Exosomes Derived from Umbilical Cord Mesenchymal Stem Cells： Mediated Restoration of Endometrial Receptivity and Their Potential to Improve IVF–ET Outcomes

Exosomes Derived from Umbilical Cord Mesenchymal Stem Cells： Mediated Restoration of Endometrial Receptivity and Their Potential to Improve IVF–ET Outcomes | 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 Exosomes Derived from Umbilical Cord Mesenchymal Stem Cells： Mediated Restoration of Endometrial Receptivity and Their Potential to Improve IVF–ET Outcomes Wencong Li, Hong Wei, Xiaohua Ge, Siqi Guo, Meijuan Liu This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7801878/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: Intrauterine adhesion (IUA), a fibrotic disorder characterized by endometrial basal layer damage, severely impairs endometrial receptivity and fertility. Conventional hysteroscopic adhesiolysis restores uterine morphology but fails to reverse fibrosis or promote functional regeneration. Human umbilical cord mesenchymal stem cell–derived exosomes (UCMSCs-EXOs) have emerged as a promising acellular therapeutic strategy due to their potent regenerative and anti-fibrotic properties. This study aimed to evaluate the therapeutic potential of UCMSCs-EXOs in restoring endometrial receptivity in a rat model of IUA. Methods and Results: UC-MSCs were isolated from Wharton’s jelly, characterized by flow cytometry and trilineage differentiation, and their exosomes were purified via ultracentrifugation and confirmed by TEM, DLS, and Western blotting for CD9, CD63, and TSG101. A mechanical injury model of IUA was established in female Sprague–Dawley rats (n = 24), which were divided into control, model, UC-MSCs, and UCMSCs-EXOs groups. One week post-injury, intrauterine administration of UCMSCs-EXOs markedly enhanced endometrial regeneration. Compared with the model group, UCMSCs-EXOs treatment significantly increased endometrial thickness (0.40 ± 0.02 mm vs. 0.10 ± 0.04 mm), gland count (28.18 ± 3.18 vs. 16.56 ± 2.32), and reduced collagen deposition (13.49 ± 1.15% vs. 30.62 ± 4.44%). Immunohistochemistry further revealed upregulated cytokeratin expression, indicating enhanced epithelial proliferation. Conclusions: UCMSCs-EXOs effectively restore endometrial structure and function by promoting epithelial proliferation and inhibiting fibrosis, demonstrating therapeutic efficacy comparable to their parent cells. As a cell-free alternative, UCMSCs-EXOs present a novel and promising strategy for IUA treatment, with the potential to improve IVF-ET outcomes. Intrauterine Adhesions Exosomes Mesenchymal Stem Cells Endometrium Embryo Implantation/physiology Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 INTRODUCTION Intrauterine adhesions (IUA) is a fibrotic condition caused by injury to the basal layer of the endometrium. The underlying pathology involves the disruption of the normal, scar-free cyclical repair mechanism of the endometrium, which is then replaced by the abnormal proliferation of fibrous connective tissue, ultimately leading to partial or complete occlusion of the uterine cavity[ 1 – 3 ]. As a leading cause of secondary infertility, IUA severely compromises endometrial receptivity, thereby impeding embryo implantation[ 4 ]. Clinically, intrauterine procedures or infections are the principal precipitants of IUA[ 5 ], accounting for approximately 15%–20% of cases, whereas postpartum curettage confers an even higher risk of 21%–40%[ 6 ]. Although hysteroscopic adhesiolysis remains the standard treatment, its limitations are increasingly evident: the procedure restores uterine cavity morphology but does not reverse tissue fibrosis. As a result, postoperative recurrence rates in moderate-to-severe cases can be as high as 62.5%[ 7 ], and the subsequent regeneration and functional recovery of the endometrium remain significant challenges. Endometrial receptivity (ER) is the capacity of the endometrium to support embryo apposition, adhesion, and invasion, thereby enabling implantation[ 8 ]. Current theories propose that intrauterine procedures injure or deplete endometrial stem/progenitor cells in the basal layer, constituting a key initiating event in IUA [9, 10] . Loss of these cells disrupts endometrial homeostasis and epithelial regeneration, driving post-injury repair toward pathological fibrosis rather than physiologic regeneration[ 11 – 13 ]. This pathological process directly leads to a significant decline in endometrial receptivity. In vitro fertilization–embryo transfer (IVF–ET) is among the most established assisted reproductive technologies, yet implantation failure still occurs in 32%–51% of transfer cycles[ 14 ].Approximately two-thirds of these failures are attributable to suboptimal endometrial receptivity[ 15 ].Thus, addressing the therapeutic bottleneck in intrauterine adhesions requires moving beyond simple mechanical separation toward strategies that promote high-quality, functional endometrial regeneration to restore receptivity. Regenerative medicine strategies utilizing mesenchymal stem cells (MSCs) have emerged as a promising approach to address this challenge. MSCs exhibit substantial potential in tissue repair owing to their multipotency, immunomodulatory properties, and robust paracrine activity[ 16 , 17 ]. However, the clinical translation of direct MSCs transplantation is hampered by several limitations, including low cell survival rates, potential immune rejection, and ethical concerns [18] . Evidence suggests that the therapeutic effects of MSCs are primarily mediated by their secreted extracellular vesicles, particularly exosomes[ 19 ]. As key mediators of intercellular communication, exosomes are enriched with bioactive molecules, including proteins, RNA, and miRNAs, which facilitates the transfer of parental cell functions to recipient cells[ 20 , 21 ]. Among these, exosomes derived from human umbilical cord mesenchymal stem cells (UCMSCs-EXOs) are of particular interest due to their abundant availability, low immunogenicity, and potent capabilities for tissue regeneration and wound healing, as demonstrated in multiple studies[ 22 , 23 ]. Therefore, this study systematically evaluates the effects of UCMSCs-EXOs on endometrial repair and receptivity in a rat model of IUA and explores their potential to improve IVF-ET outcomes. These findings may inform a safer, more effective approach to the clinical management of IUA and provide theoretical and experimental support for applying regenerative medicine in gynecology. MATERIALS AND METHODS Extraction and Identification of Umbilical Cord Mesenchymal Stem Cells(UC-MSCs): This study was approved by the Ethics Committee of Yuhuangding Hospital, Affiliated to Qingdao University (Approval No. 2024 − 544). Umbilical cord tissues were collected from healthy, full-term infants following vaginal delivery, after obtaining written informed consent from all parental donors. Under aseptic conditions, Wharton’s jelly was dissected and minced into tissue fragments of approximately 1 mm³. Fragments were plated in culture dishes and maintained in UC-MSC growth medium(Procell,Wuhan China) at 37°C in a humidified 5% CO₂ incubator. UC-MSCs between passages 4 and 6 were utilized for all subsequent experiments. For phenotypic characterization, cells were prepared as a single-cell suspension at a concentration of 1.0 × 10⁶ cells/mL, and 100 µL aliquots were distributed into nine flow cytometry tubes. Add CD90-FITC, CD44-PE, CD105-PerCP-Cy5.5, and CD73-APC from the BD Human MSC Analysis Kit (BD Biosciences, USA) to Tubes 1–4, respectively; Tube 5 serves as a blank control; Tube 6 contains an isotype-control cocktail (mIgG1-FITC, mIgG1-PerCP-Cy5.5, mIgG1-APC, mIgG1-PE, mIgG2a-PE); Tube 7 contains a positive-marker cocktail (CD90-FITC, CD105-PerCP-Cy5.5, CD73-APC) together with a negative-marker cocktail (CD34-PE, CD11b-PE, CD19-PE, CD45-PE, HLA-DR-PE). Acquire data by flow cytometry and analyze cell phenotypes in FlowJo, following the manufacturer’s instructions for the BD kit (e.g., staining, compensation, and gating). All antibodies were used as provided in the BD Human MSC Analysis Kit. The multipotent differentiation potential of the UC-MSCs was evaluated by seeding the cells into 6 cm culture dishes. Upon reaching 90%–100% confluence, the medium is replaced with adipogenic, osteogenic, and chondrogenic induction media (HyCyte,Suzhou China). The induction medium was refreshed every three days, interspersed with a 24-hour incubation in maintenance medium. This differentiation protocol was continued for a total of 21 days. Following induction, cells are fixed in 4% paraformaldehyde and stained with