Intrauterine infusion of autologous endometrial stem cells for the treatment of moderate and severe intrauterine adhesions: a before-and-after study.

The study was a single-centre and one-group pretest‒posttest design clinical trial approved by the Reproductive Medicine Ethics Committee of Xiangtan Central Hospital in March 2018 (s20180322), and registered in the Chinese Clinical Trial Registry (registration number: CHICTR1800016769). The inclusion criteria for this study were a history of infertility due to IUA or multiple failed transplants; age 20–40 years; recurrent moderate-to-severe IUA by hysteroscopy; normal liver, heart and kidney function; no infectious diseases such as HIV, hepatitis B or C, or syphilis; and reproductive intent. The degree of IUA was assessed in accordance with the American Fertility Society (AFS) (1998) scoring criteria. The exclusion criteria were chromosomal karyotype abnormalities, adenomyosis of the uterus, a history of malignant tumour, the presence of a serious physical disease, surgical site infection, coagulation dysfunction, and severe mental or psychological illness. Each participant was informed of the content of the study and signed an informed consent form. The work has been reported in line with the STROCSS criteria [ 22 ]. A total of 15 eligible patients with recurrent moderate to severe IUA (AFS score ≥ 6) were recruited between 2018 and 2020, and their clinical characteristics were summarized in Table  1 . Table 1 Patients information and clinical data Patients Age Causes of disease Past treatment Endometrial condition The number of endometrial stem cell treatments 1 29 abortion curettage multiple HAs thin and moderate IUA 1 2 31 abortion curettage HA; G-CSF thin and severe IUA 2 3 34 history of hydatidiform mole HA; saccule; IUD; sodium hyaluronate thin and severe IUA 1 4 31 abortion curettage HA; saccule; sodium hyaluronate moderate IUA 3 5 35 history of extra-pelvic tuberculosis HA; IUD thin and moderate IUA 4 6 31 abortion curettage HA thin and moderate IUA 2 7 28 separation of uterus and mediastinum saccule; sodium hyaluronate severe IUA 2 8 28 abortion curettage multiple HAs; IUD thin and severe IUA 5 9 37 abortion curettage HA moderate IUA 2 10 40 abortion curettage HA thin and moderate IUA 4 11 29 recurrent abortion HA; IUD thin and moderate IUA 2 12 33 uterine manipulation; history of ectopic pregnancy HA thin and moderate IUA 2 13 33 uterine manipulation; history of ectopic pregnancy HA thin and moderate IUA 2 14 30 abortion curettage HA moderate IUA 2 15 31 pelvic inflammation; abortion curettage multiple HAs; IUD; sodium hyaluronate moderate IUA 2 HA, hysteroscopic adhesiolysis; IUA, intrauterine adhesions; G-CSF, granulocyte colony-stimulating factor; IUD, intrauterine device; Patients information and clinical data HA, hysteroscopic adhesiolysis; IUA, intrauterine adhesions; G-CSF, granulocyte colony-stimulating factor; IUD, intrauterine device; Endometrial tissue was acquired by hysteroscopy three to seven days after menstruation. The IUA patient was placed in the lithotomy position, the adhesion in the uterine cavity was mechanically separated under hysteroscopic observation to restore the uterine anatomy structure, and the IUD was applied after surgery. A small amount of endometrial tissue was obtained and sent to the laboratory for endometrial stem cell culture during the operation. Then, in a sterile environment, the endometrial tissue was immediately washed 5 times with phosphate buffer saline (PBS) buffer containing 100 IU/mL penicillin and streptomycin to remove blood stains and cut to a diameter of < 1 mm. Subsequently, 0.1% type I collagenase (approximately 3 times the volume of the tissue) was added to the sample, and the sample was mixed and oscillated on a constant-temperature shaking table at 37℃ for digestion for 60 min. DMEM/F12 medium containing 10% Fetal Bovine Serum (FBS) was added to terminate the digestion. After blowing the cell suspension, the suspension was filtered