The regulatory effect of electroacupuncture on endometrial M1-type macrophages in rats with intrauterine adhesions. / ç”µé’ˆå¯¹å®«è ”ç²˜è¿žå¤§é¼ å­å®«å† è†œM1型巨噬细胞的调控作用.

OBJECTIVES: To observe the effect of electroacupuncture(EA) on endometrial fibrosis and M1-type macrophages in rats with intrauterine adhesions(IUA), so as to explore the possible mechanism of EA in the treatment of IUA. METHODS: Fifteen female SD rats were randomly divided into blank group, model group and EA group, with 5 rats in each group. The IUA rat model was established by double damage method using mechanical scraping combined with lipopolysaccharide infection. Rats in the EA group were treated with acupuncture at "Guanyuan"(CV4), and EA at bilateral "Zusanli"(ST36) and "Sanyinjiao"(SP6)for 20 minutes each time, once a day, for 3 consecutive cycles of estrus. Five rats in each group were sampled during the estrous period, and the endometrial morphology, endometrial thickness and the number of blood vessels and glands were observed after HE staining. The fibrotic area of the uterus was observed after Masson staining. The positive expressions of Runt-related transcription factor(RUNX1), transforming growth factor-ß1(TGF-ß1), connective tissue growth factor(CTGF), α-smooth muscle actin(α-SMA), collagen type I(Col-Ⅰ), cluster of differentiation 86(CD86), interleukin-1ß(IL-1ß), and tumor necrosis factor-α(TNF-α) in endometrial tissue were detected by immunohistochemistry. Western blot was used to detect relative protein expressions of RUNX1, TGF-ß1, α-SMA, CD86, and TNF receptor 2 (TNFR2), and real-time fluorescence quantitative PCR was used to detect mRNA expressions of RUNX1, TGF-ß1, α-SMA, CD86, and TNF-α in the endometrium. RESULTS: During the estrous phase, the endometrial layer in the model group was damaged, with reduced folds, disordered arrangement of epithelial cells, loose fibrous connective tissue, significant narrowing and adhesions in the uterine cavity, interstitial congestion, edema, and a significant infiltration of inflammatory cells with sparse glands. While uterine tissue structure of the EA group was basically intact, resembling a normal uterus, with more newly formed glands and a small amount of inflammatory cell infiltration. In comparison with the blank group, the endometrial thickness, the number of blood vessels, and the number of glands were significantly decreased(P<0.001) in the model group, while the ratio of uterine fibrosis area, the positive expressions of RUNX1, TGF-ß1, CTGF, α-SMA, Col-Ⅰ, CD86, IL-1ß, and TNF-α, the protein relative expressions of RUNX1, TGF-ß1, α-SMA, CD86 and TNFR2, and the mRNA relative expression levels of RUNX1, TGF-ß1, α-SMA, CD86 and TNF-α in the endometrium were significantly increased (P<0.001, P<0.01). Compared to the model group, the endometrial thickness, the number of blood vessels, and the number of glands were significantly increased(P<0.01, P<0.05) in the EA group, while the ratio of uterine fibrosis area, the positive expressions of RUNX1, TGF-ß1, CTGF, α-SMA, Col-Ⅰ, CD86, IL-1ß and TNF-α in the endometrial tissue, the protein expressions of RUNX1, TGF-ß1, α-SMA, CD86 and TNFR2, and the mRNA relative expressions of RUNX1, TGF-ß1, α-SMA, CD86 and TNF-α in the endometrium were significantly decreased (P<0.001, P<0.01, P<0.05). CONCLUSIONS: EA can improve endometrial fibrosis in IUA rats, which may be related to its function in decreasing the level of endometrial M1-type macrophages and the secretion of related inflammatory factors.

Impaired fertility in adenomyosis: a murine model reveals endometrial receptivity and progesterone resistance imbalances.

In brief: The impact of adenomyosis on reproductive health needs to be fully understood. By using a murine model, this study provides novel insights into the nuanced mechanisms associated with fertility challenges and offers a foundation for targeted interventions. Abstract: This study investigates the intricate relationship between adenomyosis and reproductive health using a murine model, offering novel insights into this prevalent gynecological disorder. Adenomyosis, characterized by the invasive growth of endometrial tissue into the myometrium, is believed to negatively impact fertility. However, the challenge lies in disentangling this influence, as adenomyosis often coexists with other gynecological diseases. A tamoxifen-induced mice model presents a significant advantage by enabling the specific study of adenomyosis, devoid of confounding influences of concurrent gynecological diseases such as endometriosis. Focusing exclusively on adenomyosis, our study aims to elucidate pathogenic mechanisms underlying fertility issues, focusing on estrous cyclicity, ovarian follicle development, and overall fertility. Our findings uncover disruptions in estrous cyclicity, characterized by an increased duration of time spent in the estrus phase in adenomyosis-induced mice. These disturbances are potentially linked to observed compromised folliculogenesis and the remarkable reduction in litter number and size in mice affected by adenomyosis. Moreover, this study unveils potential drivers of subfertility such as progesterone resistance and altered endometrial receptivity. Within the uteri of mice with adenomyosis, reduced expression of the progesterone receptor and a decreased expression of two implantation-related markers (HoxA10 and integrin ß3) were observed. This comprehensive examination sheds light on the nuanced complexities of adenomyosis-associated reproductive challenges, providing a foundation for targeted interventions in addressing fertility issues related to this disease.

