Human embryonic stem cell-derived immunity-and-matrix-regulatory cells promote endometrial repair and fertility restoration in IUA rats

Abstract

Background

Intrauterine adhesion (IUA) refers to endometrial fibrosis resulting from endometrial injury and infection. In promoting the repair of the endometrium, mesenchymal stem cells therapy has shown great potential. However, adult-derived mesenchymal stem cells (MSCs) are associated with several challenges, including invasive manipulation, susceptibility to contamination, and low proliferative capacity. Immunity-and-matrix-regulatory cells (IMRCs) derived from human embryonic stem cells exhibit enhanced immunomodulatory and anti-fibrotic capabilities. Despite their success in treating lung injury and fibrosis, membranous nephropathy, and acute liver failure, their therapeutic potential in IUA remains undetermined.

Methods

TGF-β1-induced human endometrial stromal cells (HESCs) were utilized to construct the IUA cell model and were treated with IMRCs conditioned medium. Morphological changes in the cells were observed, and RT-qPCR and Western blot analyses were employed to detect the expression of relevant markers during the process of epithelial-mesenchymal transition (EMT) in vitro. The IUA rat model was established using the dual injury method and subsequently treated with intrauterine infusion of IMRCs. HE and Masson staining were used to assess endometrial damage, repair and the extent of fibrosis. Fertility assays were performed to compare the effectiveness of IMRCs and umbilical cord mesenchymal stem cells (UCMSCs) in improving endometrial function in IUA rats. Sequencing analysis of IMRCs-derived exosomes (Exos) was conducted to identify specific miRNAs and the pathways they target.

Results

TGF-β1 treatment induced HESCs to undergo fibrotic transformation and express fibrosis-related markers, while treatment with IMRCs conditioned medium inhibited TGF-β1-induced fibrosis. IUA rats were treated with intrauterine infusion of IMRCs. IMRCs facilitated the repair of damaged endometrium, restored the structure of the uterine cavity, and reduced collagen deposition. IMRCs reversed the process of endometrial EMT in rats with IUA, upregulated the expression of epithelial markers, and downregulated the expression of mesenchymal markers. IMRCs further exerted antifibrotic effects by reducing inflammatory responses. Fertility recovery in rats receiving intrauterine infusion of IMRCs was superior to that in rats receiving intrauterine infusion of UCMSCs. Specific miRNAs in Exos, including miR-27b-3p, miR-145-5p, and miR-16-5p, directly target Smad2, inhibit Smad2 phosphorylation, and modulate the TGF-β/Smad pathway.

Conclusions

Our study demonstrated that IMRCs inhibited TGF-β1-induced fibrosis in HESCs, suppressed the EMT process ex vivo, reduced the inflammatory response, and reversed endometrial damage and fibrosis in IUA rats. IMRCs exerted their effects through the paracrine pathway, with specific miRNAs in Exos downregulating the TGF-β/Smad signaling pathway to inhibit uterine endometrial fibrosis. IMRCs provide a new direction for the treatment of IUA.

Introduction

Intrauterine adhesion (IUA), also known as Asherman’s syndrome, is a condition characterized by partial or complete adhesion of the uterine cavity or cervical canal, resulting from damage to the basal layer of the endometrium and endometrial fibrosis. The widespread adoption of hysteroscopic techniques and the rising frequency of uterine related surgeries have led to a corresponding increase in the incidence of IUA. As the second most common cause of secondary infertility in women, IUA is a serious threat to women’s reproductive health [1]. Transcervical resection of adhesion (TCRA) remains the standard treatment for IUA [2, 3]. TCRA can partially restore uterine morphology, alleviate pelvic pain caused by impaired menstrual blood discharge, recover menstruation, and increase the postoperative pregnancy rates [4]. However, TCRA is less effective in patients with severe IUA, with recurrence rates as high as 62.5% and pregnancy rates ranging from 22.5–33.3% [5, 6]. TCRA combined with hormone therapy, hyaluronic acid gel, intrauterine balloon, amniotic membrane, biologic scaffold and IUD is currently the most common clinical treatment for IUA; however, its effectiveness in reversing endometrial fibrosis remains limited [7, 8]. Therefore, promoting endometrial regeneration, reducing recurrence rates, and improving reproductive outcomes are critical challenges for patients with IUA.

Mesenchymal stem cells (MSCs), characterized by self-renewal, multidirectional differentiation, and immunoregulatory capabilities, hold great promise in tissue repair and regenerating. They have been extensively studied in the context of neurodegenerative diseases, hematological disorders, cardiovascular disorders, diabetes mellitus, intrauterine adhesion, premature ovarian failure and other diseases [9,10,11,12]. MSCs therapy represents one of the most promising therapeutic approaches in regenerative medicine and has demonstrated considerable potential in the treatment of IUA. Current research on MSCs therapy for IUA predominantly focuses on MSCs derived from adult sources, including bone marrow mesenchymal stem cells (BMSCs), adipose-derived mesenchymal stem cells (ADMSCs), umbilical cord mesenchymal stem cells (UCMSCs), menstrual blood-derived mesenchymal stem cells (MenSCs), and human amniotic mesenchymal stromal cells (hAMSCs) [13,14,15,16,17]. MSCs from different sources may possess varying biofunctions, such as increasing the expression and sensitivity of estrogen and progesterone receptors to improve endometrial tolerance [18], modulating immunity responses to promote endometrial regeneration [19], increasing the expression of VEGF to stimulate angiogenesis and contribute to endometrial re-epithelialization [20], and secreting insulin-like growth factor and epidermal growth factor to regulate endometrial cell differentiation and proliferation [21].

Immunity-and-matrix-regulatory cells (IMRCs) derived from human embryonic stem cell uniquely modulate immunity and regulate extracellular matrix production. Like MSCs, IMRCs exhibit self-renewal capacity and trilineage differentiation. Whole transcriptome analysis revealed that highly expressed genes in IMRCs were predominantly enriched in pathways associated with inflammatory response, extracellular matrix degradation, and angiogenesis. Gene expression analysis revealed significant differences between IMRCs and UCMSCs, with IMRCs exhibiting higher expression levels of genes involved in immune regulation, antifibrosis, and tissue repair, while showing lower expression of pro-inflammatory genes. Following exposure to pro-inflammatory factors, both IMRCs and UCMSCs were analyzed, with IMRCs exhibiting superior immunomodulatory capacity [22]. To date, IMRCs have been applied to lung injury and fibrosis [23], Alzheimer’s disease [24], membranous nephropathy [25], chronic cerebral hypoperfusion [26], acute liver failure [27], and meniscus injuries [28]. However, their potential in treating IUA remains unexplored. MSCs primarily exert their effects via the paracrine pathway, with exosomes serving as key paracrine factors through which miRNAs facilitate intercellular communication [1, 29]. MiRNAs are small RNA molecules, ranging from 18 to 25 nucleotides in length, that regulate downstream target gene expression post-transcriptionally by binding complementary to the 3’ untranslated region (3’UTR) of mRNAs. Numerous studies have shown that stem cell-derived exosomal miRNAs exert favorable anti-fibrotic effects in IUA [30,31,32].