Oil Red O, Alizarin Red S, and Alcian Blue, respectively (all from HyCyte). Differentiation is evaluated by light microscopy. Exosome Extraction and Characterization: For the isolation of exosomes, the culture medium was replaced with serum-free medium(Procell,Wuhan China) once the UC-MSCs reached approximately 80% confluence. After 48–72 hours of incubation, the conditioned medium was collected for exosome purification. Centrifuge the medium sequentially at 2,000 × g for 30 min and 10,000 × g for 45 min at 4°C to remove cells and debris. The resulting supernatant was passed through a 0.22 µm filter and then subjected to ultracentrifugation at 100,000 × g for 70 minutes at 4°C to pellet the exosomes. Resuspend the pellet in phosphate-buffered saline (PBS) ( Sparkjade,Shandong China) to yield the final preparation of UCMSCs-EXOs. Protein concentration is determined by a bicinchoninic acid(BCA)( Sparkjade,Shandong China) assay, and samples are stored at − 80°C. Deposit 20 µL of the exosome suspension onto a copper grid, incubate at room temperature for 10 min, and wick off excess liquid with filter paper. Negative-stain with 2% phosphotungstic acid for 10 min, blot, and air-dry at 37°C. The morphology of the exosomes was then examined using a transmission electron microscope. Separately, dilute exosomes 1:1000 in PBS to a final volume of 1 mL for dynamic light scattering (DLS) on a Zetasizer Nano ZS to determine particle-size distribution. Extract exosomal proteins in radioimmunoprecipitation assay ( Sparkjade,Shandong China) buffer supplemented with protease and phosphatase inhibitors(Beyotime,China), then quantify by a BCA assay. Proteins are separated on 10% SDS–PAGE and transferred to PVDF membranes. Membranes are blocked with 5% nonfat dry milk for 1 h, then incubated with anti-CD9(Proteintech,Wuhan China), anti-CD81(Proteintech,Wuhan China), and anti-TSG101(Proteintech,Wuhan China) primary antibodies (overnight at 4°C) followed by HRP-conjugated secondary antibodies(Sparkjade,Shandong China). Signals are visualized by enhanced chemiluminescence. Animal Model Establishment and Treatment: Female, specific pathogen-free grade Sprague-Dawley rats were procured from Jinan Pengyue Laboratory Animal Co., Ltd. and housed at 20–26°C, 40–70% relative humidity, under a 12-h light–dark cycle with ad libitum access to food and water. All animal experiments were conducted in strict accordance with the NIH Guidelines for the Care and Use of Laboratory Animals. Following a 1- to 2-week acclimatization period, the estrous cycles of the rats were monitored daily via vaginal smear analysis. Rats in estrus phase were anesthetized with isoflurane delivered via a small-animal inhalation anesthesia machine (carrier gas oxygen), and the uterus was exposed via laparotomy. IUA was induced by mechanical injury; the endometrial lining of both uterine horns was gently scraped with a surgical curette to create a standardized injury. A total of twenty-four rats exhibiting regular estrous cycles were randomly allocated into four experimental groups (n = 6 ): control (no intervention), model (injury without treatment), UC-MSCs treatment, and UCMSCs-EXOs treatment. One week post-surgery, animals in the treatment groups received a single intrauterine injection of either 2 × 10⁷ UC-MSCs or 150–200 µg of UC-MSC-EXOs, respectively, each suspended in a 200 µL volume. Four to five estrous cycles following treatment, the animals were euthanized, and uterine tissues were harvested for subsequent analysis. Histology and Immunohistochemistry: Uterine tissues are fixed in 4% paraformaldehyde, dehydrated through graded solvents, and embedded in paraffin. Sections 4-µm thick are cut and stained with hematoxylin and eosin (H&E) and Masson’s trichrome according to the manufacturer’s protocols,Four high-power fields were randomly selected from each section for analysis. Endometrial thickness, glandular count, and the fibrosis area ratio (fibrous area/total area of stroma and glands) were measured using ImageJ software. For immunohistochemistry, sections are deparaffinized, rehydrated, and incubated overnight at 4°C with primary antibodies (Solarbio,Beijing China). The following day, the sections were incubated with the secondary antibody (Solarbio,Beijing China) at room temperature for 1 hour. Signals are developed with DAB and counterstained with hematoxylin. Images are acquired on a light microscope and analyzed in ImageJ. Statistical Analysis: Data are presented as the mean ± standard deviation (SD) from independently conducted experiments. Statistical analysis was performed using one-way ANOVA, with t-tests employed for comparisons between two groups. Statistical significance was assessed using GraphPad Prism 10, with p-values considered significant at p < 0.05, p < 0.01, and p < 0.001. RESULTS Isolation, Culture, and Characterization of UC-MSCs: UC-MSCs were isolated from Wharton's jelly and expanded in culture. Primary cultures showed adherent, spindle-shaped cells arranged in parallel or whirl-like patterns (Fig. 1 a, 1 b). Flow cytometry analysis revealed that the cultured cells were positive for the mesenchymal stem cell surface markers CD44, CD73, CD90, and CD105, but negative for the hematopoietic and endothelial markers CD9, CD11b, CD34, and HLA-DR (Fig. 1 c). This immunophenotype is consistent with the minimal criteria for defining MSCs as established by the International Society for Cellular Therapy[ 24 ]. Furthermore, the multipotent capacity of the isolated cells was confirmed by their ability to differentiate into osteogenic, adipogenic, and chondrogenic lineages. Following induction, the formation of calcium deposits, lipid droplets, and acidic mucopolysaccharides was verified by Alizarin Red, Oil Red O, and Alcian Blue staining, respectively (Fig. 2 ). Collectively, the morphology, immunophenotype, and tri-lineage differentiation support successful isolation of high-purity UC-MSCs. Characterization of UCMSCs-EXOs: UCMSCs-EXOs were isolated from conditioned medium by ultracentrifugation. Transmission electron microscopy (TEM) imaging revealed that the isolated vesicles presented a characteristic cup-shaped or "saucer-like" morphology (Fig. 3 a). Furthermore, dynamic light scattering indicated a particle-size distribution predominantly within 30–200 nm (Fig. 3 b), consistent with exosomes. Finally, Western blot analysis confirmed the presence of the canonical exosome marker proteins CD9, CD63, and TSG101 (Fig. 3 c). Collectively, these data on vesicle morphology, size distribution, and protein expression confirmed the successful isolation of UCMSCs-EXOs suitable for downstream applications. Repair Effects of UCMSC-Exos on Damaged Endometrium: UCMSC-EXOs Promote the Structural and Functional Repair of Injured Endometrium: To evaluate therapeutic efficacy, we assessed uterine morphology across groups by H&E staining. Control group uterine displayed a normal endometrial architecture with a thick luminal epithelium and abundant glands (Fig. 4 a). In contrast, the model group exhibited severe endometrial atrophy, characterized by a thinned endometrium, discontinuous luminal epithelium, and a marked reduction in glandular number (Fig. 4 b). Following treatment, the UCMSCs-EXOs–treated group showed marked restoration of endometrial structure(Fig. 4 d). Endometrial thickness (0.40 ± 0.02 mm) and gland count (28.18 ± 3.18) approached control levels (0.44 ± 0.05 mm; 31.05 ± 1.62) and were significantly higher than the model group (0.10 ± 0.04 mm,P<0.001; 16.56 ± 2.32,P < 0.01). As a positive control, the UC-MSCs–treated group also improved (thickness 0.36 ± 0.06 mm; glands 27.46 ± 1.95). These data indicate that UCMSCs-EXOs promote structural regeneration of the injured endometrium. UCMSCs-EXOs Inhibit Progression of Endometrial Fibrosis: To evaluate endometrial fibrosis, a key pathological hallmark of intrauterine adhesions [25] , tissue sections were subjected to Masson's trichrome staining. In control animals, Masson's staining revealed minimal collagen deposition (stained blue), which was appropriately restricted to the endometrial stroma (Fig. 5 a). By contrast, the model group displayed dense collagen replacing the endometrial layer, and the collagen-deposition ratio increased from 12.68 ± 3.09% (control) to 30.62 ± 4.44% (Table 1 ). Treatment with UC-MSC-Exos markedly reduced this fibrotic response, decreasing the collagen area ratio to 13.49 ± 1.15%, a level comparable to that of the control group. A similar anti-fibrotic effect was observed in the UC-MSCs treatment group (15.64 ± 1.98%). Collectively, these findings demonstrate that UCMSCs-EXOs suppress excessive