through a screen (40-µm aperture), and the filtrate was the stem cell suspension. Then the mixture was centrifuged at 1500 r/min for 5 min, the supernatant was discarded, an appropriate amount of PBS was added, the mixture was resuspended, the mixture was centrifuged again, the supernatant was discarded, DMEM/F12 medium containing 10% FBS was added for resuspension, and the mixture was cultured at 37℃ in a 5% CO 2 incubator. After 24 h, the culture medium was changed every 2 days, cell morphology and cell growth were observed with an inverted microscope every day, and images of the cells were obtained. When the degree of cell fusion reached approximately 90%, cell passage was performed. After isolation and culture from endometrial tissue, the endometrial stem cells exhibited a strong proliferation ability, their morphology was uniform with long spindles, and they grew in a whirlpool shape when growing to the fusion state, reflecting the typical characteristics of clonal growth (Fig.  1 A). Fig. 1 Identification of endometrial stem cells. ( A ) The morphology of endometrial stem cells under microscope. ( B ) The phenotype identification of second generation endometrial stem cells by Flow cytometry Identification of endometrial stem cells. ( A ) The morphology of endometrial stem cells under microscope. ( B ) The phenotype identification of second generation endometrial stem cells by Flow cytometry Second-generation endometrial stem cells with 90% confluence were digested with 0.25% trypsin-EDTA and prepared as single-cell suspensions. The suspension was centrifuged at 1500 r/min for 5 min, and the supernatant was discarded. The corresponding antibody working solutions (FITC-CD45, FITC-CD44, PE-CD29, PE-CD34, APC-CD90 and APC-HLA-DR) were added, and the negative controls were FITC-IgG1k, PE-IgG1k and APC-IgG1k, respectively. The mixture was subsequently centrifuged at 1500 r/min for 5 min, the supernatant was discarded, and 0.5 ml of PBS was added for resuspension. Flow cytometry revealed that common stem cell-positive (CD29, CD44, and CD90) and negative (CD34, CD45, and HLA-DR) surface markers were identified in second-generation endometrial stem cells (Fig.  1 B). Since the stem cells did not reach the ovary immediately after being injected into the uterine cavity, sodium hyaluronate gel was injected to increase its residence time to avoid loss. To avoid the interference of sodium hyaluronate gel on stem cell growth, a CCK8 assay was used to measure the proliferation of endometrial stem cells in sodium hyaluronate gel at different concentrations. The endometrial stem cells were inoculated into 96-well plates at a concentration of 1 × 10 4 per well and cultured with a mixture of sodium hyaluronate gel (12.5%, 25% and 37.5%) and complete medium after 2 h of cell adhesion. Then 10 µL of CCK8 detection solution was added at 24, 48 and 72 h. After incubation at 37℃ and 5% CO 2 for 2 h, the absorbance in each well was measured at a wavelength of 450 nm. As the proportion of sodium hyaluronate gel increased, the proliferation rate of the endometrial stem cells was slowed and 12.5% sodium hyaluronate gel was determined to be the best choice (Fig.  2 ). Fig. 2 The proliferation of endometrial stem cells with different concentrations of sodium hyaluronate gel(* P <0.05) The proliferation of endometrial stem cells with different concentrations of sodium hyaluronate gel(* P <0.05) Endometrial stem cell transplantation was performed on the first day after menstruation. A total of 1 × 10 7 fifth-generation endometrial stem cells were resuspended in 1 ml of autologous plasma and taken to the operating room for intrauterine infusion. The stem cells were infused by means of a 1 ml injector, and then 2 ml of 12.5% sodium hyaluronate gel was poured into the uterine cavity. After lying down and resting for 30 min, the patient got out of bed and performed some activities. At the same time, the patient was given Oestradiol valerate tablets (3 mg/ day) and acetylsalicylic