This study constructed cellular and animal models of IUA to investigate the effectiveness and potential mechanisms of IMRCs for the treatment of IUA.

Materials and methods

Generation of IMRCs

IMRCs was prepared and generated by the National Stem Cell Resource Center, Institute of Zoology, Chinese Academy of Sciences, Beijing [22]. The purchase of IMRCs was commissioned to GENELINE Co. Ltd., who acquired them from the National Stem Cell Resource Center, Institute of Zoology, Chinese Academy of Sciences. Human embryonic stem cells were cultured in Essential 8™ basal medium and dissociated to form human embryoid bodies (hEBs). IMRCs were obtained by using serum-free medium to hEBs outgrowth cells to dissociate and continue passaging. IMRCs were passaged when they were cultured to 80% confluence. IMRCs expressed MSC-specific surface markers, whereas typical hematopoietic markers appeared negative. All the cells were maintained in a humidified incubator at 37 °C, with 5% CO₂.

Isolation and culture of human endometrial stromal cells

Our study was approved by the Ethical Committee of Beijing Obstetrics and Gynecology Hospital (Approval number: 2022-KY-056-01), and all patients provided written informed consent. The patient underwent hysteroscopy at the Beijing Obstetrics and Gynecology Hospital for reasons unrelated to endometrial disease, and normal endometrial tissue was obtained by curettage. As previously described, human endometrial stromal cells (HESCs) were extracted from human endometrium [29, 30]. Briefly, the endometrial tissues were minced and digested with 0.2% collagenase type IV in a water bath at 37 °C for 60 min. The cells suspension was passed through a 200 mesh sieve, centrifuged at 1000 rpm for 5 min, and the supernatant was discarded before resuspending the cells. The resuspended cells were cultured and non-adherent cells were removed. HESCs were cultured with DMEM/F12(Gibco, USA) medium containing 10% FBS (Gibco, USA) and 100 U/mL penicillin/streptomycin (Beyotime, CHN).

Isolation and culture of UCMSCs

The UCMSCs utilized in this study were commercially procured through Beijing Huapu Jingrui Biogenetic Technology Co. Ltd., with the cells obtained from Wuhan Pricella Biotechnology Co., Ltd (CP-CL11, CHN). UCMSCs were cultured in α-MEM medium (Gibco, USA) supplemented with 10% FBS (Gibco, USA), 100 U/mL penicillin/streptomycin (Beyotime, CHN), and 2 mM L-glutamine (Gibco, USA).

IUA cell model

Our previous experiments used different concentrations (0, 1, 5 or 10 ng/ml) of TGF-β1 and durations (0, 12, 24, 48 h) to induce HESCs to construct IUA cell model. The IUA cell model constructed by exposing HESCs to 10ng / ml TGF-β1 for 48 h was identified as the most effective [29]. After constructing the IUA cell model in this study, the HESCs were treated with or without IMRCs conditioned medium (IMRCs-CM), respectively. After growing to 80%, the IMRCs were cultured with serum-free medium for 48 h. After 48 h incubation, the conditioned medium was collected and undergone differential centrifugation at 4 °C. The experiment was assigned into four groups: HESCs group, HESCs + TGF-β1 (10 ng/ml, 48 h) group, HESCs + TGF-β1 (10 ng/ml, 48 h) + IMRCs-CM (24 h) group, and HESCs + IMRCs-CM (24 h) group (Fig. 1a). Morphologic changes in HESCs were observed. The mRNA and protein expression levels of α-SMA, Vimentin, N-Cadherin, E-Cadherin were detected by RT-qPCR and Western blot.

Fig. 1

IMRCs inhibit fibrosis in TGF-β1-induced HESCs ex vivo. (a) Schematic diagram for the group of IUA cell experiment procedure. HESCs group, HESCs + TGF-β1group, HESCs + TGF-β1 IMRCs-CM group, and HESCs + IMRCs-CM group. (b) Representative morphology of HESCs, with or without the conditioned media of IMRCs, along with TGF-β1 treatment. Scale bar, 100 μm. (c) RT-qPCR for the relative expression of α-SMA, Vimentin, N-Cadherin, and E-Cadherin in different experimental groups. (d) Western blot for the relative expression of α-SMA, Vimentin, N-Cadherin, and E-Cadherin in different experimental groups. (e) Statistical analysis of Western blot. ∗p < 0.05, ∗∗∗p < 0.01, ∗∗∗∗p < 0.001, ∗∗∗∗p < 0.0001; data are represented as the mean ± SD

RT-qPCR

Total RNA was extracted from different groups of HESCs and rat uterine tissue using TRIzol^®Reagent (Ambion, USA) according to the manufacturer’s instructions, and quantified using a Nano- 300 microspectrophotometer (ALLSHENG, CHN). cDNA was obtained by reverse transcription of the extracted RNA using HiScript III RT SuperMix for qPCR (+ gDNA wiper) (Vazyme, CHN). RT-qPCR was performed using 2×Universal SYBR Green Fast qPCR Mix (ABclonal) on a CFX96 Real-Time System (BIO-RAD). The relative expression of gene was normalized with GAPDH as an internal control. All reactions were performed in triplicates and calculated using the 2-△△Ct method. The primer sequences were listed in Additional file 1: Table 1.

Western blot

Proteins were extracted from different groups of HESCs and rat uterine tissues and concentrations were estimated using the BCA protein assay. Protein samples were separated using 10% SDS-PAGE and transferred to PVDF membranes. The membranes were blocked using 5% skimmed milk and subsequently incubated with certain concentrations of primary (anti-α-SMA, anti-Vimentin, anti-N-Cadherin, anti-E-Cadherin, anti-IL-2, anti-Smad2, anti-p-Smad2, anti-smad3, anti-p-Smad3, anti-fibronectin (FN), anti-collagen1A1 (COL1A1), anti-GAPDH, anti-β-actin, Additional file 1: Table 2) and HRP-conjugated secondary antibodies (anti-rabbit IgG, anti-mouse IgG, Additional file 1: Table 2). The bands were visualized using an enhanced chemiluminescence detection system and analyzed with ImageJ software.