collagen deposition, thereby mitigating endometrial fibrosis during uterine repair. Table 1 Characteristics of the study group(Mean ± SD) group n H&E Stained Endometrial Thickness (mm) Number of Glands in H&E Staining (count) Masson Stained Fibrosis Area Ratio (%) Average Optical Density of CK Staining Normal group 6 0.44 ± 0.05 31.05 ± 1.62 12.68 ± 3.09 0.49 ± 0.08 Model group 6 0.10 ± 0.04 16.56 ± 2.32 30.62 ± 4.44 0.30 ± 0.06 UC-MSCs treatment group 6 0.36 ± 0.06 27.46 ± 1.95 15.64 ± 1.98 0.40 ± 0.08 UCMSCs- EXOs treatment group 6 0.40 ± 0.02 28.18 ± 3.18 13.49 ± 1.15 0.42 ± 0.06 UCMSCs-EXOs Promote Proliferation of Endometrial Epithelial Cells: To investigate the cellular basis of structural recovery, we assessed cytokeratin expression by immunohistochemistry as an epithelial cell proliferation–associated marker. Immunohistochemical analysis revealed strong cytokeratin expression in the endometrium of the control group, indicating active epithelial cell proliferation (Fig. 6 a). In contrast, expression levels in the model group were significantly reduced to an average optical density of 0.30 ± 0.06, reflecting impaired cell proliferation. Following treatment, cytokeratin expression in the UCMSCs-EXOs group significantly increased, with the average optical density reaching 0.42 ± 0.06. This level was statistically significantly higher than that of the model group (P < 0.01), indicating that UCMSCs-EXOs effectively promote the proliferation of endogenous epithelial cells. The UC-MSCs–treated group also showed partial recovery (0.40 ± 0.08). These findings suggest a cellular mechanism whereby UCMSCs-EXOs facilitate functional endometrial regeneration. DISCUSSION IUA, characterized by recurrent endometrial fibrosis, impair endometrial receptivity and can lead to pregnancy failure, severely compromising reproductive outcomes. Conventional treatments exhibit limited efficacy in promoting functional endometrial regeneration, often resulting in developmental asynchrony between the repaired endometrium and the embryo, thereby failing to effectively restore reproductive function. Therefore, the development of novel therapies that can effectively promote both structural and functional regeneration of the endometrium is a critical and unmet need in reproductive medicine. In recent years, MSCs have been widely investigated for use in cell therapy owing to their potent self-renewal, multipotent differentiation, and immunomodulatory capabilities [26] . In particular, umbilical cord-derived MSCs (UC-MSCs) have emerged as an ideal cellular source for treating IUA due to their unique advantages, including abundant availability, a lack of ethical concerns, and low immunogenicity[ 27 , 28 ]. In particular, UC-MSCs have emerged as an ideal cellular source for treating IUA due to their unique advantages, including abundant availability, a lack of ethical concerns, and low immunogenicity. Furthermore, recent evidence indicates that the therapeutic effects of MSCs are primarily mediated by paracrine mechanisms, particularly via secreted exosomes. These exosomes function as intercellular messengers that promote cell proliferation and angiogenesis and attenuate fibrosis by modulating gene expression in recipient cells[ 29 – 31 ]. On this basis, we hypothesize that UCMSCs-EXOs constitute a safer, more efficient cell-free therapeutic strategy with strong potential for clinical translation in IUA. The development of IUA is closely linked to intrauterine surgical procedures[ 32 ]. Surgical injury that extends to the basal layer or myometrium can induce local ischemia and hypoxia, thereby impairing epithelial and stromal proliferation and delaying endometrial repair [12] . Current animal models of IUA primarily employ physical, chemical, or combined injury methods; yet their fidelity to human disease remains limited. In this study, we established a rat IUA model through mechanical injury. This model demonstrates high stability and reproducibility, supporting investigations of IUA pathogenesis and providing a platform to examine the effects of UCMSCs-EXOs on injured endometrium. Using this stable rat model, we systematically evaluated the therapeutic efficacy of UCMSCs-EXOs. Our results indicate that UCMSCs-EXOs promote repair of the damaged endometrium through multiple, potentially synergistic mechanisms. At the structural level, UCMSCs-EXOs increased endometrial thickness and promoted glandular regeneration. Endometrial thickness is a key clinical indicator of ER and correlates positively with pregnancy rates[ 33 , 34 ]. An excessively thin endometrium is often associated with a reduced likelihood of embryo implantation. In this study, both endometrial thickness and glandular number in the UCMSCs-EXOs-treated group recovered to levels comparable to those of the control group. This structural restoration provides the physical foundation and nutritional support essential for subsequent embryo implantation and development. Mechanistically, UCMSCs-EXOs effectively inhibited endometrial fibrosis. Fibrosis is a principal driver of endometrial dysfunction in IUA, often precipitated by inflammation, hypoxia, and epithelial injury [35] . Consistent with this mechanism, Masson staining revealed severe collagen deposition in the model group, whereas treatment with UCMSCs-EXOs significantly reduced the severity of fibrosis. This finding demonstrates the potent anti-fibrotic effect of UCMSCs-EXOs in reversing key pathological changes associated with IUA. At the cellular level, UCMSCs-EXOs enhanced the proliferative capacity of endometrial epithelial cells. As a key component of the epithelial cytoskeleton, cytokeratin expression reflects epithelial integrity and proliferative activity[ 36 ]. The significant upregulation of cytokeratin expression following UCMSCs-EXO treatment provides a cellular basis for the observed increases in endometrial thickness and glandular regeneration, suggesting that UCMSCs-EXOs facilitate repair by promoting the proliferation of endogenous epithelial cells. CONCLISION In conclusion, this study provides robust experimental evidence for the therapeutic potential of UCMSCs-EXOs in repairing the damaged endometrium, establishing them as a promising novel strategy for treating IUA. Specifically, UCMSCs-EXOs were shown to restore endometrial structure, inhibit the core pathological process of fibrosis, and promote epithelial cell proliferation. Notably, as a cell-free therapy, UCMSCs-EXOs demonstrate efficacy comparable to, if not superior to, that of UC-MSCs while avoiding the potential risks of cell transplantation, such as immune rejection and tumorigenicity. This cell-free approach offers significant advantages for standardized production, storage, and clinical application. Future studies should focus on elucidating the key bioactive molecules (e.g., miRNAs, proteins) within UCMSCs-EXOs responsible for these therapeutic effects to provide more precise guidance for clinical translation. Ultimately, such research could lead to improved reproductive outcomes for patients with IUA. Abbreviations IUA Intrauterine adhesion MSCs Mesenchymal stem cells UC-MSCs Umbilical cord mesenchymal stem cells UCMSCs-EXOs Umbilical cord mesenchymal stem cells drived exosomes H&E Hematoxylin&Eosin IVF-ET In Vitro Fertilization-Embryo Transfer Declarations Funding: This work was supported by Shandong Provincial Science (Grant numbers：No.ZR2021MH046). Competing Interests: The authors have no relevant financial or non-financial interests to disclose. Author Contributions: M.L,W.L,S.G and H.W designed and planned the study. W.L,S.G, H.W and X.G performed the experiments. H.W collected the data and analyzed the data.W.L was a major contributor in writing the manuscript. M.L amended the manuscript. All authors read and approved the final manuscript. Ethical approval: This study was performed in line with the principles of the Declaration of Helsinki.The Ethics Committee of Yuhuangding Hospital, Affiliated to Qingdao University approved the experimental procedures(Approval No. 2024-544). All experiments were conducted following national and institutional guidelines for the care and use of laboratory animals. Data availability: The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. Consent to participate: Not applicable. Consent for publication: Not applicable. References Takai IU, Kwayabura AS, Ugwa EA et al (2015) A 10-year review of the clinical presentation and treatment outcome of asherman's syndrome at a center with limited resources. 