acid (50 mg/ day) for endometrial support. After stem cell transplantation, menstruation in patients was recorded regularly every month. The degree of IUA and endometrial thickness (10–16 days after menstruation) were monitored by hysteroscopy and ultrasonography during the second month after stem cell transplantation; if the endometrial thickness was not greater than 6 mm, a second round of autologous endometrial stem cell transplantation was performed. Monitoring by means of hysteroscopy and ultrasonography was subsequently performed every 2–3 months. When the uterine adhesions were significantly relieved, 2–3 Grade I embryos were thawed and transplanted with the patient’s consent. After transplantation, the patient was given hormonal support consisting of progesterone (600 mg/ day) and oestradiol (6 mg/ day). The concentration of human chorionic gonadotropin (HCG) in peripheral blood was measured 14 days after embryo transplantation to determine whether the pregnancy was successful. Pregnancy status was then followed up by telephone every 3 months until the end of pregnancy, and all patients were followed up until May 2021. Primary endometrial stem cells from 4 healthy individuals (G0), the primary generation of endometrial stem cells from 7 IUA patients (IUA-G0), and the fifth generation of endometrial stem cells from 6 IUA patients (IUA-G5) were used for RNA sequencing analysis. Once the number of cells met the requirements, standard RNA-seq procedures were initiated, including reverse transcription into cDNA, adapter ligation, amplification, and sequencing, were initiated to obtain the raw data. After quality control of the raw data, the DEGs of IUA-G0/G0 and IUA-G5/IUA-G0 were analysed with Deseq2 software. The screening conditions were p values  1. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were performed with R-package clusterProfiler. The GO analysis included cellular component (CC), molecular function (MF), and biological process (BP). All the data in this study were analysed with SPSS 18.0 software and the R software (version 4.3.3). The differences in endometrial thickness and AFS score before and after endometrial stem cell treatment were analysed by means of paired t-test, and P  < 0.05 was considered to indicate a significant difference.

The degrees of IUA, maximum endometrial thickness and pregnancy outcomes of all patients before and after stem cell transplantation were presented in Table  2 . After stem cell transplantation, the AFS scores of all patients decreased. Moreover, the results of hysteroscopy revealed that the patient’s uterine condition improved and the symptoms of uterine adhesions were relieved to varying degrees (Fig.  3 ), indicating that the effective rate of stem cell transplantation was 100%. The maximum endometrial thickness of 10 patients with signs of a thin endometrium, and among them, 8 patients had a maximum endometrial thickness reaching transplantable thickness (≥ 6 mm). In Patient 5, the endometrium did not reach transplantable thickness, so did not undergo embryo transfer, but had a natural pregnancy, although the patient eventually miscarried. In terms of clinical outcome, 9 of the 15 patients had successful pregnancies, with 8 live births and 1 spontaneous abortion (endometrial thickness < 6 mm), Additionally, 3 of the remaining 6 patients had biochemical pregnancyies, 2 were not pregnant, and 1 had an ectopic pregnancy. The total pregnancy rate was 60.0% and the live birth rate was 53.3%. Table 2 Clinical outcomes of patients with intrauterine adhesions Patients Maximum endometrial thickness before treatment Maximum endometrial thickness after treatment AFS score before treatment AFS score after treatment Pregnancy Outcomes 1 2 6.8 8 normal uterine cavity give birth to twins at term 2 4.8 6.3 10 2 ectopic pregnancy 3 4 5 5 normal uterine cavity biochemistry 4 6.7 7.2 8 normal uterine cavity give birth at term 5 5.1 5.5 8 normal uterine cavity spontaneous abortion of pregnancy 6 5 5.8 