Construction of the IUA animal model

All animal experiments were approved by the animal ethics committee of Capital Medical University (Approval number: AEEI-2023-201). Healthy Sprague-Dawley (SD) rats of 8–10 weeks old (200–220 g) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. and housed in a specific pathogen-free environment. The temperature in the environment was 23–25 °C, with a 12/12 h light/dark cycle, and the rats had free access to food and water.

A total of 80 healthy 10-week-old SD rats were selected for the whole experiment, of which 72 females (200–220 g) were used for the construction and treatment of the IUA model and 8 males (200–220 g) were used for the fertility test. Rats were assigned into Sham, PBS, IMRCs, UCMSCs, Normal, IUA, Exos groups, using random number method. To reduce bias, the group allocation of the experiments was shown only to the experimental designers, ensuring blinding during experimental procedures and data analysis. The estrous cycle of the female rats was observed continuously and 7 females with unstable estrous cycles were removed. During the course of the experiment, 8 rats succumbed to complications related to anesthesia and infection. The remaining 57 animals were allocated as follows: Sham, PBS, IMRCs, and UCMSCs groups (n = 9 per group); Normal, IUA, and Exo groups (n = 3 per group); with an additional 12 rats designated for in vivo biodistribution assessment of IMRCs.

Rats with stable estrous cycles were selected and the experiment was conducted on the day of the estrous period. In the Sham group, only a laparotomy was performed without any manipulation of the injury. In this experiment, a dual injury approach was taken to construct the IUA rat model. Rats were anesthetized using 2% pentobarbital sodium injected intraperitoneally. A 3 cm incision was made in the middle of the rat’s lower abdomen to fully expose both sides of the uterus. An incision of 0.3 cm was made on the upper left side of the uterine bifurcation, and the endometrium was scraped with a miniature curette. Cotton thread soaked in 6 mg/L LPS solution for 24 h was placed into the left uterine cavity and the LPS thread was secured to the skin and removed after 48 h. The same treatment was given to the right uterus. The model of mechanical injury combined with infection mimics the clinical etiology of IUA in women. After one week, we injected 50 µL PBS, 5 × 10⁶ IMRCs suspended in 50 µL PBS, and 5 × 10⁶ UCMSCs suspended in 50 µL PBS into each side of the uterine cavity for the PBS, IMRCs, and UCMSCs group, respectively. For the IUA and Exos groups, 50 µL of PBS and 50 µL of Exos suspension were respectively infused into each uterine cavity of the rats. Experiments in mice with lung injury and fibrosis showed a positive correlation between IMRC concentration and therapeutic efficacy, with optimal effects observed at 5 × 10⁶ IMRCs. Short- and long-term safety assessments in crab-eating monkeys revealed that intravenous infusion of high doses of IMRCs (1 × 10⁸) had no adverse effects on vital organ function or overall metabolism. Based on these findings, a unilateral intrauterine infusion of 5 × 10⁶ IMRCs was selected for the treatment of IUA rats. To minimize the effect of confounders, the order of our treatments was consistent with the order of IUA model construction.

Histological analysis

The rats were euthanized by induction using carbon dioxide at specific time intervals (n = 3, at the different stages of the experiment), and the uterine tissues were fixed with 4% paraformaldehyde, dehydrated, embedded, and sectioned into 4 μm slides. HE and Masson staining were performed on the prepared sections. Endometrial thickness, fibrotic area percentage and gland count were measured and counted by ImageJ.

Labeling IMRCs with DiR

According to the instructions of the Cell Tracker DiR (MCE, USA), IMRCs were incubated in DiR staining solution at 37 °C for 30 min. After labeling, IMRCs were washed twice with PBS and resuspend. DiR-labeled IMRCs (5 × 10⁶ cells in 50 µL PBS) were injected into each uterine cavity.

Retention and distribution of IMRCs

Rats (n = 3, at the different stages of the experiment) were anesthetized and bioluminescence imaged (Multi-mode In Vivo Animal Imaging System, BLT, China, 750/780 nm) on days 3, 7, 14, and 21 after IMRCs intrauterine infusion to observe the retention time of IMRCs fluorescent signals in vivo. Meanwhile, fluorescence signals were detected in the major organs (heart, lungs, liver, spleen, kidneys, uterus and ovaries) ex vivo. The uterine tissues were fixed, embedded and sectioned. Sections were stained using DAPI (Solarbio, CHN) to visualize the location and migration of IMRCs within the uterus.

Fertility test

To compare the efficacy of IMRCs, UCMSCs, which are commonly used in regenerative medicine, were selected as a reference. The IUA rat model was constructed as described above and treated in different ways. At the fourth week after treatment, female rats (n = 3 in each group) of the Sham, PBS, IMRC and USMSC groups were mated at 3:1 ratio with healthy male rats, respectively. Vaginal smears were performed daily at 8:00 a.m. The sight of sperm on the smear or a copulation plug was recognized as day 0.5 of gestation. Pregnant rats were euthanized day 15 of gestation and the number of embryos on each uterine cavity were recorded.

RNA sequencing analysis

Total RNA was extracted from the uteri of the Sham, PBS, and IMRCs groups and the purity, concentration and integrity of the RNA was assessed. RNA-sequencing was conducted using an Illumina Novaseq 6000 platform (PE150) by Berry Genomics Company. RNA-sequencing library was prepared following the Hieff NGS Ultima Dual-mode mRNA Library Prep Kit for Illumina. To assess gene expression levels in different groups of uteri, the sequence reads obtained were aligned with the Rattus norvegicus genome using Hisat2, and the expression levels of all genes in each sample were calculated and compared. Carry out differential expression analysis for each experimental group. Enrich the differentially expressed genes in each experimental group and study the biological terms, pathways and functions.

Collection and characterization of IMRCs-derived exosomes (Exos)

When cell proliferation reached approximately 80%, the serum-free medium was replaced, and the cells were incubated for an additional 72 h. The supernatant was then collected and centrifuged at 4 °C. The centrifugation steps were performed in the following sequence: 3000 g for 20 min, followed by 10,000 g for 30 min. Exos were subsequently harvested by centrifugation at 9000 g for 90 min after filtration through a 0.22 μm filter membrane. Exos particle morphology and size distribution were analyzed using transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA).

Treatment and transfection

In this study, 10 ng/mL of TGF-β1 was used to induce fibrosis in HESCs, and Exos or miRNA mimics were co-administered. The miR-27b-3p mimic, miR-145-5p mimic, miR-16-5p mimic, and negative control (NC) were synthesized by GenePharma (Shanghai, China). Cells were harvested 48 h post-transfection and analyzed for proliferation and migration capabilities.