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Nat Rev Drug Discov 14:693-720. https://doi.org/10.1038/nrd4592 Zhang Q, Fu L, Liang Y et al (2018) Exosomes originating from MSCs stimulated with TGF-beta and IFN-gamma promote treg differentiation. J Cell Physiol 233:6832-6840. https://doi.org/10.1002/jcp.26436 Additional Declarations No competing interests reported. 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-7801878","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":533456690,"identity":"603561e3-7204-4432-ae68-ee3be774f999","order_by":0,"name":"Wencong Li","email":"","orcid":"","institution":"School of Medical Imaging,Binzhou Medical University","correspondingAuthor":false,"prefix":"","firstName":"Wencong","middleName":"","lastName":"Li","suffix":""},{"id":533456691,"identity":"825538b5-787d-46ed-8229-145dc5eba0b7","order_by":1,"name":"Hong Wei","email":"","orcid":"","institution":"School of Medical Imaging,Binzhou Medical University","correspondingAuthor":false,"prefix":"","firstName":"Hong","middleName":"","lastName":"Wei","suffix":""},{"id":533456692,"identity":"b7f49e3e-ea14-4bf2-98a3-192f5ca4dd36","order_by":2,"name":"Xiaohua Ge","email":"","orcid":"","institution":"School of Medical Imaging,Binzhou Medical University","correspondingAuthor":false,"prefix":"","firstName":"Xiaohua","middleName":"","lastName":"Ge","suffix":""},{"id":533456693,"identity":"ba083617-f035-4d9f-9147-d21126311817","order_by":3,"name":"Siqi Guo","email":"","orcid":"","institution":"Department of ultrasound,Yantai Yuhuangding Hospital,Affiliated Hospital of Qingdao Medical University","correspondingAuthor":false,"prefix":"","firstName":"Siqi","middleName":"","lastName":"Guo","suffix":""},{"id":533456695,"identity":"5c85a82f-30af-4650-af31-55299c459ccd","order_by":4,"name":"Meijuan 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20:02:27","extension":"xml","order_by":17,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":94100,"visible":true,"origin":"","legend":"","description":"","filename":"06e4ccea5e394013a9f3e27807820d2b1structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7801878/v1/37f1dbd22b2860b8ae8cc81c.xml"},{"id":94227629,"identity":"2a6a0347-b79e-41e6-8f74-4b9447e0c56e","added_by":"auto","created_at":"2025-10-23 20:18:27","extension":"html","order_by":18,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":104605,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7801878/v1/faf04bc73aedb243d0dc4d88.html"},{"id":94227051,"identity":"c452995b-251f-48ad-88df-19a0207b49d2","added_by":"auto","created_at":"2025-10-23 20:02:26","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":407096,"visible":true,"origin":"","legend":"\u003cp\u003eIsolation, culture, and characterization of UC-MSCs. (a) Representative images of primary UC-MSCs after 7 days of culture. (b) Morphology of the cultured UC-MSCs. (c) Flow cytometric analysis. Scale bar: 50 μm.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7801878/v1/2ea18e27d57873ee8187e2fc.png"},{"id":94227627,"identity":"810cf941-f5ee-4c75-90a8-1811d36128e7","added_by":"auto","created_at":"2025-10-23 20:18:26","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":410231,"visible":true,"origin":"","legend":"\u003cp\u003eMultilineage differentiation potential of UC-MSCs (a) Osteogenic differentiation assessed by Alizarin Red S staining of calcium nodules (red). (b) Adipogenic differentiation shown by Oil Red O staining of lipid droplets (red). (c) Chondrogenic differentiation indicated by Alcian Blue staining of acidic mucopolysaccharides (blue-green). Scale bar: 50 μm.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7801878/v1/2f13ec647fcc4febdfe4fa51.png"},{"id":94227052,"identity":"f2981aa7-b0ed-45c9-94b3-13e0b3f0bae5","added_by":"auto","created_at":"2025-10-23 20:02:26","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":175750,"visible":true,"origin":"","legend":"\u003cp\u003eCharacterization of UCMSCs-EXOs. (a) Morphology of UCMSCs-EXOs observed by transmission electron microscopy (TEM). (b) Size distribution of UCMSCs-EXOs measured by dynamic light scattering (DLS). (c) Detection of the exosomal surface marker proteins CD63, TSG101, and CD9 by Western Blot analysis. Scale bar: 500 nm. Black arrows:the cup-shaped morphology of exosomes.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7801878/v1/439c33aa2870851cf076e9a1.png"},{"id":94227383,"identity":"0c3edcb6-33a3-4357-ae01-74c6bfb0e4e6","added_by":"auto","created_at":"2025-10-23 20:10:26","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1052547,"visible":true,"origin":"","legend":"\u003cp\u003eHistological evaluation of uterine morphology and endometrial regeneration. Representative H\u0026amp;E-stained sections of uterine tissues from the (a) Control, (b) Model, (c) UC-MSCs treatment, and (d) UCMSCs-EXOs treatment groups. Insets show higher-magnification views of the boxed areas. Scale bar: 50 μm.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7801878/v1/19ea26db77947df506c4aa5b.png"},{"id":94227064,"identity":"857b4d25-d055-4596-b1d6-1278e767c753","added_by":"auto","created_at":"2025-10-23 20:02:27","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1062426,"visible":true,"origin":"","legend":"\u003cp\u003eAssessment of endometrial fibrosis. Representative Masson's trichrome-stained sections (collagen deposition shown in blue) from the (a) Control, (b) Model, (c) UC-MSCs treatment, and (d) UCMSCs-EXOs treatment groups. Insets show higher-magnification views. Scale bar: 50 μm.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-7801878/v1/c95bd580b1c45f4227c5045d.png"},{"id":94227068,"identity":"062095e6-4ca4-4ded-a5e4-b7c0a569437d","added_by":"auto","created_at":"2025-10-23 20:02:27","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1041842,"visible":true,"origin":"","legend":"\u003cp\u003eAnalysis of endometrial epithelial cell proliferation.Immunohistochemical staining for Cytokeratin in endometrial tissues from the (a) Control, (b) Model, (c) UC-MSCs treatment, and (d) UCMSCs-EXOs treatment groups. Insets show higher-magnification views. Scale bar: 50 μm.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-7801878/v1/fba4f489de2e3b5bb2ae09f6.png"},{"id":94227056,"identity":"a7769cf0-cfa7-4a53-8918-619a33dcf165","added_by":"auto","created_at":"2025-10-23 20:02:26","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":116821,"visible":true,"origin":"","legend":"\u003cp\u003eQuantitative analysis of the therapeutic effects of UCMSCs-EXOs and UC-MSCs on endometrial repair.(a) Measurement of endometrial thickness.(b) Count of endometrial glands.(c) Quantification of fibrosis area ratio.(d) Analysis of Cytokeratin expression.(***P \u0026lt; 0.001, **P \u0026lt; 0.01, *P \u0026lt; 0.05)\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-7801878/v1/4557da933fc78fecbaca6332.png"},{"id":98434050,"identity":"c0339f38-be38-437a-b18e-de554ee4d838","added_by":"auto","created_at":"2025-12-17 16:51:27","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5234592,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7801878/v1/d4fb5482-4aee-4267-8956-92efa4326de2.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Exosomes Derived from Umbilical Cord Mesenchymal Stem Cells： Mediated Restoration of Endometrial Receptivity and Their Potential to Improve IVF–ET Outcomes","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eIntrauterine adhesions (IUA) is a fibrotic condition caused by injury to the basal layer of the endometrium. The underlying pathology involves the disruption of the normal, scar-free cyclical repair mechanism of the endometrium, which is then replaced by the abnormal proliferation of fibrous connective tissue, ultimately leading to partial or complete occlusion of the uterine cavity[\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. As a leading cause of secondary infertility, IUA severely compromises endometrial receptivity, thereby impeding embryo implantation[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Clinically, intrauterine procedures or infections are the principal precipitants of IUA[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e], accounting for approximately 15%\u0026ndash;20% of cases, whereas postpartum curettage confers an even higher risk of 21%\u0026ndash;40%[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Although hysteroscopic adhesiolysis remains the standard treatment, its limitations are increasingly evident: the procedure restores uterine cavity morphology but does not reverse tissue fibrosis. As a result, postoperative recurrence rates in moderate-to-severe cases can be as high as 62.5%[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], and the subsequent regeneration and functional recovery of the endometrium remain significant challenges.