capacity\u003c/h2\u003e \u003cp\u003eThe characteristics of the stem cells[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e] were identified by flow cytometry. After 3 generations of culture, the hESCs cells were incubated with labeled surface antibodies: PerCP-CyTM5.5 Mouse Anti-Human CD45, APC Mouse Anti-Human CD146, PE Mouse Anti-Human CD140b on ice for 30 minutes. Then, the cells were resuspended in PBS for FCM analysis.\u003c/p\u003e \u003cp\u003ehESCs were inoculated in a 6-well plate and cultured in DEME/F12 medium for 24 hours until reaching 70% confluence. Then, an osteogenic differentiation medium was used to induce osteogenesis. The culture medium was changed every 7 days, and alizarin red staining solution was used to detect calcium deposition on the 28th day. Then, an osteogenic differentiation medium was used to induce osteogenesis. The culture medium was changed every 7 days, and alizarin red staining solution was used to detect calcium deposition on the 28th day.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eWound healing assay\u003c/h2\u003e \u003cp\u003eThe third generation of hESCs in the logarithmic growth phase were seeded into a 6-well plate with a density of 5 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells/well. When the cell density reached 80 to 90%, a scratch was made in the monolayer using a 100\u0026micro;l pipette tip in the middle of each well. Wound healing within the same scratched line was then observed and photographed at specific time points (0 h, 24 h, or 48 h). Each experiment was performed three times.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eTranswell\u003c/h2\u003e \u003cp\u003e5 \u0026times; 104 cells were seeded into the upper Transwell chamber using an 8\u0026micro;m pore size filter membrane and culture medium supplemented with 10% FBS. Macrophage conditioned medium was added to the lower chamber at a ratio of 1:1 as a chemoattractant. The cells that had migrated to the lower surface of the filter were fixed in a 4% paraformaldehyde and stained with crystal violet for 30 minutes. The natural air-dried filter membrane was used to count the migrated cells. Three independent experiments were conducted.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eColony Formation Assay\u003c/h2\u003e \u003cp\u003eThe third generation hESCs were digested and seeded directly in 6-well plates (3\u0026times; 10\u003csup\u003e3\u003c/sup\u003e cells/well) for the colony formation assay and cultured in the presence of 10% FBS at 37\u0026deg;C with 5% CO2. Two weeks later, the cells were fixed with 4% paraformaldehyde and stained with crystal violet for 30 minutes. Count the number of colonies with diameter\u0026thinsp;\u0026gt;\u0026thinsp;0.5mm.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eIsolation of exosomes from uterine fluid\u003c/h2\u003e \u003cp\u003eThe uterine fluid collected during the operation was thawed at room temperature and centrifuged at 3000 \u0026times; g for 20min to remove cells. Then, the supernatant is passed through a 0.22\u0026micro;m filter to remove cellular debris. The exosomes were extracted by Exosome Purification kit-exoupur (ECHO BIOTECH, Beijing, China), then concentrated by ultrafilter tube at 30000 rpm for 30min. The isolated exosomes were characterized by transmission electron microscopy, nanoparticle tracking analysis (NTA) and western blot.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eTreatment of macrophages with uterine fluid-derived exosomes\u003c/h2\u003e \u003cp\u003eUterine fluid-derived exosomes were labeled using a PKH67 Fluorescent Cell Linker Kit (Sigma-Aldrich, Saint Louis, MO, USA) to detect the direct transfer of the exosomes into macrophages (Thp1). The purified exosomes were resuspended in PBS and labeled with PKH67. PBS without exosomes was used as control. The internalization of exosomes was conducted by incubating Thp1 cells at 37\u0026deg;C with a final concentration of 100\u0026micro;g/ml. Fluorescent signals were detected after 48h.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eCell transfection\u003c/h2\u003e \u003cp\u003eThe miR-210-3p mimics, inhibitor and respective negative controls (scrambled oligos) were obtained from RiboBio. According to the manufacturer's instructions, transfections were performed using Lipofectamine 3000.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eTransfection of RAW264.7 with miR-210-3p by lentiviral vector\u003c/h2\u003e \u003cp\u003emiR-210-3p short hairpin RNA lentivirus (miR-210-3p inhibitor) and corresponding negative control lentiviruses (miRNA-NC) were purchased from Genechem Co. Ltd (Shanghai, China). Cells were infected with lentiviruses at 100 MOI (multiplicity of infection) for 6h and replaced with fresh medium for another 48h. The green fluorescent protein signal was detected by a fluorescence microscope and gene transfection efficiency was verified by PCR.\u003c/p\u003e \u003cp\u003e \u003cb\u003eEstablishment of the endometriosis mouse model.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eSix-week-old female nonpregnant C57BL6 (20g) were obtained from the Laboratory Animal Research Center of the Second Xiangya Hospital of Central South University. All animal handling and experimental procedures were approved by the Animal Experimental Ethics Committee of the Second Xiangya Hospital of Central South University (#20220059).\u003c/p\u003e \u003cp\u003eDonor mice were initially treated with estradiol benzoate (3\u0026micro;g/mouse) and sacrificed after 7 days. The uterus was removed and immediately placed in saline solution. The two uterine horns were excised and split longitudinally with a surgical blade, and the endometrium was carefully separated from muscles. Each endometrium was identically processed and disrupted into segments smaller than 1 mm. On the 0th day of modeling, the fragment was injected into the abdominal cavity of recipient mice. Overall, segments of endometrium from one uterus were placed in 400\u0026micro;l PBS and injected into two recipient mice. The mice were sacrificed by cervical dislocation 28 days after endometrial tissue injection, measure and record the size and volume of the lesions. In the experimental group, mice received intraperitoneal injections of 2mg/kg LPS, every 3 days. (control group, injection of PBS).