6 3 premature twins 7 6.5 7.5 10 normal uterine cavity give birth at term 8 4 6.7 12 2 give birth at term 9 8 8 6 normal uterine cavity biochemistry 10 4.5 9 8 3 not pregnant 11 5 9.6 7 2 give birth at term 12 8 8.2 8 normal uterine cavity give birth at term 13 5.5 6.3 8 2 give birth to twins at term 14 6.5 8 8 2 biochemistry 15 6.5 8.3 6 normal uterine cavity not pregnant Mean 5.47 ± 1.60 7.21 ± 1.32 8.33 ± 1.91 1.07 ± 1.22 P - 0.0011 - <0.0001 - AFS, the American Fertility Society Clinical outcomes of patients with intrauterine adhesions AFS, the American Fertility Society Fig. 3 Hysteroscopic images of patients with IUA before and after endometrial stem cell treatment Hysteroscopic images of patients with IUA before and after endometrial stem cell treatment Since treatment with endometrial stem cells can promote endometrial repair, RNA sequencing was performed to explore the underlying molecular mechanisms. As shown in Fig.  4 A, when the IUA-G0 group was compared with the G0 group, a total of 1577 DEGs, including 440 upregulated genes and 1137 downregulated genes, were identified (Fig.  4 B, Supplementary Table 1). There were 909 DEGs between IUA-G5 and IUA-G0, of which 509 were upregulated and 400 were downregulated (Fig.  4 C, Supplementary Table 2). There were 322 overlapping DEGs in the IUA-G0/G0 and IUA-G5/IUA-G0 groups. GO analysis showed that the BP categories associated with the DEGs in the IUA-G0/G0 group were related primarily to wound healing, synapse organization and cell junction assembly; the CC categories included apical plasma membrane, collagen-containing extracellular matrix and apical part of cell, and the MF categories gated channel activity, ion gated channel activity, ion channel activity and metal ion transmembrane transporter activity (Fig.  4 D, Supplementary Table 3). Furthermore, the BP categories associated with DEGs in the IUA-G5/IUA-G0 group were enriched in regulation of neuron projection development, axonogenesis and regulation of nervous system development; the CC involved external side of plasma membrane, glutamatergic synapse and collagen-containing extracellular matrix; and the MF categories were gated channel activity, ion gated channel activity and ion channel activity (Fig.  4 E, Supplementary Table 4). In addition, the DEGs in the IUA-G0/G0 group were involved mainly in the Pl3K‒Akt signalling pathway, human papillomavirus infection and cytokine-cytokine receptor interaction (Fig.  4 F, Supplementary Table 5), whereas the DEGs in the IUA-G5/IUA-G0 group were involved in the Pl3K-Akt signaling pathway, cytokine-cytokine receptor interaction and alcoholism (Fig.  4 G, Supplementary Table 6), suggesting that the molecular differences between primary endometrial stem cells in healthy individuals and those in IUA patients may be somewhat similar to the differences between primary and fifth-generation stem cells in IUA patients. These GO and KEGG results, suggest that endometrial stem cells extracted from the endometrial tissue of IUA patients, after being removed from the IUA disease state environment, can be transformed into a normal endometrial stem cell growth pattern in vitro and promote endometrial repair. Fig. 4 Functional analysis of DEGs in IUA-G0/G0 and IUA-G5/IUA-G0 groups. ( A ) Venn diagram depicting variation and overlap of DEGs in IUA-G0/G0 and IUA-G5/IUA-G0 groups. ( B - C ) Volcano plot of DEGs in IUA-G0/G0 ( B ) and IUA-G5/IUA-G0 ( C ) groups. ( D - E ) GO enrichment analysis of DEGs in IUA-G0/G0 (D) and IUA-G5/IUA-G0 ( E ) groups, including top 10 of BP, CC, and MF. ( F - G ) KEGG analysis of DEGs in IUA-G0/G0 ( F ) and IUA-G5/IUA-G0 ( G ) groups Functional analysis of DEGs in IUA-G0/G0 and IUA-G5/IUA-G0 groups. ( A ) Venn diagram depicting variation and overlap of DEGs in IUA-G0/G0 and IUA-G5/IUA-G0 groups. ( B - C ) Volcano plot of DEGs in IUA-G0/G0 ( B ) and IUA-G5/IUA-G0 ( C ) groups. ( D - E ) GO enrichment analysis of DEGs in IUA-G0/G0 (D) and IUA-G5/IUA-G0 ( E ) groups, including top 10 