Cell proliferation and migration assays

HESCs, following transfection with specific miRNAs, were seeded into 96-well plates. After 12 h of cell culture, 10 µL of CCK-8 reagent (Additional file 1: Table 3)) was added to each well. Cell proliferation was assessed by measuring the absorbance at 450 nm after incubation at 37 °C for 1, 2, 3, and 4 days, respectively. To evaluate the migration of EECs post-transfection, a cell scratch assay was performed. Once the cells formed a monolayer, a scratch was made at the center of the monolayer, and the migration of cells into the scratched area was monitored and quantified over 48 h using ImageJ software.

Dual-luciferase reporter assay

To determine whether miR-27b-3p, miR-145-5p, and miR-16-5p could target SMAD2, pMIR-Smad2-WT and pMIR-Smad2-Mut vectors were constructed by cloning the SMAD2 3’ untranslated region (UTR), which includes the wild-type (WT) and mutant (Mut) binding sites for miR-27b-3p, miR-145-5p, and miR-16-5p. The pMIR-Smad2-WT or pMIR-Smad2-Mut vector was co-transfected into cells along with miRNA mimics or NC. Luciferase activity was measured 48 h post-transfection using the Dual-Luciferase Reporter Assay System (Beyotime, RG005).

Exosome sequencing

Total RNA was extracted from the collected exosomes using a specialized extraction kit for exosomal RNA. Small RNAs were fragmented, and complementary DNA (cDNA) was synthesized using reverse transcriptase. The cDNA was then amplified by PCR, and the fragment size of the library was assessed using the Illumina NovaSeq platform. A minimum of 10 million reads were obtained for each sample. Sequencing quality was assessed to remove low-quality sequences (Phred < 20), adapter sequences, and short fragments (< 18 nucleotides), as well as non-miRNA sequences such as rRNA, tRNA, and snRNA (refer to the RNA database). Sequences of low quality (Phred < 20), splice sequences, and short fragments (< 18 nucleotides), along with non-miRNA sequences (e.g., rRNA, tRNA, snRNA, as per the Rfam database), were excluded. The remaining data were compared to database annotations of known miRNAs to eliminate sequencing depth discrepancies. Differential miRNAs were identified based on a negative binomial distribution model. The top 30 miRNAs were selected for further analysis using DIANA-mirPath, which identified the top 30 potential mechanism pathways. Gene Ontology (GO) analysis was conducted to examine the biological processes and molecular functions of target genes, and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis was performed to identify relevant signaling pathways. A miRNA-target gene-pathway network was constructed by integrating miRNAs and their target genes into the competing endogenous RNA (ceRNA) network.

Statistical analysis

All statistical analyses in this study were performed using GraphPad Prism 8.0 software. Student’s t-test and one-way ANOVA were taken to analyze the data of the study. The experimental data in the study were expressed as mean ± SD, ∗p < 0.05, ∗∗∗p < 0.01, ∗∗∗∗p < 0.001, ∗∗∗∗p < 0.0001, and ns indicating no significant difference. The work has been reported in line with the ARRIVE guidelines 2.0. ARRIVE checklist.

Results

IMRCs reverse the pro-fibrotic effect of TGF-β1 on HESCs

TGF-β1, a central pro-fibrotic factor, is involved in the pathological process of IUA by inducing the transformation of various cells into myofibroblasts (MFs) and promoting excessive deposition of extracellular matrix (ECM) as collagen through multiple pathways [31]. High expression of TGF-β1 was verified in both IUA patients and animal models of IUA [32]. An IUA cell model was established using TGF-β1-induced HESCs to explore the role of IMRCs on fibrosis. It was observed that the morphology of HESCs shifted toward a myofibroblast-like phenotype after 48 h of treatment with 10 ng/mL TGF-β1 (Fig. 1b). These morphological changes were accompanied by a decrease in the expression of the epithelial marker E-Cadherin and an increase in the expression of myofibroblast markers α-SMA, Vimentin and N-Cadherin (Fig. 1c-e). The morphological changes, along with alterations in epithelial and mesenchymal markers expression, demonstrated that TGF-β1 successfully induced the transdifferentiation of HESCs to myofibroblasts. Meanwhile, HESCs were cultured with or without IMRCs-CM. RT-qPCR (Fig. 1c) and Western blot (Fig. 1d-e) analyses proved that IMRCs-CM could reduce the expression of α-SMA, Vimentin and N-Cadherin and promote the expression of E-Cadherin. These results confirmed that IMRCs-CM might reverse the pro-fibrotic effect of TGF-β1 on HESCs.

Location of IMRCs in the endometrium in IUA rats

Uteri from the IMRCs group were sampled and sectioned at designated time points (day 3, 7, 14 and 21), then observed under a fluorescence microscope to assess the implantation of IMRCs in the endometrium (Fig. 2a). DiR-labeled IMRCs were detected in all uterine sections of the IMRCs group at all time points, indicating the integration of IMRCs into the rat endometrium. Further observation revealed that DiR-labeled IMRCs were distributed throughout the uterus, rather than being confined to the endometrial layer. Red fluorescence was observed in both the myometrium and the perimetrium.

Fig. 2

Localization, distribution and residence of IMRCs in IUA rats. (a) Localization of of IMRCs in the rat endometrium. Immunofluorescence images of the localization of DiR-labeled IMRCs (red) in the endometrium at day 3, 7, 14, and 21 after treatment. Scale bar: 100 μm. (b) Residence and distribution of IMRCs in IUA rat. In vivo bioimaging imaging at day 3, 7, 14, and 21 after intrauterine injection of IMRCs labeled with DIR. Ex vivo imaging of major organs at day 3, 7, 14, and 21 after uterine infusion of IMRCs labeled with DiR. The isolated organs in order from top to bottom are heart, lungs, liver, spleen, kidneys, ovaries and uteri. (c) Schematic diagram for the construction and treatment of the IUA rat. (d) Surgical procedures for the construction and treatment of the IUA rat model

Residence and distribution of IMRCs in IUA rats

To track IMRCs, the IMRCs were labeled with DiR. After the injection of DiR-labeled IMRCs, bioluminescence imaging and ex vivo organ imaging of rats were performed at different time points (day 3, 7, 14, and 21) to track the retention and distribution of IMRCs in rats (Fig. 2b). The fluorescence intensity of DiR was strongest on day 3 and weakest on day 21, but still present. These results demonstrate that the fluorescence intensity of DiR continues to decrease over time and is not permanently present in vivo. No fluorescent signal of DiR was detected in the Sham group. Clear fluorescence signals were detected bilaterally in the uterus at 3, 7, 14 and 21 days after IMRCs were injected into the uterus, whereas the signal was not observed in other major organs such as the heart, lungs, liver, spleen, and kidneys. The data suggested that IMRCs remain at the injection site and are less likely to be transplanted into other organs.