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eFCM analysis\u003c/h2\u003e \u003cp\u003eAfter euthanizing the animals, peritoneal cells were retrieved by peritoneal lavage with 7ml of ice-cold washing buffer PBS. After shaking the mice, peritoneal cells were collected. CD16/32 was used to block Fc receptors, F4/80 and CD11b were used as pan-macrophage markers, iNOS and CD206 were used to detect M1 and M2 macrophages. The peritoneal macrophages were incubated with antibodies. After washing with PBS, the cells were resuspended in PBS for FCM analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eRNA extraction and quantitative real-time PCR\u003c/h2\u003e \u003cp\u003eTotal RNA was extracted using TRIzol reagent. RNA was reverse-transcribed into cDNA using the HiScript II Q RT SuperMix for qPCR (+\u0026thinsp;gDNA wiper) (No. R223-01;Vazyme), followed by qRT-PCR using ChamQ Universal SYBR qPCR Master Mix (No. Q711-02; Vazyme) under the following thermal cycling conditions: initial denaturation at 95\u0026deg;C for 30 s, followed by 40 cycles of 95\u0026deg;C for 5 s and 60\u0026deg;C for 30 s, and lastly 95\u0026deg;C for 15 s, 60\u0026deg;C for 1 min, and 95\u0026deg;C for 15 s. All PCR primers were designed and synthesized by Sangon Biotech (Shanghai, China; Supplemental table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). The threshold cycle method (2\u0026thinsp;\u0026minus;\u0026thinsp;ΔΔCT) was used to calculate the relative expression levels normalized to GAPDH levels.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eWestern blot analysis\u003c/h2\u003e \u003cp\u003eThe total protein was extracted, and the protein concentration was determined by BCA. The proteins separated via SDS-PAGE and transferred to PVDF membranes, and then blocked in 5% non-fat milk at room temperature for 2h. The membranes were incubated overnight at 4\u0026deg;C with primary antibodies. According to the manufacturer's instructions, HRP conjugated secondary antibodies and ECL kits were used to detect immunoreactive protein.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eTMT Proteomics\u003c/h2\u003e \u003cp\u003eTotal proteins of Thp1 transfected with miR-210-3p were extracted and their concentrations were quantified by the BCA assay (Thermo Fisher Scientific, Waltham, MA). Peptides were labeled with TMT according to the manufacturer's protocol. For each sample, 2ug of total peptides were separated and analyzed with a nano\u0026shy;UPLC(EASY\u0026shy;nLC1200) coupled to a Q Exactive HFX Orbitrap instrument (Thermo Fisher Scientific) with a nano\u0026shy;electrospray ion source. Data dependent acquisition (DDA) was performed in profile and positive mode with Orbitrap analyzer.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eThe data are presented as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD from at least three independent experiments. Statistical significance was measured using Student\u0026rsquo;s t test (two-tailed). A value of p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 indicates that the difference was statistically. All statistical analyses were performed with GraphPad Prism 8.0.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eAbdominal inflammatory microenvironment promotes the development of endometriosis\u003c/h2\u003e \u003cp\u003eEstablishment of a mouse endometriosis model, and simulation of different abdominal environments by injecting LPS and PBS into the peritoneal cavity. Typical endometriosis-like lesions were formed in the mesentery or peritoneum 28 days after implantation (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea, b). The results showed that the volume of lesions in LPS group was significantly larger than that in PBS group, and the number of lesions showed the same trend (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec). We conducted immunocytochemical staining for Ki-67 (proliferation marker) and vimentin (mesenchymal marker) followed by fluorescence microscopy, and then observed that abdominal inflammation significantly promoted the proliferation of endometrial stromal cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed). At the same time, the peritoneal fluid of mice was collected for FCM analysis, it was found that the number of macrophages in LPS group was more than that in PBS group, and M2 macrophages were dominant in both groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee, f).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003ePeritoneal macrophages promote the proliferation and migration of hESCs\u003c/h2\u003e \u003cp\u003ePrimary isolated hESCs expressed high levels of mesenchymal stem cell surface markers CD140b and CD146, and have the potential for osteogenic and adipogenic differentiation (Supplemental Fig.\u0026nbsp;1a, b, c).\u003c/p\u003e \u003cp\u003ePeritoneal fluid macrophages conditioned medium from patients with endometriosis (EMs) and patients without endometriosis (Control) was used to interfere with hESCs. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea, b, the results of wound healing assays and transwell migration were performed to assess the influence of macrophages on the migration of hESCs. As expected, macrophages from EMs remarkably enhanced the migration capacity of hESCs. In addition, Western blot analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec, d) showed that compared to the Control group, macrophages from EMs enhanced the expression of MMP9, MMP2 and N-Cadherin in hESCs. Subsequently, FCM analysis and colony formation revealed that macrophages from EMs promoted the proliferative capacity of hESCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee, f).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo assess the influence of macrophages on progesterone resistance of hESCs, we induced decidualization in vitro. Then we examined the expression of estrogen (ER) and progesterone receptors (PR) in hESCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eg). Western blot analysis revealed that peritoneal macrophages in patients with endometriosis reduced the expression of PR and epithelial marker E-cadherin and enhanced the expression of mesenchymal marker N-Cadherin, which means that the differentiation ability of hESCs into epithelial cells and decidualization was impaired. Consistent with the findings, ELISA result showed (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eh) macrophages from EMs decreased the expression of the decidualization markers IGFBP-1 in hESCs.