\u003c/p\u003e\u003cp\u003eEndometrial receptivity (ER) is the capacity of the endometrium to support embryo apposition, adhesion, and invasion, thereby enabling implantation[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Current theories propose that intrauterine procedures injure or deplete endometrial stem/progenitor cells in the basal layer, constituting a key initiating event in IUA\u003csup\u003e[9, 10]\u003c/sup\u003e. Loss of these cells disrupts endometrial homeostasis and epithelial regeneration, driving post-injury repair toward pathological fibrosis rather than physiologic regeneration[\u003cspan additionalcitationids=\"CR12\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. This pathological process directly leads to a significant decline in endometrial receptivity. In vitro fertilization\u0026ndash;embryo transfer (IVF\u0026ndash;ET) is among the most established assisted reproductive technologies, yet implantation failure still occurs in 32%\u0026ndash;51% of transfer cycles[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].Approximately two-thirds of these failures are attributable to suboptimal endometrial receptivity[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].Thus, addressing the therapeutic bottleneck in intrauterine adhesions requires moving beyond simple mechanical separation toward strategies that promote high-quality, functional endometrial regeneration to restore receptivity.\u003c/p\u003e\u003cp\u003eRegenerative medicine strategies utilizing mesenchymal stem cells (MSCs) have emerged as a promising approach to address this challenge. MSCs exhibit substantial potential in tissue repair owing to their multipotency, immunomodulatory properties, and robust paracrine activity[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. However, the clinical translation of direct MSCs transplantation is hampered by several limitations, including low cell survival rates, potential immune rejection, and ethical concerns\u003csup\u003e[18]\u003c/sup\u003e. Evidence suggests that the therapeutic effects of MSCs are primarily mediated by their secreted extracellular vesicles, particularly exosomes[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. As key mediators of intercellular communication, exosomes are enriched with bioactive molecules, including proteins, RNA, and miRNAs, which facilitates the transfer of parental cell functions to recipient cells[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Among these, exosomes derived from human umbilical cord mesenchymal stem cells (UCMSCs-EXOs) are of particular interest due to their abundant availability, low immunogenicity, and potent capabilities for tissue regeneration and wound healing, as demonstrated in multiple studies[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eTherefore, this study systematically evaluates the effects of UCMSCs-EXOs on endometrial repair and receptivity in a rat model of IUA and explores their potential to improve IVF-ET outcomes. These findings may inform a safer, more effective approach to the clinical management of IUA and provide theoretical and experimental support for applying regenerative medicine in gynecology.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eExtraction and Identification of Umbilical Cord Mesenchymal Stem Cells(UC-MSCs):\u003c/h2\u003e\u003cp\u003e This study was approved by the Ethics Committee of Yuhuangding Hospital, Affiliated to Qingdao University (Approval No. 2024\u0026thinsp;\u0026minus;\u0026thinsp;544). Umbilical cord tissues were collected from healthy, full-term infants following vaginal delivery, after obtaining written informed consent from all parental donors. Under aseptic conditions, Wharton\u0026rsquo;s jelly was dissected and minced into tissue fragments of approximately 1 mm\u0026sup3;. Fragments were plated in culture dishes and maintained in UC-MSC growth medium(Procell,Wuhan China) at 37\u0026deg;C in a humidified 5% CO₂ incubator. UC-MSCs between passages 4 and 6 were utilized for all subsequent experiments.\u003c/p\u003e\u003cp\u003eFor phenotypic characterization, cells were prepared as a single-cell suspension at a concentration of 1.0 \u0026times; 10⁶ cells/mL, and 100 \u0026micro;L aliquots were distributed into nine flow cytometry tubes. Add CD90-FITC, CD44-PE, CD105-PerCP-Cy5.5, and CD73-APC from the BD Human MSC Analysis Kit (BD Biosciences, USA) to Tubes 1\u0026ndash;4, respectively; Tube 5 serves as a blank control; Tube 6 contains an isotype-control cocktail (mIgG1-FITC, mIgG1-PerCP-Cy5.5, mIgG1-APC, mIgG1-PE, mIgG2a-PE); Tube 7 contains a positive-marker cocktail (CD90-FITC, CD105-PerCP-Cy5.5, CD73-APC) together with a negative-marker cocktail (CD34-PE, CD11b-PE, CD19-PE, CD45-PE, HLA-DR-PE). Acquire data by flow cytometry and analyze cell phenotypes in FlowJo, following the manufacturer\u0026rsquo;s instructions for the BD kit (e.g., staining, compensation, and gating). All antibodies were used as provided in the BD Human MSC Analysis Kit.\u003c/p\u003e\u003cp\u003eThe multipotent differentiation potential of the UC-MSCs was evaluated by seeding the cells into 6 cm culture dishes. Upon reaching 90%\u0026ndash;100% confluence, the medium is replaced with adipogenic, osteogenic, and chondrogenic induction media (HyCyte,Suzhou China). The induction medium was refreshed every three days, interspersed with a 24-hour incubation in maintenance medium. This differentiation protocol was continued for a total of 21 days. Following induction, cells are fixed in 4% paraformaldehyde and stained with Oil Red O, Alizarin Red S, and Alcian Blue, respectively (all from HyCyte). Differentiation is evaluated by light microscopy.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eExosome Extraction and Characterization:\u003c/h3\u003e\n\u003cp\u003eFor the isolation of exosomes, the culture medium was replaced with serum-free medium(Procell,Wuhan China) once the UC-MSCs reached approximately 80% confluence. After 48\u0026ndash;72 hours of incubation, the conditioned medium was collected for exosome purification. Centrifuge the medium sequentially at 2,000 \u0026times; g for 30 min and 10,000 \u0026times; g for 45 min at 4\u0026deg;C to remove cells and debris. The resulting supernatant was passed through a 0.22 \u0026micro;m filter and then subjected to ultracentrifugation at 100,000 \u0026times; g for 70 minutes at 4\u0026deg;C to pellet the exosomes. Resuspend the pellet in phosphate-buffered saline (PBS) ( Sparkjade,Shandong China) to yield the final preparation of UCMSCs-EXOs. Protein concentration is determined by a bicinchoninic acid(BCA)( Sparkjade,Shandong China) assay, and samples are stored at \u0026minus;\u0026thinsp;80\u0026deg;C.\u003c/p\u003e\u003cp\u003eDeposit 20 \u0026micro;L of the exosome suspension onto a copper grid, incubate at room temperature for 10 min, and wick off excess liquid with filter paper. Negative-stain with 2% phosphotungstic acid for 10 min, blot, and air-dry at 37\u0026deg;C. The morphology of the exosomes was then examined using a transmission electron microscope.\u003c/p\u003e\u003cp\u003eSeparately, dilute exosomes 1:1000 in PBS to a final volume of 1 mL for dynamic light scattering (DLS) on a Zetasizer Nano ZS to determine particle-size distribution.\u003c/p\u003e\u003cp\u003eExtract exosomal proteins in radioimmunoprecipitation assay ( Sparkjade,Shandong China) buffer supplemented with protease and phosphatase inhibitors(Beyotime,China), then quantify by a BCA assay. Proteins are separated on 10% SDS\u0026ndash;PAGE and transferred to PVDF membranes. Membranes are blocked with 5% nonfat dry milk for 1 h, then incubated with anti-CD9(Proteintech,Wuhan China), anti-CD81(Proteintech,Wuhan China), and anti-TSG101(Proteintech,Wuhan China) primary antibodies (overnight at 4\u0026deg;C) followed by HRP-conjugated secondary antibodies(Sparkjade,Shandong China). Signals are visualized by enhanced chemiluminescence.\u003c/p\u003e\n\u003ch3\u003eAnimal Model Establishment and Treatment:\u003c/h3\u003e\n\u003cp\u003eFemale, specific pathogen-free grade Sprague-Dawley rats were procured from Jinan Pengyue Laboratory Animal Co., Ltd. and housed at 20\u0026ndash;26\u0026deg;C, 40\u0026ndash;70% relative humidity, under a 12-h light\u0026ndash;dark cycle with ad libitum access to food and water. All animal experiments were conducted in strict accordance with the NIH Guidelines for the Care and Use of Laboratory Animals.