of BP, CC, and MF. ( F - G ) KEGG analysis of DEGs in IUA-G0/G0 ( F ) and IUA-G5/IUA-G0 ( G ) groups Furthermore, to explore the potential mechanism by which endometrial stem cells cultured in vitro return to normal patterns, we selected genes with inverse relationships between the IUA-G0/G0 and IUA-G5/IUA-G0 groups for analysis. By intersecting the upregulated DEGs in IUA-G0/G0 with the downregulated DEGs in IUA-G5/IUA-G0 and the downregulated DEGs in IUA-G0/G0 with the upregulated DEGs in IUA-G5/IUA-G0, 211 reversed DEGs were screened (Fig.  5 A and B). The BP categories of these DEGs were mainly gliogenesis, urogenital system development and morphogenesis of an epithelium; the CC categories were glutamatergic synapse, synaptic membrane and atrioventricular valve development; the MF categories were metallopeptidase activity, anion transmembrane transporter activity and DNA − binding transcription activator activity, RNA polymerase II − specific (Fig.  5 C, Supplementary Table 7). KEGG analysis showed that these DEGs were related to cytokine − cytokine receptor interaction, the estrogen signaling pathway and the chemokine signaling pathway (Fig.  5 D, Supplementary Table 8). Fig. 5 Functional analysis of DEGs with inverse relationships between IUA-G0/G0 and IUA-G5/IUA-G0 groups. ( A ) Venn diagram depicting variation and overlap of upregulation DEGs in IUA-G0/G0 and downregulation DEGs IUA-G5/IUA-G0 groups. ( B ) Venn diagram depicting variation and overlap of downregulation DEGs in IUA-G0/G0 and upregulation DEGs IUA-G5/IUA-G0 groups. ( C ) GO enrichment analysis of reverse trend DEGs in IUA-G0/G0 and IUA-G5/IUA-G0. (D) KEGG analysis of reverse trend DEGs in IUA-G0/G0 and IUA-G5/IUA-G0 Functional analysis of DEGs with inverse relationships between IUA-G0/G0 and IUA-G5/IUA-G0 groups. ( A ) Venn diagram depicting variation and overlap of upregulation DEGs in IUA-G0/G0 and downregulation DEGs IUA-G5/IUA-G0 groups. ( B ) Venn diagram depicting variation and overlap of downregulation DEGs in IUA-G0/G0 and upregulation DEGs IUA-G5/IUA-G0 groups. ( C ) GO enrichment analysis of reverse trend DEGs in IUA-G0/G0 and IUA-G5/IUA-G0. (D) KEGG analysis of reverse trend DEGs in IUA-G0/G0 and IUA-G5/IUA-G0

Intrauterine adhesions (IUAs) interfere with embryo implantation by decreasing the surface area of the endometrium and even obstructing the fallopian tube orifice, resulting in infertility, recurrent miscarriages or other conditions that seriously affect the woman’s physical and reproductive health [ 1 , 2 ]. The main causes of IUAs are destruction of the uterine lining by intrauterine surgery and damage caused by intrauterine infection [ 3 ]. The current treatment methods used to address these factors include hysteroscopic adhesiolysis (HA), postoperative placement of an intrauterine device (IUD), the placement of a Foley catheter or the use of biological materials to prevent readhesion [ 4 – 6 ]. However, in the clinic, IUA is a poor therapeutic uterine cavity disease, especially among patients with severe IUA; among these patients, the recurrence rate of IUA after HA is still as high as 62.5%, and the pregnancy rate among IUA patients is only 33.3% [ 7 – 9 ]. Therefore, new therapeutic methods are urgently needed to improve the reproductive function of women with IUA. In recent studies, stem cells with self-renewal and differentiation abilities are shown to have good application prospects in regenerative medicine [ 10 , 11 ], including for endometrial repair and reconstruction [ 12 ]. The repair process after endometrial injury is mediated by endometrial stem/progenitor cells [ 13 ]. The types of stem cells that can promote endometrial repair and regeneration include bone marrow-derived stem cells, umbilical cord-derived mesenchymal stromal cells, adipose-derived stem cells, amniotic epithelial cells, oral mucosal epithelial cells and menstrual blood-derived mesenchymal stem cells [ 14 – 19 ]. Therefore, stem cell therapy can be a valuable treatment approach