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eThe effect of macrophages treated with exosomes on hESCs\u003c/h2\u003e \u003cp\u003eEMs-exosomes (EMs-exo) from uterine fluid of patients with endometriosis and Control-exosomes (Control-exo) from uterine fluid of patients without endometriosis showed similar characteristics of 100nm diameter, biconcave morphological features (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003ea, b), and positive staining for exosomal makers TSG101, HSP70 and CD63(Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003ec). To elucidate the role of exosomes in the macrophages, we used the Thp1 cell line as model of macrophages in vitro. The exosomes were labeled with PKH67 and incubated with the macrophages. Uterine fluid-derived exosomes with green fluorescence were observed in the macrophages (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003ed). The results show that we successfully extracted uterine-derived exosomes and that macrophages can phagocytose uterine-derived exosomes.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo investigate the function of macrophages treated with uterine-derived exosomes in hESCs. Thp1 were incubated with EMs-exo and Control-exo for 48 hours, conditioned medium was collected and co-cultured with hESCs. Wound healing assays and transwell assays revealed that EMs-exo group enhanced the migration of hESCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003ea, b). Similarly, western blot analysis showed that EMs-exo group increased the protein level of MMP9, MMP2 and N-Cadherin in hESCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003ec, d). Moreover, FCM analysis and colony formation indicated that compared to the Control-exo group, EMs-exo group promoted the proliferative capacity of hESCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003ee, f).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFurthermore, co-culturing EMs-exo conditioned medium with hESCs resulted in a downregulation of PR and E-cadherin expression and an upregulation of N-cadherin in hESCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eg). Similarly, ELISA results showed that the secretion of IGFBP1 in hESCs was significantly decreased in the EMs-exo group compared to the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eh). These results suggested that macrophages co-incubated with EMs-exo can promote the proliferation and migration of hESCs, resulting in progesterone resistance and impairs decidualization.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003emiR-210-3p is overexpressed in macrophages treated with EMs-exo\u003c/h2\u003e \u003cp\u003eTo assess endometriosis-induced changes in the expression of exosomal miRNAs, we conducted sequencing of small RNAs from endometriosis and non-endometriosis uterine-derived exosomes and eutopic endometrium, 9 differentially expressed miRNAs were identified in exosomes, while 22 differentially expressed miRNAs were identified in endometrium. The miRanda and TargetScan databases were used to predict the target genes of the differentially expressed miRNAs. Functional enrichment analysis showed that the target genes of exosomal miRNA were mainly involved in the positive regulation of macrophage differentiation, cytokine activity, ATP binding and glycogen metabolic process (Supplemental Fig.\u0026nbsp;2a, b). miR-210-3p expression was significantly upregulated in uterine-derived exosomes and eutopic endometrium from patients with endometriosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003ee, f), and qRT-PCR results also confirmed that it was markedly elevated in EMs-exo and eutopic endometrium[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Therefore, we selected this miRNA for further analyses. Notably, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eg, miR-210-3p levels were significantly higher in peritoneal fluid macrophages from endometriosis, compared with control group. Similarly, qRT-PCR results also confirmed that miR-210-3p expression of Thp1 was obviously upregulated after EMs-exo treatment for 48h (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eh).\u003c/p\u003e \u003cp\u003eBased on the above results, we speculated that the uterine-derived exosomes of patients with endometriosis may promote the development of endometriosis by upregulating the expression of miR210-3p in macrophages.\u003c/p\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003e\u003cb\u003emiR-210-3p promotes the proliferation and migration of hESCs in vitro\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eTo clarify the effect of miR-210-3p on endometriosis, we first silenced miR-210-3p by transfecting miR-210-3p inhibitor in Thp1. Next, transwell migration and wound healing assays were performed to assess whether silenced miR-210-3p could reverse the effect of peritoneal macrophages on the migration of hESCs. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea, b, c, d, the low-expression of miR-210-3p inhibited the migration capacity in hESCs. Subsequently, the results of colony formation and FCM assays revealed that the low-expression of miR-210-3p in macrophages inhibited the proliferative capacity of hESCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ee, f). Western blot analysis revealed that downregulating miR-210-3p enhanced the expression of epithelial marker E-cadherin and reduced the expression of mesenchymal marker N-cadherin (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eg). The results showed that the ability of mesenchymal stem cells to differentiate into epithelial cells was enhanced and the decidualization ability was improved after the inhibition of macrophage miR-210-3p, and the decidualization marker IGFBP1 showed the same trend (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eh). These results suggested that inhibiting the expression of miR-210-3p in macrophages can inhibit the migration and proliferation of hESCs, and improve the progesterone resistance of hESCs.