\u003c/p\u003e\u003cp\u003eFollowing a 1- to 2-week acclimatization period, the estrous cycles of the rats were monitored daily via vaginal smear analysis. Rats in estrus phase were anesthetized with isoflurane delivered via a small-animal inhalation anesthesia machine (carrier gas oxygen), and the uterus was exposed via laparotomy. IUA was induced by mechanical injury; the endometrial lining of both uterine horns was gently scraped with a surgical curette to create a standardized injury.\u003c/p\u003e\u003cp\u003eA total of twenty-four rats exhibiting regular estrous cycles were randomly allocated into four experimental groups (n\u0026thinsp;=\u0026thinsp;6 ): control (no intervention), model (injury without treatment), UC-MSCs treatment, and UCMSCs-EXOs treatment. One week post-surgery, animals in the treatment groups received a single intrauterine injection of either 2 \u0026times; 10⁷ UC-MSCs or 150\u0026ndash;200 \u0026micro;g of UC-MSC-EXOs, respectively, each suspended in a 200 \u0026micro;L volume. Four to five estrous cycles following treatment, the animals were euthanized, and uterine tissues were harvested for subsequent analysis.\u003c/p\u003e\n\u003ch3\u003eHistology and Immunohistochemistry:\u003c/h3\u003e\n\u003cp\u003eUterine tissues are fixed in 4% paraformaldehyde, dehydrated through graded solvents, and embedded in paraffin. Sections 4-\u0026micro;m thick are cut and stained with hematoxylin and eosin (H\u0026amp;E) and Masson\u0026rsquo;s trichrome according to the manufacturer\u0026rsquo;s protocols,Four high-power fields were randomly selected from each section for analysis. Endometrial thickness, glandular count, and the fibrosis area ratio (fibrous area/total area of stroma and glands) were measured using ImageJ software.\u003c/p\u003e\u003cp\u003eFor immunohistochemistry, sections are deparaffinized, rehydrated, and incubated overnight at 4\u0026deg;C with primary antibodies (Solarbio,Beijing China). The following day, the sections were incubated with the secondary antibody (Solarbio,Beijing China) at room temperature for 1 hour. Signals are developed with DAB and counterstained with hematoxylin. Images are acquired on a light microscope and analyzed in ImageJ.\u003c/p\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003eStatistical Analysis:\u003c/h2\u003e\u003cp\u003eData are presented as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD) from independently conducted experiments. Statistical analysis was performed using one-way ANOVA, with t-tests employed for comparisons between two groups. Statistical significance was assessed using GraphPad Prism 10, with p-values considered significant at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, and p\u0026thinsp;\u0026lt;\u0026thinsp;0.001.\u003c/p\u003e\u003c/div\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003eIsolation, Culture, and Characterization of UC-MSCs:\u003c/h2\u003e\u003cp\u003eUC-MSCs were isolated from Wharton's jelly and expanded in culture. Primary cultures showed adherent, spindle-shaped cells arranged in parallel or whirl-like patterns (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea,\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). Flow cytometry analysis revealed that the cultured cells were positive for the mesenchymal stem cell surface markers CD44, CD73, CD90, and CD105, but negative for the hematopoietic and endothelial markers CD9, CD11b, CD34, and HLA-DR (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec). This immunophenotype is consistent with the minimal criteria for defining MSCs as established by the International Society for Cellular Therapy[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Furthermore, the multipotent capacity of the isolated cells was confirmed by their ability to differentiate into osteogenic, adipogenic, and chondrogenic lineages. Following induction, the formation of calcium deposits, lipid droplets, and acidic mucopolysaccharides was verified by Alizarin Red, Oil Red O, and Alcian Blue staining, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Collectively, the morphology, immunophenotype, and tri-lineage differentiation support successful isolation of high-purity UC-MSCs.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eCharacterization of UCMSCs-EXOs:\u003c/h3\u003e\n\u003cp\u003eUCMSCs-EXOs were isolated from conditioned medium by ultracentrifugation. Transmission electron microscopy (TEM) imaging revealed that the isolated vesicles presented a characteristic cup-shaped or \"saucer-like\" morphology (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). Furthermore, dynamic light scattering indicated a particle-size distribution predominantly within 30\u0026ndash;200 nm (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb), consistent with exosomes. Finally, Western blot analysis confirmed the presence of the canonical exosome marker proteins CD9, CD63, and TSG101 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec). Collectively, these data on vesicle morphology, size distribution, and protein expression confirmed the successful isolation of UCMSCs-EXOs suitable for downstream applications.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eRepair Effects of UCMSC-Exos on Damaged Endometrium:\u003c/h2\u003e\u003cp\u003eUCMSC-EXOs Promote the Structural and Functional Repair of Injured Endometrium:\u003c/p\u003e\u003cp\u003eTo evaluate therapeutic efficacy, we assessed uterine morphology across groups by H\u0026amp;E staining. Control group uterine displayed a normal endometrial architecture with a thick luminal epithelium and abundant glands (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). In contrast, the model group exhibited severe endometrial atrophy, characterized by a thinned endometrium, discontinuous luminal epithelium, and a marked reduction in glandular number (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). Following treatment, the UCMSCs-EXOs\u0026ndash;treated group showed marked restoration of endometrial structure(Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed). Endometrial thickness (0.40\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02 mm) and gland count (28.18\u0026thinsp;\u0026plusmn;\u0026thinsp;3.18) approached control levels (0.44\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05 mm; 31.05\u0026thinsp;\u0026plusmn;\u0026thinsp;1.62) and were significantly higher than the model group (0.10\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04 mm,P\u0026lt;0.001; 16.56\u0026thinsp;\u0026plusmn;\u0026thinsp;2.32,P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). As a positive control, the UC-MSCs\u0026ndash;treated group also improved (thickness 0.36\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06 mm; glands 27.46\u0026thinsp;\u0026plusmn;\u0026thinsp;1.95). These data indicate that UCMSCs-EXOs promote structural regeneration of the injured endometrium.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eUCMSCs-EXOs Inhibit Progression of Endometrial Fibrosis:\u003c/p\u003e\u003cp\u003eTo evaluate endometrial fibrosis, a key pathological hallmark of intrauterine adhesions\u003csup\u003e[25]\u003c/sup\u003e, tissue sections were subjected to Masson's trichrome staining. In control animals, Masson's staining revealed minimal collagen deposition (stained blue), which was appropriately restricted to the endometrial stroma (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). By contrast, the model group displayed dense collagen replacing the endometrial layer, and the collagen-deposition ratio increased from 12.68\u0026thinsp;\u0026plusmn;\u0026thinsp;3.09% (control) to 30.62\u0026thinsp;\u0026plusmn;\u0026thinsp;4.44% (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Treatment with UC-MSC-Exos markedly reduced this fibrotic response, decreasing the collagen area ratio to 13.49\u0026thinsp;\u0026plusmn;\u0026thinsp;1.15%, a level comparable to that of the control group. A similar anti-fibrotic effect was observed in the UC-MSCs treatment group (15.64\u0026thinsp;\u0026plusmn;\u0026thinsp;1.98%). Collectively, these findings demonstrate that UCMSCs-EXOs suppress excessive collagen deposition, thereby mitigating endometrial fibrosis during uterine repair.