IMRCs restore endometrial histology in IUA rats

The inhibition of TGF-β1-induced HESCs fibrosis by IMRCs was verified through in vitro experiments. To further validate the therapeutic effects of IMRCs on IUA, a rat model of IUA was established using mechanical injury combined with infection. A schematic of the IUA animal experimental procedure was illustrated in Fig. 2c. The specific surgical steps for modelling and treatment of IUA rats were demonstrated by Fig. 2d. Anatomical observation of the rat uterus in each group revealed that the uterus of the Sham group appeared smooth and elastic, with a plasma layer rich in blood vessels, while the uterus of the PBS group showed segmental stenosis, hyperplasia, and stiff tissues, which were alleviated following IMRCs infusion treatment (Fig. 3a-b). Endometrial morphology, thickness, and gland counts were compared among each experimental groups at days 14 and 21 after PBS and IMRCs transplantation.

Fig. 3

IMRCs facilitate endometrial morphological recovery and attenuate fibrosis in IUA rats. The uteri of each group were sampled and HE stained at different time intervals to measure the thickness of the endometrium and count the number of glands. (a-b) Morphological characteristics and HE staining of the uterus in each group at day 14 and 21. In HE staining, unidirectional red arrows indicate glands and bidirectional red arrows indicate endometrial thickness. Unidirectional black arrows indicate adhesion bands. Scale bars: 50 μm. (c-d) Statistical analysis of endometrial thickness at day 14 and 21. (e-f) Statistical analysis of number of glands at day 14 and 21. (g-h) Masson staining of the uterus in each group at day 14 and 21. (i-j) Statistical analysis of endometrial fibrosis at day 14 and 21. ∗p < 0.05, ∗∗∗p < 0.01, ∗∗∗∗p < 0.001, ∗∗∗∗p < 0.0001; data are represented as the mean ± SD

Proliferation and glandular recovery of the endometrium are crucial processes in the repair of endometrial damage and prerequisites for the restoration of endometrial function. HE staining (Fig. 3a-b) showed that the endometrium of Sham group exhibited intact borders, tightly arranged epithelial cells, and clear glandular organization. In contrast, the PBS group demonstrated severely damaged endometrium with a narrow uterine cavity, discontinuous luminal surface, massive inflammatory cells infiltration, and an absence of glands. On day 14, endometrial thickness in the IMRCs group (849.67 ± 5.08 μm) was prominently higher than that in the PBS group (399.27 ± 32.76 μm) and even higher than that in the Sham group (623.16 ± 43.58 μm) (P < 0.0001 vs. the PBS group, P < 0.001 vs. the Sham group; Fig. 3c). Until day 21, endometrial thickness in the IMRCs group (755.63 ± 16.29 μm) remained significantly greater than that of the PBS group (425.52 ± 29.83 μm) (P < 0.001; Fig. 3d). There was no statistically significant difference in endometrial thickness between the Sham group (650.80 ± 50.51 μm) and the IMRCs group at 21 (P >0.05; Fig. 3d). The number of glands in the IMRCs group (23.33 ± 3.68; 15 ± 0.82) was markedly higher than in the PBS group (0.67 ± 0.94; 5 ± 1.63) at day 14 and 21 (p < 0.001; p < 0.05 Fig. 3e-f). At day 14, no significant difference was observed in the number of glands between the IMRCs and Sham groups (16.33 ± 2.05). Whereas at day 21, the Sham group had significantly more glands (33.67 ± 4.99) than the IMRCs group (P >0.01; Fig. 3e-f). IMRCs transplantation treatment repaired normal uterine structure, characterized by a continuous, regular endometrium and the presence of a certain number of glands.

IMRCs inhibit endometrial fibrosis in IUA rats

Excessive deposition of ECM, leading to the replacement of functional endometrium by fibrotic tissue, is a hallmark pathological feature of IUA. To assess whether IMRCs improve collagen remodeling in the endometrium, Matson staining was performed on days 14 and 21 after a transplantation of PBS and IMRCs to calculate the degree of endometrial fibrosis by semi-quantifying collagen-positive areas. Masson staining (Fig. 3g-h) presented minimal collagen deposition in the endometrium of the Sham group, while the PBS group displayed severe fibrosis. At days 14 and 21 after transplantation of IMRCs, the proportion of endometrial fibrosis in the IMRCs group (18.10% ± 2.40%; 16.61% ± 1.95%) was significantly reduced compared with the PBS group (39.68% ± 2.42%; 38.83% ± 1.97%) (Fig. 3i-j). These results demonstrated that IMRCs effectively mitigated abnormal ECM deposition and decreased endometrial fibrosis.

IMRCs reduce expression of fibrosis markers in IUA rats

Epithelial-mesenchymal transition (EMT) is a process in which endothelial cells lose cell-to-cell junctions and original features, subsequently acquiring mesenchymal cell markers (α-SMA, vimentin). EMT is pivotal in the progression of most fibrotic diseases and constitutes one of the key pathogenic mechanisms of IUA. During EMT, the expression of endothelial and mesenchymal markers undergoes dynamic alterations [33]. We detected changes in the expression of epithelial and mesenchymal related genes and proteins expression by RT-qPCR and Western blot, respectively.

α-SMA and vimentin are typical fibrosis markers [34]. RT-qPCR analysis revealed a significant increase in the mRNA expression of the mesenchymal markers α-SMA (P < 0.05, Fig. 4a) and vimentin (P < 0.01, Fig. 4a) in the PBS group on day 14 following intrauterine injection, compared with the Sham group. Consistent with these findings, the Western blot results revealed a marked increase in α-SMA (P < 0.05, p<0.001 Fig. 4b-e) and vimentin (P < 0.01, p<0.01 Fig. 4b-e) protein expression in the PBS group at both day 14 and 21, relative to the Sham group. Results showed that in the IMRCs group, the expression of α-SMA (P < 0.05 vs. the PBS group, Fig. 4a) and vimentin (P < 0.01 vs. the PBS group, Fig. 4a) mRNA were significantly declined at day 14. Similarly, Western blot confirmed significant decreases in the expression levels of these markers in the IMRCs group at days 14 and 21 (Fig. 4b-e).