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003emiR-210-3p promotes the development of endometriosis in vivo\u003c/h2\u003e \u003cp\u003eMacrophage-depleting liposomes containing clodronate were used to deplete mouse peritoneal macrophages, then the miR-210-3p of the mouse macrophage cell line RAW264.7 was knocked out using a lentivirus and intraperitoneally injected (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea). At sacrifice, 28 days after intraperitoneal injection of syngeneic endometrial tissue, lesions had developed in all mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb, c). Endometriotic lesions with low-expression of miR-210-3p in mouse macrophages were significantly bigger, which was reflected by their total volume and number (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ed). Immunofluorescent staining of Ki-67 and vimentin were performed that downregulation of miR-210-3p in macrophages inhibited the proliferation capacity of endometrial stromal cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ee). These results implied that inhibiting miR-210-3p in macrophages effectively protected mice from endometriosis, and show that miR-210-3p may be a valuable biomarker for non-invasive diagnosis and treatment of endometriosis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec25\" class=\"Section3\"\u003e \u003ch2\u003emiR-210-3p induces M2 macrophage polarization by ATP5D\u003c/h2\u003e \u003cp\u003eTo identify the molecular mechanism of miR-210-3p in macrophages, we transfected miR-210-3p mimics and inhibitors, and then conducted proteomic sequencing. According to the results, we identified a total of 360 differentially expressed proteins between miR-210-3p overexpression and the control group, and 37 differentially expressed proteins between miR-210-3p underexpression and the control group.(Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea, b). Subsequently, functional analysis of the differential proteins was performed. It was found that inhibition of miR-210-3p mainly affected ATP metabolic pathways, such as oxidative phosphorylation pathway, ATP metabolism and ATP synthase activity (Supplemental Fig.\u0026nbsp;3b, d). KEGG showed that miR-210-3p overexpression mainly affected immunoinflammatory pathways (Supplemental Fig.\u0026nbsp;3a, c), such as IL-17 signaling pathway, TNF signaling pathway and Nod-like receptor signaling pathway. We screened out differential proteins with consistent expression trends in the two groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ec), and found that the expression of ATP5F1D, FAU and MST1L was inhibited in macrophages when miR-210-3p was overexpressed, while the three proteins were up-regulated in macrophages when miR-210-3p was underexpressed.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe qRT-PCR and WB results confirmed that ATP5F1D (ATP5D) expression was consistent with the proteomic results (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ed, e). The expression of ATP5D was significantly reduced in abdominal macrophages of patients with endometriosis as compared to non-endometriosis patients (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ef). Therefore, we hypothesize that the excessive expression of miR-210-3p in macrophages inhibits the expression of ATP5D, affecting the function of macrophages.\u003c/p\u003e \u003cp\u003eBy knocking down ATP5D expression in macrophages, the expressions of CD206, CD163 and IL10 in macrophages were significantly increased, while the expressions of iNOS and CCR7 were significantly decreased (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eg), suggesting that inhibiting ATP5D expression drive the M2 polarization of macrophages. Subsequently, the supernatant of macrophages with low-expression of ATP5D was co-cultured with hESCs for 48 hours, wound healing assays and transwell indicated that macrophages with decreased expression of ATP5D remarkably enhanced the migration capacity of hESCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eh, i).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eEndometriosis is a common benign gynecological disease; however, its biological behavior regarding distant metastasis and invasion is similar to that of malignant tumors[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. The pathogenesis of endometriosis is still unclear[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], despite a large body of evidence suggests that, in addition to hormonal and intrinsic abnormalities of the endometrium, the immune system plays a key role in its development[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eImmune dysfunction is believed to play a role in both the initiation and progression of endometriosis by creating a microenvironment that encourages the ectopic survival and implantation of endometrial cells[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Our study has demonstrated that the inflammatory abdominal microenvironment is conducive to the implantation and growth of ectopic endometrial cells, and that peritoneal macrophages are primarily M2 macrophages. Furthermore, we found that peritoneal macrophages from endometriosis patients can stimulate the migration, invasion, and proliferation of hESCs while also inducing their progesterone resistance. This evidence suggests that the changes of macrophages establish an \"immune tolerance\" microenvironment locally in the abdominal cavity, providing a suitable \"soil\" for the implantation and growth of ectopic lesions[\u003cspan additionalcitationids=\"CR33\" citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Consequently, there is a new research focus on regulating the abdominal immune microenvironment, reprogramming macrophage epigenetic, and disrupting the \"peaceful coexistence\" between ectopic endometrial tissue and the local microenvironment.\u003c/p\u003e \u003cp\u003eExosomes act as natural carriers of intercellular information and play a crucial role in immune regulation, particularly in remote regulation[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Our results demonstrate that uterine-derived exosomes from endometriosis patients, when administered to peritoneal macrophages, stimulate the proliferation and migration of hESCs while also inducing their resistance to progesterone. Our earlier study revealed that miR-210-3p expression was upregulated in both uterine-derived exosomes and eutopic endometrial tissue from women with endometriosis[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Our present study shows that miR-210-3p expression is upregulated in peritoneal macrophages from women with endometriosis. Therefore, we hypothesize that exosomes from the uterine cavity may cause the ectopic implantation and growth of endometrial cells by elevating miR-210-3p expression in peritoneal macrophages.