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eCharacteristics of the study group(Mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD)\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"6\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003egroup\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003en\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eH\u0026amp;E Stained Endometrial Thickness (mm)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eNumber of Glands in H\u0026amp;E Staining (count)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eMasson Stained Fibrosis Area Ratio (%)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eAverage Optical Density of CK Staining\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNormal group\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e0.44\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e31.05\u0026thinsp;\u0026plusmn;\u0026thinsp;1.62\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e12.68\u0026thinsp;\u0026plusmn;\u0026thinsp;3.09\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e\u003cp\u003e0.49\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eModel group\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e0.10\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e16.56\u0026thinsp;\u0026plusmn;\u0026thinsp;2.32\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e30.62\u0026thinsp;\u0026plusmn;\u0026thinsp;4.44\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e\u003cp\u003e0.30\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eUC-MSCs treatment group\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e0.36\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e27.46\u0026thinsp;\u0026plusmn;\u0026thinsp;1.95\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e15.64\u0026thinsp;\u0026plusmn;\u0026thinsp;1.98\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e\u003cp\u003e0.40\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eUCMSCs- EXOs treatment group\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e0.40\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e28.18\u0026thinsp;\u0026plusmn;\u0026thinsp;3.18\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e13.49\u0026thinsp;\u0026plusmn;\u0026thinsp;1.15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e\u003cp\u003e0.42\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eUCMSCs-EXOs Promote Proliferation of Endometrial Epithelial Cells:\u003c/p\u003e\u003cp\u003eTo investigate the cellular basis of structural recovery, we assessed cytokeratin expression by immunohistochemistry as an epithelial cell proliferation\u0026ndash;associated marker. Immunohistochemical analysis revealed strong cytokeratin expression in the endometrium of the control group, indicating active epithelial cell proliferation (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea). In contrast, expression levels in the model group were significantly reduced to an average optical density of 0.30\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06, reflecting impaired cell proliferation. Following treatment, cytokeratin expression in the UCMSCs-EXOs group significantly increased, with the average optical density reaching 0.42\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06. This level was statistically significantly higher than that of the model group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01), indicating that UCMSCs-EXOs effectively promote the proliferation of endogenous epithelial cells. The UC-MSCs\u0026ndash;treated group also showed partial recovery (0.40\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08). These findings suggest a cellular mechanism whereby UCMSCs-EXOs facilitate functional endometrial regeneration.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eIUA, characterized by recurrent endometrial fibrosis, impair endometrial receptivity and can lead to pregnancy failure, severely compromising reproductive outcomes. Conventional treatments exhibit limited efficacy in promoting functional endometrial regeneration, often resulting in developmental asynchrony between the repaired endometrium and the embryo, thereby failing to effectively restore reproductive function. Therefore, the development of novel therapies that can effectively promote both structural and functional regeneration of the endometrium is a critical and unmet need in reproductive medicine.\u003c/p\u003e\u003cp\u003eIn recent years, MSCs have been widely investigated for use in cell therapy owing to their potent self-renewal, multipotent differentiation, and immunomodulatory capabilities\u003csup\u003e[26]\u003c/sup\u003e. In particular, umbilical cord-derived MSCs (UC-MSCs) have emerged as an ideal cellular source for treating IUA due to their unique advantages, including abundant availability, a lack of ethical concerns, and low immunogenicity[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. In particular, UC-MSCs have emerged as an ideal cellular source for treating IUA due to their unique advantages, including abundant availability, a lack of ethical concerns, and low immunogenicity. Furthermore, recent evidence indicates that the therapeutic effects of MSCs are primarily mediated by paracrine mechanisms, particularly via secreted exosomes. These exosomes function as intercellular messengers that promote cell proliferation and angiogenesis and attenuate fibrosis by modulating gene expression in recipient cells[\u003cspan additionalcitationids=\"CR30\" citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. On this basis, we hypothesize that UCMSCs-EXOs constitute a safer, more efficient cell-free therapeutic strategy with strong potential for clinical translation in IUA.\u003c/p\u003e\u003cp\u003eThe development of IUA is closely linked to intrauterine surgical procedures[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Surgical injury that extends to the basal layer or myometrium can induce local ischemia and hypoxia, thereby impairing epithelial and stromal proliferation and delaying endometrial repair\u003csup\u003e[12]\u003c/sup\u003e. Current animal models of IUA primarily employ physical, chemical, or combined injury methods; yet their fidelity to human disease remains limited. In this study, we established a rat IUA model through mechanical injury. This model demonstrates high stability and reproducibility, supporting investigations of IUA pathogenesis and providing a platform to examine the effects of UCMSCs-EXOs on injured endometrium.\u003c/p\u003e\u003cp\u003eUsing this stable rat model, we systematically evaluated the therapeutic efficacy of UCMSCs-EXOs. Our results indicate that UCMSCs-EXOs promote repair of the damaged endometrium through multiple, potentially synergistic mechanisms.\u003c/p\u003e\u003cp\u003eAt the structural level, UCMSCs-EXOs increased endometrial thickness and promoted glandular regeneration. Endometrial thickness is a key clinical indicator of ER and correlates positively with pregnancy rates[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. An excessively thin endometrium is often associated with a reduced likelihood of embryo implantation. In this study, both endometrial thickness and glandular number in the UCMSCs-EXOs-treated group recovered to levels comparable to those of the control group. This structural restoration provides the physical foundation and nutritional support essential for subsequent embryo implantation and development.\u003c/p\u003e\u003cp\u003eMechanistically, UCMSCs-EXOs effectively inhibited endometrial fibrosis. Fibrosis is a principal driver of endometrial dysfunction in IUA, often precipitated by inflammation, hypoxia, and epithelial injury\u003csup\u003e[35]\u003c/sup\u003e. Consistent with this mechanism, Masson staining revealed severe collagen deposition in the model group, whereas treatment with UCMSCs-EXOs significantly reduced the severity of fibrosis. This finding demonstrates the potent anti-fibrotic effect of UCMSCs-EXOs in reversing key pathological changes associated with IUA.\u003c/p\u003e\u003cp\u003eAt the cellular level, UCMSCs-EXOs enhanced the proliferative capacity of endometrial epithelial cells. As a key component of the epithelial cytoskeleton, cytokeratin expression reflects epithelial integrity and proliferative activity[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. The significant upregulation of cytokeratin expression following UCMSCs-EXO treatment provides a cellular basis for the observed increases in endometrial thickness and glandular regeneration, suggesting that UCMSCs-EXOs facilitate repair by promoting the proliferation of endogenous epithelial cells.\u003c/p\u003e"},{"header":"CONCLISION","content":"\u003cp\u003eIn conclusion, this study provides robust experimental evidence for the therapeutic potential of UCMSCs-EXOs in repairing the damaged endometrium, establishing them as a promising novel strategy for treating IUA. Specifically, UCMSCs-EXOs were shown to restore endometrial structure, inhibit the core pathological process of fibrosis, and promote epithelial cell proliferation. Notably, as a cell-free therapy, UCMSCs-EXOs demonstrate efficacy comparable to, if not superior to, that of UC-MSCs while avoiding the potential risks of cell transplantation, such as immune rejection and tumorigenicity. This cell-free approach offers significant advantages for standardized production, storage, and clinical application. Future studies should focus on elucidating the key bioactive molecules (e.g., miRNAs, proteins) within UCMSCs-EXOs responsible for these therapeutic effects to provide more precise guidance for clinical translation. Ultimately, such research could lead to improved reproductive outcomes for patients with IUA.