Fig. 4

IMRCs inhibit the mRNA and protein expression of fibrotic markers in IUA rats. (a) RT-qPCR for the relative expression of α-SMA, Vimentin, N-Cadherin, and IL-2 in different experimental groups at day 14. (b) Western blot for the relative expression of α-SMA, Vimentin, N-Cadherin, E-Cadherin, and IL-2 in different experimental groups at day 14. Full-length blots/gels are presented in Supplementary Fig. 8. (c) Statistical analysis of Western blot at day 14. (d) Western blot for the relative expression of α-SMA, Vimentin, N-Cadherin, and E-Cadherin in different experimental groups at day 21. Full-length blots/gels are presented in Supplementary Fig. 9. (e) Statistical analysis of Western blot at day 21. ∗p < 0.05, ∗∗∗p < 0.01, ∗∗∗∗p < 0.001, ∗∗∗∗p < 0.0001; data are represented as the mean ± SD

During the course of EMT, there is a decrease in the expression of E-Cadherin and a corresponding increase in the expression of N-Cadherin in epithelial cells. The results confirmed that E-Cadherin and N-Cadherin levels changed in opposite directions across all experimental groups. The RT-qPCR results revealed that in the IMRCs group, the expression of N-Cadherin was significantly declined (P < 0.001 vs. the PBS group, Fig. 4a). Western blot analysis indicated a reduction in the mesenchymal marker N-Cadherin (P < 0.001, p<0.001 Fig. 4b-e) and a concurrent increase in the epithelial marker E-Cadherin (P < 0.05, p<0.05 Fig. 4b-e) in the IMRCs group at days 14 and 21,compared with the PBS group. It was indicated that IMRCs effectively reverse the process of EMT.

IMRCs inhibit the expression of inflammatory factors in IUA rats

Studies have shown that inflammation precedes fibrosis in the formation of IUA and that inflammation of the endometrium exacerbates fibrosis progression [35]. Consistent with these findings, our results confirmed the involvement of inflammation in IUA formation. IL-2 is a classic inflammatory factor. The mRNA and protein expression of IL-2 was significantly higher in the PBS group compared to the Sham group at day 14 (P < 0.05, p<0.05 Fig. 4a-c). Treatment with intrauterine IMRCs infusion effectively reduced the expression of the pro-inflammatory factor IL-2 (Fig. 4a-c).

Transcriptomic analysis of IMRCs for the treatment of IUA

RNA sequencing was performed on three groups of tissues (i.e., Sham, PBS, and IMRCs groups) to detect differentially expressed genes (DEGs) and explore the possible mechanisms of IMRCs in the treatment of IUA. A total of 4589 genes were detected in the samples from the three groups, with a fold change (FC) > ± 1.5 | and p < 0.05 used as the screening criterion for DEGs (Fig. 5a). A total of 359 DEGs were found in the IMRCs and PBS groups, including 245 up-regulated genes and 150 down-regulated genes (Fig. 5b). DEGs function enrichment analysis and cluster analysis revealed that the up-regulated genes in IMRCs group were mainly involved in cytoplasmic membrane components and their interactions. Conversely, the down-regulated genes were predominantly associated with extracellular matrix, organelle endomembrane components, mitochondrial endomembrane and interactions (Fig. 5c). Consulting gene databases, we found that these DEGs are related to smooth muscle formation and focal adhesions and contribute to various ECM component compositions (e.g., mitochondria, lysine, collagen). These findings suggests that IMRCs reduces the redox response and fibrosis formation while promoting IUA in repairing damaged endometrium.

Fig. 5

RNA-seq analysis of IUA rats after IMRCs infusion treatment to investigate the associated biological process. a Venn diagram of DEGs between comparison groups. b Volcano plot of DEGs between IMRCs and PBS groups. Red (or blue) color indicates up-regulated (or down-regulated) genes. c KEGG analysis demonstrated their biological processes and related signaling pathways, respectively

IMRCs treatment of IUA is superior to UCMSCs

As a leading candidate in regenerative medicine, UCMSCs have been widely used in IUA research, demonstrating notable success. To assess the efficacy of IMRCs, we compared it with UCMSCs for the treatment of IUA rats (Fig. 6a-b). Our results demonstrated that intrauterine infusion of both IMRCs and UCMSCs significantly ameliorate endometrial thinning and glandular reduction due to injury (Fig. 6a 6c 6d). Although the area of endometrial fibrosis was lower in the IMRCs group compared to the UCMSCs group, there was no statistical difference between the two groups (Fig. 6b 6e).

Fig. 6

IMRCs are superior to UCMSCs in repairing endometrial tissue structure and function. (a) HE staining of the uterus in IMRCs group and UCMSCs group at day 28. In HE staining, unidirectional red arrows indicate glands and bidirectional red arrows indicate endometrial thickness. Scale bars: 50 μm. (b) Matson staining of the uteri to assess the extent of fibrosis in IMRCs group and UCMSCs group at day 28. Scale bars: 50 μm. (c-d) Statistical analysis of endometrial thickness and number of glands. (e) Statistical analysis of endometrial fibrosis. Comparison of different treatments for improvement of fertility in IUA. (f) Embryo implantation in the different groups on day 15 of gestation. (g) Embryo implantation rate in each group. (h) The number of embryo implantation in each group at E15. ∗p < 0.05, ∗∗∗p < 0.01, ∗∗∗∗p < 0.001, ∗∗∗∗p < 0.0001; data are represented as the mean ± SD

We assessed endometrial structural damage and recovery using uterine tissue sections, while RT-qPCR and Western blot analyzed expression of fibrosis markers, and further determined the repair of endometrial function by fertility testing. Endometrial damage and repair can directly affect embryo implantation rate. On the 28th day after intrauterine treatment, female rats were mated with male rats, and sampling was performed on the 15th day post-mating. As shown in the results, IUA severely impaired the fertility of the rats, and the embryo implantation rate in the PBS group significantly declined compared to the Sham group, where no embryo implantation was observed (Fig. 6f-g). The number of embryo implantations was highest in the Sham group (n = 3), with 17, 17, and 20 embryos (18 ± 1.41), respectively (Fig. 6f). Embryo implantation rates in the IMRCs group (16.67 ± 1.25) were obviously higher than in the PBS group (1.67 ± 2.36) and slightly lower than in the Sham group, 17, 18, and 15, respectively (Fig. 6f 6h). The number of embryos in the UCMSCs group (11.33 ± 0.47) was lower than that in the IMRCs group, which was 12,11, and 11, respectively (Fig. 6f), and there was a statistical difference between the two groups (Fig. 6h). The results proved that IMRCs were superior to UCMSCs in improving endometrial function and fertility after endometrial damage and fibrosis.

Structural characteristics of exos

Observations revealed that the cellular Exos exhibited varying morphologies, primarily consisting of vesicular structures combined with membrane sheets (Supplementary Fig. 11A) and vesicular structures (Supplementary Fig. 11B). The average diameter of the Exos was 116.8 nm, with a concentration of 1.2 × 10¹² particles/ml (Supplementary Fig. 11C). Notably, vesicular exosomes accounted for a larger proportion of the total population.