\u003c/p\u003e \u003cp\u003eIn the present study, we show that upregulation of macrophage miR-210-3p inhibits ATP5D expression, thereby enhancing the invasion and migration of hESCs. Moreover, downregulating macrophage ATP5D expression induces M2 polarization, which may be a mechanism by which uterine-derived exosomes promote endometriosis development. ATP5D is a subunit of mitochondrial ATP synthase that plays an important role in catalyzing the synthesis of ATP in the mitochondria[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Decreased mitochondrial ATP generation typically increases AMP concentration and shifts metabolism from synthetic to catabolic[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. The metabolic state of macrophages not only reflects the intrinsic stability of the local microenvironment but also affects the local microenvironment by altering their own function[\u003cspan additionalcitationids=\"CR38 CR39\" citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Therefore, exosomes derived from the uterine cavity may affect the function of macrophages by disrupting their metabolism through the alteration of ATP synthase in peritoneal macrophages.\u003c/p\u003e \u003cp\u003eSimilar to the \"tumor niche\" hypothesis[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e], the immune microenvironment of the abdomen (\"soil\") undergoes changes before endometrial cells (\"seeds\") reach the peritoneal cavity, promoting the escape of the \"seeds\" from immune surveillance and aiding in their settlement and growth. Uterine-derived exosomes may function as a mode of information transfer. In fact, endometriotic exosomes may retrogradely enter the pelvic area and adjust local cell functions, shaping a suitable \"soil\" for ectopic endometrial growth, which might be key molecules in the pathogenesis of endometriosis[\u003cspan additionalcitationids=\"CR43\" citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Our data suggest that uterine-derived exosomes inhibit macrophage ATP5D expression, driving macrophages towards M2 polarization, establishing a suitable soil of local immune inhibition and promoting ectopic endometrial settlement. Additionally, study found that exosomes induce macrophage polarization to create a favorable microenvironment for angiogenesis, regulating the development of endometriosis[\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOur study demonstrates that uterine-derived exosomes can regulate the expression of miR-210-3p in peritoneal macrophages to induce the formation of an immunosuppressive microenvironment. These findings provide new insights into the role of uterine-derived exosomes in mediating the establishment of an immunosuppressive peritoneal microenvironment and show that miR-210-3p may be a valuable biomarker, and that inhibiting the expression of miR-210-3p in peritoneal macrophages may become a novel immunotherapeutic strategy for endometriosis.\u003c/p\u003e \u003cp\u003eHowever, our study has some limitations. Although the isolated extracellular vesicles from the uterus cavity may reflect the initial state of the cavity, it is hard to identify a specific source of exosomes. Because of species variation, we were unable to directly investigate the impacts of exosomes that are derived from the uterus of endometriosis patients on the implantation and growth of ectopic lesions and peritoneal macrophages in a mouse model. It is crucial to investigate the particular regulatory mechanism of uterine-derived exosomes on the function of macrophages, to gain a more profound understanding of the role they plays in the growth and metastasis of hESCs.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn summary, we presented evidence of the mediation of growth and migration of hESCs by exosomes derived from the uterine cavity in endometriosis patients. We discovered that uterine-derived exosomes upregulate the expression of miR-210-3p in peritoneal macrophages to inhibit ATP5D, thereby promoting M2 polarization and reshaping the peritoneal immune microenvironment, which leads to the development of endometriosis.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eEMs\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eEndometriosis\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ehESCs\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eHuman Endometrial Stromal Cells\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eEMSCs\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eEndometrium-derived Mesenchymal Stem Cells\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eATP5D\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eATP synthase, H\u0026thinsp;+\u0026thinsp;transporting, mitochondrial F1 complex, δ subunit\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePBS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePhosphate buffer saline\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eLPS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eLipopolysaccharide\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePMA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePhorbol-12-myristate-13-acetate\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eWB\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eWestern blot analysis\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePR\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eProgesterone Receptor\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eER\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eEstrogen Receptor\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eE-Ca\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eEpithelial Cadherin\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eN-Ca\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eNeural Cadherin\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eEMs-exo\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eexosomes from uterine fluid of patients with