for IUA patients. To date, multiple clinical trials of stem cell therapy for IUA have been performed worldwide [ 20 , 21 ]. In 2024, the results of a randomized controlled trial revealed that IUA patients treated with a combination of an autologous bone marrow stem cell scaffold plus a Foley balloon catheter had significantly higher rates of sustained pregnancy and live birth than did those treated with Foley balloon catheters alone [ 21 ]. However, importantly, different stem cell origins and different combinations of therapies have different clinical effects on reproductive improvement in IUA patients, so more effective and safer stem cell therapies need to be developed. The aim of study was to evaluate the efficacy of intrauterine infusion with autologous endometrial stem cells and sodium hyaluronate gel intrauterine perfusion in combination with estrogen and acetylsalicylic acid support therapy in improving the degree of IUA, endometrial thickness and pregnancy rate in patients with recurrent moderate-to-severe IUA and to explore the possible mechanism through RNA sequencing.

The incidence of IUA is increasing gradually, and although HA is the preferred treatment, with an effective rate of 100% for mild IUA, the treatment of severe IUA needs improvement [ 9 ]. At present, a variety of stem cells from different sources have been used in the clinical treatment of IUA patients, and have shown varying levels of clinical efficacy and safety [ 15 , 20 , 21 ]. Here, the focus of the study was on the use of autologous endometrial stem cells as a source of stem cells to treat moderate-to-severe IUA. With the treatment strategy of autologous endometrial stem cells and sodium hyaluronate gel intrauterine perfusion in combination with estrogen and acetylsalicylic acid support therapy, 15 IUA patients had an overall pregnancy rate of 60% and an overall live birth rate of 53.3% within two years. Moreover, 15 patients did not have any adverse reactions. With respect to pregnancy rates and live birth rates in other stem cell treatments, research from one study showed that 26 recurrent patients underwent umbilical cord-derived mesenchymal stromal cells transplantation after HA; among them, 10 patients (38.46%) were pregnant, and 8 (30.77%) had live births [ 15 ]. Furthermore, Zhu et al. found an ongoing pregnancy rate of 62.5% and a live birth rate of 56.9% in 72 moderate-to-severe IUA participants treated with an autologous bone marrow stem cells-scaffold plus Foley balloon catheter [ 21 ]. Hence, the overall clinical outcome of the treatment strategy in this study was good and similar to that in Zhu’s study, although the population in this study was small. Compared with other stem cells, autologous endometrial tissue as a stem cell source has the advantages of simple preparation and nonimmunogenicity. In the development of new treatments, in addition to promoting endometrial repair, the use of materials with a certain supportive strength and good tissue receptivity to block the abnormal connection of endometrial tissue is also an important strategy for the treatment of IUAs. Currently, the clinical use of antisticking materials includes mainly intrauterine devices, intrauterine support balloons and bioglue materials [ 23 ]. Studies have shown that the readhesion rate in patients with moderate-to-severe IUA can be reduced to 35% and 30%, respectively, after IUD or supportive balloon placement [ 24 , 25 ]. However, as an intrauterine foreign body, the intrauterine barrier device can easily cause an excessive inflammatory reaction, resulting in endometrial ischaemia and necrosis, consequently affecting endometrial regeneration and repair [ 26 ]. Hyaluronic acid is an elastic natural water-soluble glycosaminoglycan material. Compared with the uterine barrier device, it has better histocompatibility and can effectively support the intrauterine space and prevent endometrial resticking [ 27 ]. In this study, we confirmed that 12.5% hyaluronic acid did not inhibit