The anti-fibrotic mechanism of exos

HESCs were seeded into well plates, and 10 ng/mL of TGF-β1 was used to induce the formation of the IUA cell model, which was subsequently treated with or without Exos. The expression of p-Smad2/3 in HESCs was significantly increased following TGF-β1 treatment, but it was notably reduced after treatment with Exos, as shown in Fig. 7A and B. These results suggest that Exos can modulate the TGF-β/Smad signaling pathway, a key pathway in IUA development.

Fig. 7

Exos participate in the regulation of the TGF-β/Smad signaling pathway. (a) Western blot analysis of Smad2 and Smad3 phosphorylation levels in cells following induction. (b) Statistical analysis of Western blot. (c) KEGG pathway analysis of the top 30 miRNAs. (d) miRNA-Gene network map. (e) RT-qPCR for the relative expression of miR-27b-3p, miR-145-5p, and miR-16-5p. (f) Western blot detection of fibronectin expression (FN, COL1A1and α-SMA). (g) Statisticalanalysis of Western blot (FN, COL1A1and α-SMA). (h) CCK8 assay to assess HESCs proliferation. （i-j） Scratch assay to evaluate cell migration.

To further investigate the therapeutic mechanism of Exos, miRNA sequencing was performed. KEGG pathway analysis of the top 30 miRNAs in Exos, conducted using DIANA-mirPath, revealed a significant association with the TGF-β/Smad pathway (Fig. 7C). TargetScan and GO analysis were then employed to predict target genes and related functions of these miRNAs, and to construct miRNA-mRNA interaction maps. Among the analyzed miRNAs, 19 were found to directly target TGFB1, SMAD2, SMAD3, and other related genes (Fig. 7D). Based on these findings, miR-27b-3p, miR-145-5p, and miR-16-5p were selected for further investigation (Fig. 7E).

To validate their effects, miRNA mimics were transfected into HESCs. The results demonstrated that these three miRNA mimics effectively attenuated TGF-β1-induced fibrosis, reducing the protein expression levels of α-SMA, COL1A1, and FN (Fig. 7F, G). Furthermore, CCK-8 and scratch assays showed that transfection with these miRNA mimics significantly enhanced the proliferation and migration abilities of HESCs (Fig. 7H, I, J).

Dual luciferase reporter assays demonstrated that miR-27b-3p, miR-145-5p, and miR-16-5p could bind to the 3’ UTR of SMAD2 (Fig. 8A). Western blot (WB) analysis revealed a decrease in the expression of both SMAD2 and p-SMAD2 following transfection with these three miRNA mimics (Fig. 8B, C).

Fig. 8

Specific miRNAs in Exos, including miR-27b-3p, miR-145-5p, and miR-16-5p, inhibit Smad2 phosphorylation and exert anti-fibrotic effects. (a) Dual luciferase reporter assay. (b) Western blot detection of p-Smad2 in HESCs. (c) Statistical analysis of Western blot. (d) RT-qPCR analysis of miR-27b-3p, miR-145-5p, and miR-16-5p expression in uterine tissues from rats. (e) Western blot detection of smad2 and smad3 expression in uterine tissues of rats. (f) Statistical analysis of Western blot

To further confirm whether Exos could target the TGF-β/Smad pathway, we conducted in vivo experiments and analyzed uterine tissues from rats (n = 3) using RT-qPCR. The results showed that the expression levels of miR-27b-3p, miR-145-5p, and miR-16-5p were significantly reduced in the IUA group. However, treatment with Exos led to significantly higher expression levels of these three miRNAs (Fig. 8D). Additionally, Western blot analysis of uterine tissues showed results consistent with the cellular findings, indicating that Exos treatment effectively reduced the phosphorylation of SMAD2 and SMAD3 (Fig. 8E, F).

Discussion

IUA is endometrial fibrosis caused by endometrial damage and infection, which seriously undermines women’s reproductive health. A great deal of research has been carried out by gynaecologists worldwide to overcome this problem. According to statistics, 88 studies on BMMSCs, ADMSCs, hAMSCs, UCMSCs, MenMSCs, and others MSCs for the treatment of endometrial pathologies have been conducted since 2011 [36]. Autologous and allogeneic adult-derived MSCs have shown encouraging results in preclinical and clinical studies of IUA [37,38,39,40]. In the treatment of IUA, MSCs can reduce endometrial fibrosis, stimulate angiogenesis, promote endometrial regeneration, restore the structure of the uterine cavity and physiological function of the endometrium, and improve pregnancy outcomes through mechanisms such as immunosuppression and modulation, promotion of endogenous stem cells differentiation, paracrine effects, and homing properties [41, 42].

Despite the fact that the vast majority of studies have shown that adult-derived MSCs are safe and effective, the application of MSCs therapy has been hampered in many ways. The invasive acquisition of BMSCs and ADMSCs causes great physical and psychological harm to the donor [43]. The acquisition process of UCMSCs, hAMSCs and MenSCs is susceptible to contamination [44]. Adult-derived MSCs have limited proliferative capacity and require expansion of cells from multiple donor sources to obtain sufficient quantities for clinical use, thus not guaranteeing uniformity of cell quality [37]. Higher passage MSCs have the potential to trigger blood-mediated inflammatory responses, which can affect the survival and function of infused cells [45]. Different from adult-derived MSCs, the non-invasive acquisition and unlimited proliferative differentiation capacity of human embryonic stem cells allows for a constant supply of IMRCs. A batch of human embryonic stem cells-derived IMRCs ensures homogeneity of stem cell quality and minimizes batch-to-batch variation. The GMP-compliant IMRCs not only guarantees quality but also meets the needs of mass production.

Besides efficacy and standardized production systems, safety is the first and foremost indicator to consider, especially when it comes to clinical translation. Whole genome sequencing analysis showed that IMRCs demonstrated no mutations in proto-oncogenes or tumor suppressor genes and exhibited no demonstrable potential for tumor formation. Tumor formation was not observed in either in vivo and in vitro tumor formation assays of IMRCs. In systemic evaluations of both rabbits and cynomolgus monkeys, IMRCs showed no significant toxicity and demonstrated a favorable safety profile for in vivo administration. In the phase I clinical trial of IMRCs for the treatment of meniscal injuries, there were no serious adverse events associated with IMRCs and no abnormal blood biochemical indices, and the treatment with IMRCs was safe [28].

In this research, we constructed both a cellular model and an animal model of IUA, respectively, to investigate the effects of IMRCs in reversing endometrial fibrosis, restoring the structure of the uterine cavity and promoting the process of endometrial regeneration.