endometriosis\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eControl-exo\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eexosomes from uterine fluid of patients without endometriosis\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eNTA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eNanoparticle Tracking Analysis\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTEM\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eTransmission Electron Microscope\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eThp1\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eHuman myeloid leukemia mononuclear cells\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMMP\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMatrix Metalloproteinase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eIGFBP1\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eInsulin-like Growth Factor Binding Protein 1\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003emimics NC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMimics negative control\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eInhibitor NC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eInhibitor negative control\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003esiRNA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eSmall interfering RNA\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eGO\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eGene Ontology\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eKEGG\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eKyoto Encyclopedia of Genes\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e "},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was approved by the Medical Ethics Committee of the Second Xiangya Hospital of Central South University (approval number:2020-065). Informed consent was obtained from each patient. All animal handling and experimental procedures were approved by the Animal Experimental Ethics Committee of the Second Xiangya Hospital of Central South University (approval number:20220059).\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data produced or analyzed in this study are contained in this published article and its supplementary information files. All data used in the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests in this work. \u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by a grant from the National Natural Science Foundation of China (#81873826). \u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSN.C and XQ.W conceived and designed the experiments; SN.C, Y.J and XQ.W prepared the manuscript; SN.C, Y.J and XS.C performed the experiments; ZY.C, H.T, M.L, TY.Z, WW.SG collected the samples; SN.C and XQ.W analyzed the data. All authors read and approved the final manuscript.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNone\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eZondervan KT, Becker CM, Missmer SA. Endometriosis. Longo DL, editor. N Engl J Med [Internet]. 2020 [cited 2023 May 31];382:1244\u0026ndash;56. Available from: https://pubmed.ncbi.nlm.nih.gov/32212520/\u003c/li\u003e\n\u003cli\u003eGiudice LC, Kao LC. Endometriosis. Lancet [Internet]. 2004 [cited 2023 May 31];364:1789\u0026ndash;99. Available from: https://pubmed.ncbi.nlm.nih.gov/15541453/\u003c/li\u003e\n\u003cli\u003eHalme J, HAMMOND M, \u0026hellip; JH-O\u0026amp;, 1984 undefined. Retrograde menstruation in healthy women and in patients with endometriosis. journals.lww.com [Internet]. [cited 2023 May 31]; Available from: https://journals.lww.com/greenjournal/Abstract/1984/08000/Retrograde_Menstruation_in_Healthy_Women_and_in.1.aspx\u003c/li\u003e\n\u003cli\u003eKjerulff KH, Erickson BA, Langenberg PW. Chronic gynecological conditions reported by US women: findings from the National Health Interview Survey, 1984 to 1992. Am J Public Health [Internet]. 1996 [cited 2023 May 31];86:195\u0026ndash;9. Available from: https://pubmed.ncbi.nlm.nih.gov/8633735/\u003c/li\u003e\n\u003cli\u003eZhang Z, Suo L, Chen Y, Zhu L, International GW-SC, 2019 undefined. Endometriotic peritoneal fluid promotes myofibroblast differentiation of endometrial mesenchymal stem cells. hindawi.com [Internet]. [cited 2023 May 31]; Available from: https://www.hindawi.com/journals/sci/2019/6183796/\u003c/li\u003e\n\u003cli\u003eKhatun M, Sorjamaa A, Kangasniemi M, Sutinen M, Salo T, Liakka A, et al. Niche matters: The comparison between bone marrow stem cells and endometrial stem cells and stromal fibroblasts reveal distinct migration and cytokine profiles in response to inflammatory stimulus. PLoS One [Internet]. 2017 [cited 2023 May 31];12. Available from: https://pubmed.ncbi.nlm.nih.gov/28419140/\u003c/li\u003e\n\u003cli\u003eGargett CE, Schwab KE, Deane JA. Endometrial stem/progenitor cells: the first 10 years. Hum Reprod Update [Internet]. 2016 [cited 2023 May 31];22:137\u0026ndash;63. Available from: https://pubmed.ncbi.nlm.nih.gov/26552890/\u003c/li\u003e\n\u003cli\u003eKao AP, Wang KH, Chang CC, Lee JN, Long CY, Chen HS, et al. Comparative study of human eutopic and ectopic endometrial mesenchymal stem cells and the development of an in vivo endometriotic invasion model. Fertil Steril [Internet]. 2011 [cited 2023 May 31];95. Available from: https://pubmed.ncbi.nlm.nih.gov/21047634/\u003c/li\u003e\n\u003cli\u003eNair ARKG, Pandit H, Warty N, Madan T. Endometriotic mesenchymal stem cells exhibit a distinct immune phenotype. Int Immunol [Internet]. 2015 [cited 2023 May 31];27:195\u0026ndash;204. Available from: https://pubmed.ncbi.nlm.nih.gov/25416515/\u003c/li\u003e\n\u003cli\u003eBurney RO, Giudice LC. Pathogenesis and pathophysiology of endometriosis. Fertil Steril [Internet]. 2012 [cited 2023 May 31];98:511\u0026ndash;9. Available from: https://pubmed.ncbi.nlm.nih.gov/22819144/\u003c/li\u003e\n\u003cli\u003eMa S, Xie N, Li W, Yuan B, Shi Y, Wang Y. Immunobiology of mesenchymal stem cells. Cell Death Differ [Internet]. 2014 [cited 2023 May 31];21:216\u0026ndash;25. Available from: https://pubmed.ncbi.nlm.nih.gov/24185619/\u003c/li\u003e\n\u003cli\u003eVallv\u0026eacute;-Juanico J, Houshdaran S, Giudice LC. The endometrial immune environment of women with endometriosis. Hum Reprod Update [Internet]. 