the proliferation of endometrial stem cells by means of CCK8 experiments. Moreover, IUA patients underwent uterine perfusion combined with 12.5% hyaluronic acid during stem cell therapy to prevent stem cell overflow on the one hand and prevent adhesion on the other hand. Therefore, hyaluronic acid is a good choice of supportive material. In addition, several studies have shown that the use of estrogen after HA is helpful for the repair and regeneration of endometrial wounds, but estrogen affects the remaining endometrium. Therefore, for severe IUAs, estrogen therapy is more appropriate as an adjunctive therapy in combination with other antiadhesion therapies [ 28 ]. Vasodilators such as acetylsalicylic acid are thought to improve endometrial blood flow, and combined hormone replacement therapy has shown some efficacy in promoting endometrial growth [ 29 ]. Therefore, in this study, hormone replacement therapy and acetylsalicylic acid were used for the comprehensive treatment of patients with recurrent moderate-to-severe IUA. Stem cells have the ability to undergo multidirectional/unidirectional differentiation and have great potential in the treatment of traumatic diseases; their functions are mainly through nutritional support, cell differentiation, immunosuppression/anti-inflammation, stimulation/regulation of endogenous cells and so on [ 30 ]. By RNA sequencing, we further revealed that the re-entry of endometrial stem cells cultured in vitro into the patient’s uterine cavity to promote endometrial repair was associated with cytokines. According to the results of the KEGG analysis, the DEGs of G0 and IUA-G0, the DEGs of IUA-G0 and IUA-G5, and the DEGs of abnormal expression in IUA-G0 but recovery in IUA-G5 were all enriched in the cytokine − cytokine receptor interaction. Cytokines are closely related to the development of IUA. If cytokines are abnormally expressed in the endometrium, an imbalance between extracellular matrix (ECM) synthesis and degradation can result in excessive accumulation of ECM, inhibition of endometrial regeneration, replacement of endometrial tissue by connective tissue, and poor endometrial repair, which ultimately leads to endometrial fibrosis and the occurrence of IUA [ 31 , 32 ]. Luo et al. reported that the downregulated expression of the cytokines PAI-1, and MMP-9, and the CXCL13-CXCR5 axis are involved in the repair of IUA by menstrual blood-derived mesenchymal stem cells [ 33 ]. Therefore, the proliferative effect of fifth-generation endometrial stem cells in IUA patients may be due to the recovery of normal expression of some cytokines in vitro after they are removed from their original environment. However, these DEGs require further screening and functional verification. There were still several limitations in this study. First, this study was a before-and-after comparison without a rigorous parallel control group. Second, the sample size was relatively small, and more patients may need to be tested to further verify the effectiveness of autologous endometrial stem cells in the treatment of moderate and severe IUA. Third, this study didn’t conduct additional mechanisms research by which fifth-endometrial stem cells were able to promote endometrial repair, with DEGs and functions identified only by RNA sequencing. Thus, the molecular mechanisms of these potential functional DEGs needs to be investigated further, and we intend to do this in our next study.

In summary, autologous endometrial stem cells and sodium hyaluronate gel intrauterine perfusion in combination with estrogen and acetylsalicylic acid support therapy are effective and promising treatments for patients with moderate-to-severe IUA and can improve the pregnancy rate and live birth rates of IUA patients. Furthermore, change in cytokines may be a potential mechanism by which autologous endometrial stem cells promote endometrial repair.

Below is the link to the electronic supplementary material. Supplementary Material 1 Supplementary Material 1