TGF-β1, a key driver in the development of fibrotic diseases, is involved in the EMT process by promoting the aggregation of fibroblasts and inflammatory cells, inducing the synthesis and deposition of collagen fibers and ECM, and inhibiting their degradation. It has been implicated in a wide range of fibrotic diseases [33]. The TGF-β/Smad signaling pathway is closely associated with endometrial fibrosis, where the degree of TGF-β1 upregulation is positively correlated with the severity of IUA [46]. In our study, HESCs were treated with TGF-β1 to induce in vitro EMT and establish an IUA cell model. When HESCs were stimulated by TGF-β1, they manifested a fibrotic phenotype. HESCs lost their original features and expressed typical mesenchymal fibroblast-like features such as α-SMA, vimentin. This change was reversed once treatment with IMRCs-CM. IMRCs-CM treatment up-regulated epithelial cell marker expression and down-regulated fibrosis marker expression, suggesting that IMRCs can reverse the fibrotic process of HESCs induced by TGF-β1.

In this study, we took mechanical injury combined with LPS infection to construct a rat model of IUA, which is the closest to the clinical cause of IUA formation in women. one week after model construction, we performed intrauterine infusion of IMRCs. IMRCs were labeled with DiR to track the distribution and retention of in rats. The results showed that the fluorescent signal of IMRCs appeared only in uterine tissues and gradually diminish over time. Further analysis revealed that the fluorescence signal of IMRCs appeared not only in the endometrial layer, but also in the myometrial and plasma layers of the uterus. Tissue sections showed that the volume of the uterine cavity was significantly reduced due to severe uterine injury. The uterine cavity was marked by adhesive bands, and the endometrium was replaced by non-vascularized fibrotic tissue, accompanied by the loss of glands. IMRCs intrauterine infusion therapy reversed this damage, promoting endometrial repair and proliferation, restoring endometrial function, and reducing the area of fibrosis.

Due to their easy accessibility, high proliferative potential, strong differentiation, and migration abilities as well as low immunogenicity and non-tumorigenicity, UCMSCs are widely used in both basic and clinical studies of IUA. Previous studies have found IMRCs to be superior to UCMSCs in terms of long-term proliferative capacity, hyper-immunomodulatory and anti-fibrotic functions [22]. In this study, we constructed a rat model of IUA, performed uterine infusion treatment with IMRCs and UCMSCs, respectively, to compared their efficacy. The results showed that while both IMRCs and UCMSCs promoted endometrial regeneration, repaired the structure of the uterine cavity, and reduced the area of fibrosis. In terms of improving endometrial function and fertility, IMRCs were significantly better than UCMSCs, and there was a statistical difference between the two groups.

The process of EMT involves the transformation epithelial cells to a mesenchymal phenotype, characterized by enhanced migratory ability, invasiveness, resistance to apoptosis, and the deposition of large amounts of collagen. These changes contribute to the fibrotic process in multiple organs in vivo [47]. Several studies have shown that EMT is involved in the pathogenesis of IUA [48]. α-SMA, vimentin, N-cadherin (mesenchymal markers) and E-cadherin (epithelial marker) were selected as indicators for detecting EMT changes [32]. Tissue engineering has even focused on anti-EMT as an effective therapy against fibrosis [49]. In the cellular model of IUA, mRNA and protein expression levels of α-SMA, vimentin, N-cadherin increased, while E-cadherin expression declined. IMRCs-CM treatment up-regulated epithelial cell marker expression and down-regulated fibrosis marker expression, suggesting that IMRCs can reverse the in vitro EMT process in HESCs. Similarly, in the IUA rat model, intrauterine infusion of IMRCs reduced the expression of fibrosis marker at both the mRNA and protein levels and reversed the course of EMT. In summary, IMRCs may play an antifibrotic role in the treatment of IUA by reversing the EMT process through paracrine effects. IMRCs with immunomodulatory capacity can reduce the expression of pro-inflammatory factors. In the treatment of IUA, IMRCs play an anti-fibrotic role by suppressing the inflammatory response. RNA Sequencing analysis revealed that IMRCs regulate the expression of genes related to smooth muscle formation, focal adhesions, and the composition of ECM components, thereby reducing redox reactions and fibrosis formation and repairing damaged endometrium.

A growing body of research suggests that the paracrine pathway plays a crucial role in stem cell function, with exosomes acting as key mediators of this process [50]. In studies related to IUA, exosomes derived from stem cells exhibit similar anti-fibrotic properties as the stem cells themselves [51]. The miRNAs enriched in exosomes can target genes involved in the regulation of various signaling pathways [52]. Our results indicate that Exos treatment can inhibit Smad phosphorylation in response to TGF-β1 induction. Sequencing analysis of Exos revealed that several miRNAs, which were highly enriched, are involved in the regulation of the TGF-β/Smad signaling pathway. Specifically, miR-27b-3p, miR-145-5p, and miR-16-5p were highly expressed in Exos and were found to directly target key molecules in this pathway, including TGFBR and SMAD2.

Previous studies have demonstrated that dysregulation of the TGF-β/Smad signaling pathway is a key pathogenic mechanism in fibrotic diseases. TGF-β binds to its receptor, leading to the phosphorylation of Smad2 and Smad3, which triggers a cascade of events that promote fibrosis development. In our study, we found that miR-27b-3p, miR-145-5p, and miR-16-5p specifically targeted Smad2, inhibited the activation of this signaling pathway, and exhibited anti-fibrotic effects. Additionally, miR-27b-3p, miR-145-5p, and miR-16-5p have been shown to be involved in the regulation of the YAP/LOXL2 pathway, the EMT process, and NOTCH signaling, thereby inhibiting the progression of hepatic fibrosis, renal fibrosis, and systemic sclerosis [53,54,55,56,57]. Our study demonstrated for the first time that IMRCs can reverse the EMT process and reduce inflammation both in vivo and in vitro. Furthermore, IMRCs exert antifibrotic effects, promote the restoration of endometrial structure and function, and improve fertility in IUA rats. However, Further investigation revealed that IMRCs exert their effects through a paracrine pathway, wherein specific miRNAs enriched in Exos can inhibit the TGF-β/Smad signaling pathway, thereby exhibiting antifibrotic effects.

Conclusion

Transplantation of IMRCs has shown potential in promoting endometrial regeneration, reducing the area of fibrosis, and repairing both the structure of the uterine cavity and endometrial function. IMRCs were superior to UCMSCs in improving endometrial tolerance and pregnancy outcomes. Specific miRNAs, including miR-27b-3p, miR-145-5p, and miR-16-5p, enriched in Exos, can directly target the TGF-β/Smad pathway and inhibit the development of IUA. Our research provided theoretical evidence for the effectiveness of IMRCs in treating IUA and brought a new direction for the treatment of IUA.