2019 [cited 2023 May 31];25:565\u0026ndash;92. Available from: https://pubmed.ncbi.nlm.nih.gov/31424502/\u003c/li\u003e\n\u003cli\u003eRam\u0026iacute;rez-Pavez TN, Mart\u0026iacute;nez-Esparza M, Ruiz-Alcaraz AJ, Mar\u0026iacute;n-S\u0026aacute;nchez P, Machado-Linde F, Garc\u0026iacute;a-Pe\u0026ntilde;arrubia P. The Role of Peritoneal Macrophages in Endometriosis. Int J Mol Sci [Internet]. 2021 [cited 2023 May 31];22. Available from: https://pubmed.ncbi.nlm.nih.gov/34639133/\u003c/li\u003e\n\u003cli\u003eBerbic M, Schulke L, Markham R, Tokushige N, Russell P, Fraser IS. Macrophage expression in endometrium of women with and without endometriosis. Hum Reprod [Internet]. 2009 [cited 2023 May 31];24:325\u0026ndash;32. Available from: https://pubmed.ncbi.nlm.nih.gov/19049988/\u003c/li\u003e\n\u003cli\u003eWeinberg J, Haney A, Xu F, Ramakrishnan S. Peritoneal fluid and plasma levels of human macrophage colony-stimulating factor in relation to peritoneal fluid macrophage content. 1991 [cited 2023 May 31]; Available from: https://ashpublications.org/blood/article-abstract/78/2/513/168804\u003c/li\u003e\n\u003cli\u003eBacci M, Capobianco A, Monno A, Cottone L, Di Puppo F, Camisa B, et al. Macrophages are alternatively activated in patients with endometriosis and required for growth and vascularization of lesions in a mouse model of disease. Am J Pathol [Internet]. 2009 [cited 2023 May 31];175:547\u0026ndash;56. Available from: https://pubmed.ncbi.nlm.nih.gov/19574425/\u003c/li\u003e\n\u003cli\u003eJeljeli M, Riccio LGC, Chouzenoux S, Moresi F, Toullec L, Doridot L, et al. Macrophage Immune Memory Controls Endometriosis in Mice and Humans. Cell Rep [Internet]. 2020 [cited 2023 May 31];33. Available from: https://pubmed.ncbi.nlm.nih.gov/33147452/\u003c/li\u003e\n\u003cli\u003eXie Q, He H, Wu YH, Zou LJ, She XL, Xia XM, et al. Eutopic endometrium from patients with endometriosis modulates the expression of CD36 and SIRP-\u0026alpha; in peritoneal macrophages. J Obstet Gynaecol Res [Internet]. 2019 [cited 2023 May 31];45:1045\u0026ndash;57. Available from: https://pubmed.ncbi.nlm.nih.gov/30843336/\u003c/li\u003e\n\u003cli\u003eKalluri R, LeBleu VS. The biology , function , and biomedical applications of exosomes. Science [Internet]. 2020 [cited 2023 May 31];367. Available from: https://pubmed.ncbi.nlm.nih.gov/32029601/\u003c/li\u003e\n\u003cli\u003eWhiteside TL. Exosome and mesenchymal stem cell cross-talk in the tumor microenvironment. Semin Immunol [Internet]. 2018 [cited 2023 May 31];35:69\u0026ndash;79. Available from: https://pubmed.ncbi.nlm.nih.gov/29289420/\u003c/li\u003e\n\u003cli\u003eZhao J, Li X, Hu J, Chen F, Qiao S, Sun X, et al. Mesenchymal stromal cell-derived exosomes attenuate myocardial ischaemia-reperfusion injury through miR-182-regulated macrophage polarization. Cardiovasc Res [Internet]. 2019 [cited 2023 May 31];115:1205\u0026ndash;16. Available from: https://pubmed.ncbi.nlm.nih.gov/30753344/\u003c/li\u003e\n\u003cli\u003eXin L, Lin X, Zhou F, Li C, Wang X, Yu H, et al. A scaffold laden with mesenchymal stem cell-derived exosomes for promoting endometrium regeneration and fertility restoration through macrophage immunomodulation. Acta Biomater [Internet]. 2020 [cited 2023 May 31];113:252\u0026ndash;66. Available from: https://pubmed.ncbi.nlm.nih.gov/32574858/\u003c/li\u003e\n\u003cli\u003eSun H, Li D, Yuan M, Li Q, Zhen Q, Li N, et al. Macrophages alternatively activated by endometriosis-exosomes contribute to the development of lesions in mice. Mol Hum Reprod [Internet]. 2019 [cited 2023 May 31];25:5\u0026ndash;16. Available from: https://pubmed.ncbi.nlm.nih.gov/30428082/\u003c/li\u003e\n\u003cli\u003eCampoy I, Lanau L, Altadill T, Sequeiros T, Cabrera S, Cubo-Abert M, et al. Exosome-like vesicles in uterine aspirates: a comparison of ultracentrifugation-based isolation protocols. J Transl Med [Internet]. 2016 [cited 2023 May 31];14. Available from: https://pubmed.ncbi.nlm.nih.gov/27317346/\u003c/li\u003e\n\u003cli\u003eHarp D, Driss A, Mehrabi S, Chowdhury I, Xu W, Liu D, et al. Exosomes derived from endometriotic stromal cells have enhanced angiogenic effects in vitro. Cell Tissue Res [Internet]. 2016 [cited 2023 May 31];365:187\u0026ndash;96. Available from: https://pubmed.ncbi.nlm.nih.gov/26841879/\u003c/li\u003e\n\u003cli\u003eJiang Y, Chai X, Chen S, Chen Z, Tian H, Liu M, et al. Exosomes from the Uterine Cavity Mediate Immune Dysregulation via Inhibiting the JNK Signal Pathway in Endometriosis. Biomedicines [Internet]. 2022 [cited 2023 May 31];10. Available from: /pmc/articles/PMC9775046/\u003c/li\u003e\n\u003cli\u003eGiudice LC, Kao LC. Endometriosis. Lancet [Internet]. 2004 [cited 2023 May 31];364:1789\u0026ndash;99. Available from: https://pubmed.ncbi.nlm.nih.gov/15541453/\u003c/li\u003e\n\u003cli\u003eSymons LK, Miller JE, Kay VR, Marks RM, Liblik K, Koti M, et al. The Immunopathophysiology of Endometriosis. Trends Mol Med [Internet]. 2018 [cited 2023 May 31];24:748\u0026ndash;62. Available from: https://pubmed.ncbi.nlm.nih.gov/30054239/\u003c/li\u003e\n\u003cli\u003eAgostinis C, Balduit A, Mangogna A, Zito G, Romano F, Ricci G, et al. Immunological Basis of the Endometriosis: The Complement System as a Potential Therapeutic Target. Front Immunol [Internet]. 2021 [cited 2023 May 31];11. Available from: https://pubmed.ncbi.nlm.nih.gov/33505394/\u003c/li\u003e\n\u003cli\u003eSaunders PTK, Horne AW. Endometriosis: Etiology, pathobiology, and therapeutic prospects. Cell [Internet]. 2021 [cited 2023 May 31];184:2807\u0026ndash;24. Available from: https://pubmed.ncbi.nlm.nih.gov/34048704/\u003c/li\u003e\n\u003cli\u003eWang Y, Nicholes K, Shih IM. The Origin and Pathogenesis of Endometriosis. Annu Rev Pathol [Internet]. 2020 [cited 2023 May 31];15:71\u0026ndash;95. Available from: https://pubmed.ncbi.nlm.nih.gov/31479615/\u003c/li\u003e\n\u003cli\u003eCapobianco A, Rovere-Querini P. Endometriosis, a disease of the macrophage. Front Immunol [Internet]. 2013 [cited 2023 May 31];4. Available from: https://pubmed.ncbi.nlm.nih.gov/23372570/\u003c/li\u003e\n\u003cli\u003eWu J, Xie H, Yao S, Liang Y. Macrophage and nerve interaction in endometriosis. J Neuroinflammation [Internet]. 2017 [cited 2023 May 31];14. Available from: https://pubmed.ncbi.nlm.nih.gov/28288663/\u003c/li\u003e\n\u003cli\u003eKlemmt PA, Starzinski-Powitz A. Molecular and Cellular Pathogenesis of Endometriosis. Curr Womens Health Rev [Internet]. 