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 277px;\"\u003e\n \u003cp\u003eIUA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 277px;\"\u003e\n \u003cp\u003eIntrauterine adhesion\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 277px;\"\u003e\n \u003cp\u003eMSCs\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 277px;\"\u003e\n \u003cp\u003eMesenchymal stem cells\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 277px;\"\u003e\n \u003cp\u003eUC-MSCs\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 277px;\"\u003e\n \u003cp\u003eUmbilical cord mesenchymal stem cells\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 277px;\"\u003e\n \u003cp\u003eUCMSCs-EXOs\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 277px;\"\u003e\n \u003cp\u003eUmbilical cord mesenchymal stem cells drived exosomes\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 277px;\"\u003e\n \u003cp\u003eH\u0026amp;E\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 277px;\"\u003e\n \u003cp\u003eHematoxylin\u0026amp;Eosin\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 277px;\"\u003e\n \u003cp\u003eIVF-ET\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 277px;\"\u003e\n \u003cp\u003eIn Vitro Fertilization-Embryo Transfer\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by Shandong Provincial Science (Grant numbers：No.ZR2021MH046).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eM.L,W.L,S.G and H.W designed and planned the study. W.L,S.G, H.W and X.G performed the experiments. H.W collected the data and analyzed the data.W.L was a major contributor in writing the manuscript. M.L amended the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was performed in line with the principles of the Declaration of Helsinki.The Ethics Committee of Yuhuangding Hospital, Affiliated to Qingdao University approved the experimental procedures(Approval No. 2024-544). All experiments were conducted following national and institutional guidelines for the care and use of laboratory animals.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to participate:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eTakai IU, Kwayabura AS, Ugwa EA et al (2015) A 10-year review of the clinical presentation and treatment outcome of asherman\u0026apos;s syndrome at a center with limited resources. 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Front Endocrinol (Lausanne) 14:1146991. https://doi.org/10.3389/fendo.2023.1146991\u003c/li\u003e\n\u003cli\u003eAla M (2023) The beneficial effects of mesenchymal stem cells and their exosomes on myocardial infarction and critical considerations for enhancing their efficacy. Ageing Res Rev 89:101980. https://doi.org/10.1016/j.arr.2023.101980\u003c/li\u003e\n\u003cli\u003eLu Y, Huangfu S, Ma C et al (2024) Exosomes derived from umbilical cord mesenchymal stem cells promote healing of complex perianal fistulas in rats. Stem Cell Res Ther 15:414. https://doi.org/10.1186/s13287-024-04028-0\u003c/li\u003e\n\u003cli\u003eTeng L, Maqsood M, Zhu M et al (2022) Exosomes derived from human umbilical cord mesenchymal stem cells accelerate diabetic wound healing via promoting m2 macrophage polarization, angiogenesis, and collagen deposition. Int J Mol Sci 23. https://doi.org/10.3390/ijms231810421\u003c/li\u003e\n\u003cli\u003eDominici M, Le Blanc K, Mueller I et al (2006) Minimal criteria for defining multipotent mesenchymal stromal cells. The international society for cellular therapy position statement. Cytotherapy 8:315-317. https://doi.org/10.1080/14653240600855905\u003c/li\u003e\n\u003cli\u003eLeung RK, Lin Y, Liu Y (2021) Recent advances in understandings towards pathogenesis and treatment for intrauterine adhesion and disruptive insights from single-cell analysis. Reprod Sci 28:1812-1826. https://doi.org/10.1007/s43032-020-00343-y\u003c/li\u003e\n\u003cli\u003eChen L, Zhu S, Guo S et al (2023) Mechanisms and clinical application potential of mesenchymal stem cells-derived extracellular vesicles in periodontal regeneration. 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J Control Release 318:25-37. https://doi.org/10.1016/j.jconrel.2019.12.007\u003c/li\u003e\n\u003cli\u003eCraciunas L, Gallos I, Chu J et al (2019) Conventional and modern markers of endometrial receptivity: a systematic review and meta-analysis. Hum Reprod Update 25:202-223. https://doi.org/10.1093/humupd/dmy044\u003c/li\u003e\n\u003cli\u003eLv H, Li X, Du J et al (2020) Effect of endometrial thickness and embryo quality on live-birth rate of fresh IVF/ICSI cycles: a retrospective cohort study. Reprod Biol Endocrinol 18:89. https://doi.org/10.1186/s12958-020-00636-6\u003c/li\u003e\n\u003cli\u003eNanthakumar CB, Hatley RJD, Lemma S et al (2015) Dissecting fibrosis: therapeutic insights from the small-molecule toolbox. Nat Rev Drug Discov 14:693-720. https://doi.org/10.1038/nrd4592\u003c/li\u003e\n\u003cli\u003eZhang Q, Fu L, Liang Y et al (2018) Exosomes originating from MSCs stimulated with TGF-beta and IFN-gamma promote treg differentiation. J Cell Physiol 233:6832-6840. https://doi.org/10.1002/jcp.26436\u003c/li\u003e\n\u003c/ol\u003e"}],"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":"Intrauterine Adhesions, Exosomes, Mesenchymal Stem Cells, Endometrium, Embryo Implantation/physiology","lastPublishedDoi":"10.21203/rs.3.rs-7801878/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7801878/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIntrauterine adhesion (IUA), a fibrotic disorder characterized by endometrial basal layer damage, severely impairs endometrial receptivity and fertility. Conventional hysteroscopic adhesiolysis restores uterine morphology but fails to reverse fibrosis or promote functional regeneration. Human umbilical cord mesenchymal stem cell–derived exosomes (UCMSCs-EXOs) have emerged as a promising acellular therapeutic strategy due to their potent regenerative and anti-fibrotic properties. This study aimed to evaluate the therapeutic potential of UCMSCs-EXOs in restoring endometrial receptivity in a rat model of IUA.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods and Results:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eUC-MSCs were isolated from Wharton’s jelly, characterized by flow cytometry and trilineage differentiation, and their exosomes were purified via ultracentrifugation and confirmed by TEM, DLS, and Western blotting for CD9, CD63, and TSG101. A mechanical injury model of IUA was established in female Sprague–Dawley rats (n = 24), which were divided into control, model, UC-MSCs, and UCMSCs-EXOs groups. One week post-injury, intrauterine administration of UCMSCs-EXOs markedly enhanced endometrial regeneration. Compared with the model group, UCMSCs-EXOs treatment significantly increased endometrial thickness (0.40 ± 0.02 mm vs. 0.10 ± 0.04 mm), gland count (28.18 ± 3.18 vs. 16.56 ± 2.32), and reduced collagen deposition (13.49 ± 1.15% vs. 30.62 ± 4.44%). Immunohistochemistry further revealed upregulated cytokeratin expression, indicating enhanced epithelial proliferation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eUCMSCs-EXOs effectively restore endometrial structure and function by promoting epithelial proliferation and inhibiting fibrosis, demonstrating therapeutic efficacy comparable to their parent cells. As a cell-free alternative, UCMSCs-EXOs present a novel and promising strategy for IUA treatment, with the potential to improve IVF-ET outcomes.\u003c/p\u003e","manuscriptTitle":"Exosomes Derived from Umbilical Cord Mesenchymal Stem Cells： Mediated Restoration of Endometrial Receptivity and Their Potential to Improve IVF–ET Outcomes","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-23 20:02:22","doi":"10.21203/rs.3.rs-7801878/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":"9d76fc79-091e-4548-8426-ca1b994a16ab","owner":[],"postedDate":"October 23rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-12-15T11:25:03+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-23 20:02:22","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7801878","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7801878","identity":"rs-7801878","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}