2018 [cited 2023 May 31];14:1\u0026ndash;11. Available from: https://pubmed.ncbi.nlm.nih.gov/29861704/\u003c/li\u003e\n\u003cli\u003eOl\u0026aacute;hov\u0026aacute; M, Yoon WH, Thompson K, Jangam S, Fernandez L, Davidson JM, et al. Biallelic Mutations in ATP5F1D, which Encodes a Subunit of ATP Synthase, Cause a Metabolic Disorder. Am J Hum Genet [Internet]. 2018 [cited 2023 May 31];102:494\u0026ndash;504. Available from: https://pubmed.ncbi.nlm.nih.gov/29478781/\u003c/li\u003e\n\u003cli\u003eWeinberg SE, Sena LA, Chandel NS. Mitochondria in the regulation of innate and adaptive immunity. Immunity [Internet]. 2015 [cited 2023 May 31];42:406\u0026ndash;17. Available from: https://pubmed.ncbi.nlm.nih.gov/25786173/\u003c/li\u003e\n\u003cli\u003eHe L, Jhong JH, Chen Q, Huang KY, Strittmatter K, Kreuzer J, et al. Global characterization of macrophage polarization mechanisms and identification of M2-type polarization inhibitors. Cell Rep [Internet]. 2021 [cited 2023 May 31];37. Available from: https://pubmed.ncbi.nlm.nih.gov/34731634/\u003c/li\u003e\n\u003cli\u003eVerdeguer F, Aouadi M. Macrophage heterogeneity and energy metabolism. Exp Cell Res. 2017;360:35\u0026ndash;40. \u003c/li\u003e\n\u003cli\u003eYan J, Horng T. Lipid Metabolism in Regulation of Macrophage Functions. Trends Cell Biol [Internet]. 2020 [cited 2023 May 31];30:979\u0026ndash;89. Available from: https://pubmed.ncbi.nlm.nih.gov/33036870/\u003c/li\u003e\n\u003cli\u003eVan den Bossche J, O\u0026rsquo;Neill LA, Menon D. Macrophage Immunometabolism: Where Are We (Going)? Trends Immunol [Internet]. 2017 [cited 2023 May 31];38:395\u0026ndash;406. Available from: https://pubmed.ncbi.nlm.nih.gov/28396078/\u003c/li\u003e\n\u003cli\u003eLiu Y, Cao X. Characteristics and Significance of the Pre-metastatic Niche. Cancer Cell [Internet]. 2016 [cited 2023 May 31];30:668\u0026ndash;81. Available from: https://pubmed.ncbi.nlm.nih.gov/27846389/\u003c/li\u003e\n\u003cli\u003eHorne AW, Saunders PTK. SnapShot: Endometriosis. Cell [Internet]. 2019 [cited 2023 May 31];179:1677-1677.e1. Available from: https://pubmed.ncbi.nlm.nih.gov/31951524/\u003c/li\u003e\n\u003cli\u003eSun H, Li D, Yuan M, Li Q, Li N, Wang G. Eutopic stromal cells of endometriosis promote neuroangiogenesis via exosome pathway\u0026dagger;. Biol Reprod [Internet]. 2019 [cited 2023 May 31];100:649\u0026ndash;59. Available from: https://pubmed.ncbi.nlm.nih.gov/30295741/\u003c/li\u003e\n\u003cli\u003eQiu JJ, Lin XJ, Zheng TT, Tang XY, Zhang Y, Hua KQ. The Exosomal Long Noncoding RNA aHIF is Upregulated in Serum From Patients With Endometriosis and Promotes Angiogenesis in Endometriosis. Reprod Sci [Internet]. 2019 [cited 2023 May 31];26:1590\u0026ndash;602. Available from: https://pubmed.ncbi.nlm.nih.gov/30808247/\u003c/li\u003e\n\u003cli\u003eSun H, Li D, Yuan M, Li Q, Zhen Q, Li N, et al. Macrophages alternatively activated by endometriosis-exosomes contribute to the development of lesions in mice. Mol Hum Reprod [Internet]. 2019 [cited 2023 May 31];25:5\u0026ndash;16. Available from: https://pubmed.ncbi.nlm.nih.gov/30428082/\u003c/li\u003e\n\u003cli\u003eLi Y, Lyu P, Ze Y, Li P, Zeng X, Shi Y, et al. Exosomes derived from plasma: promising immunomodulatory agents for promoting angiogenesis to treat radiation-induced vascular dysfunction. PeerJ [Internet]. 2021 [cited 2023 May 31];9. Available from: https://pubmed.ncbi.nlm.nih.gov/33859878/\u003c/li\u003e\n\u003c/ol\u003e\n"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Endometriosis, exosome, macrophage, miR-210-3p, ATP5D","lastPublishedDoi":"10.21203/rs.3.rs-3030329/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3030329/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eEndometriosis development is associated with peritoneal immune microenvironment abnormality. However, the specific mechanism is uncertain. Investigating peritoneal immune microenvironment regulation mechanisms could introduce novel therapeutic strategies for effective endometriosis treatment.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eWound healing assay, Transwell, colony formation were carried out to assess cells invasion and proliferation. Western blotting analysis and ELISA were used to evaluate cells resistance to progesterone. Endometriosis C57BL6 mouse model was conducted to assess the impact of peritoneal inflammatory environment and macrophage miR-210-3p on ectopic lesion implantation and growth. miRNA and proteomics sequencing were carried out to verify the potential mechanisms influencing the development of endometriosis.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eMedium conditioned of peritoneal macrophages from patients with endometriosis, as well as the medium conditioned of macrophages treated with uterine-derived exosomes of endometriosis patients, promoted the proliferation, invasion and progesterone resistance of cells. Interestingly, Uterine-derived exosomes of endometriosis patients increased miR-210-3p expression in peritoneal macrophages. In vivo experiments confirmed that macrophages lentivirally transduced with miR-210-3p inhibitor can significantly decrease the number and volume of endometriotic lesions. Mechanistically, miR-210-3p significantly induced M2 macrophage polarization by inhibiting the expression of ATP5D, and promoted cells migration.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eUterine-derived exosomes of endometriosis patients upregulated the expression of miR-210-3p in peritoneal macrophages to inhibit ATP5D, driving macrophages polarization towards M2 and promoting the development of endometriosis.\u003c/p\u003e","manuscriptTitle":"Uterine-derived exosomes induce the M2 polarization of macrophages via miR210-3p to promote the development of endometriosis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-06-09 15:41:52","doi":"10.21203/rs.3.rs-3030329/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"45f7a3ce-e01b-459c-8ed6-0c787a953c01","owner":[],"postedDate":"June 9th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2023-06-10T04:29:22+00:00","versionOfRecord":[],"versionCreatedAt":"2023-06-09 15:41:52","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3030329","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3030329","identity":"rs-3030329","version":["v1"]},"buildId":"M1DPXKE8UapkOyQliHcFZ","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}