Janus adhesive microneedle patch loaded with exosomes for intrauterine adhesion treatment.

Intrauterine adhesions (IUAs) are common sequelae of cervical mucosa damage caused by uterine curettage. Establishing an anti-adhesion barrier between the damaged endometrium with a sustained-release drug capability and hence promoting endogenous regeneration of the endometrium is an available treatment for IUA. However, current therapy lacks long-term intracavitary residence, drug-delivery permeability, and tissue anti-adhesion to the endometrium. Here, we report the design of a Janus microneedle patch consisting of two layers: an adhesive inner layer with an exosomes-loaded microneedle, which endows the patch with a tissue adhesive capability as well as transdermal drug-delivery capability; and an anti-adhesion outer layer, which prevents the intrauterine membrane from postoperative adhesion. This Janus adhesive microneedle patch firmly adhered to uterine tissue, and sustainedly released ∼80% of the total loaded exosomes in 7 days, hence promoting the expression of vascular- and endothelial-related cell signals. Furthermore, the anti-adhesive layer of the microneedle patch exhibited low cell and protein adhesion performance. In rats, the microneedle patch successfully prevented uterine adhesions, improved endometrial angiogenesis, proliferation, and hormone response levels. This study provides a stable anti-adhesion barrier as well as efficient drug-release capability treatment for intrauterine adhesion treatment.

Histological study of telocytes in mice intrauterine adhesion model and their positive effect on mesenchymal stem cells in vitro.

Intrauterine adhesions (IUA), the main cause of secondary infertility in women, result from irreversible fibrotic repair of the endometrium due to inflammation or human factors, accompanied by disruptions in the repair function of endometrial stem cells. This significantly impacts the physical and mental health of women in their childbearing years. Telocytes (TCs), a distinctive type of interstitial cells found in various tissues and organs, play diverse repair functions due to their unique spatial structure. In this study, we conduct the inaugural exploration of the changes in TCs in IUA disease and their potential impact on the function of stem cells. Our results show that in vivo, through double immunofluorescence staining (CD34+/Vimentin+; CD34+/CD31-), as endometrial fibrosis deepens, the number of TCs gradually decreases, telopodes shorten, and the three-dimensional structure becomes disrupted in the mouse IUA mode. In vitro, TCs can promote the proliferation and cycle of bone mesenchymal stem cells (BMSCs) by promoting the Wnt/ß-catenin signaling pathway, which were inhibited using XAV939. TCs can promote the migrated ability of BMSCs and contribute to the repair of stem cells during endometrial injury. In addition, TCs can inhibit the apoptosis of BMSCs through the Bcl-2/Bax pathway. In conclusion, our study demonstrates, for the first time, the resistance role of TCs in IUA disease, shedding light on their potential involvement in endometrial repair through the modulation of stem cell function.

Hypoxic bone marrow mesenchymal stem cell exosomes promote angiogenesis and enhance endometrial injury repair through the miR-424-5p-mediated DLL4/Notch signaling pathway.

Background: Currently, bone marrow mesenchymal stem cells (BMSCs) have been reported to promote endometrial regeneration in rat models of mechanically injury-induced uterine adhesions (IUAs), but the therapeutic effects and mechanisms of hypoxic BMSC-derived exosomes on IUAs have not been elucidated. Objective: To investigate the potential mechanism by which the BMSCS-derived exosomal miR-424-5p regulates IUA angiogenesis through the DLL4/Notch signaling pathway under hypoxic conditions and promotes endometrial injury repair. Methods: The morphology of the exosomes was observed via transmission electron microscopy, and the expression of exosome markers (CD9, CD63, CD81, and HSP70) was detected via flow cytometry and Western blotting. The expression of angiogenesis-related genes (Ang1, Flk1, Vash1, and TSP1) was detected via RT‒qPCR, and the expression of DLL4/Notch signaling pathway-related proteins (DLL4, Notch1, and Notch2) was detected via Western blotting. Cell proliferation was detected by a CCK-8 assay, and angiogenesis was assessed via an angiogenesis assay. The expression of CD3 was detected by immunofluorescence. The endometrial lesions of IUA rats were observed via HE staining, and the expression of CD3 and VEGFA was detected via immunohistochemistry. Results: Compared with those in exosomes from normoxic conditions, miR-424-5p was more highly expressed in the exosomes from hypoxic BMSCs. Compared with those in normoxic BMSC-derived exosomes, the proliferation and angiogenesis of HUVECs were significantly enhanced after treatment with hypoxic BMSC-derived exosomes, and these effects were weakened after inhibition of miR-424-5p. miR-424-5p can target and negatively regulate the expression of DLL4, promote the expression of the proangiogenic genes Ang1 and Flk1, and inhibit the expression of the antiangiogenic genes Vash1 and TSP1. The effect of miR-424-5p can be reversed by overexpression of DLL4. In IUA rats, treatment with hypoxic BMSC exosomes and the miR-424-5p mimic promoted angiogenesis and improved endometrial damage. Conclusion: The hypoxic BMSC-derived exosomal miR-424-5p promoted angiogenesis and improved endometrial injury repair by regulating the DLL4/Notch signaling pathway, which provides a new idea for the treatment of IUAs.

Endometrial mesenchymal stromal/stem cells improve regeneration of injured endometrium in mice.

BACKGROUND: The monthly regeneration of human endometrial tissue is maintained by the presence of human endometrial mesenchymal stromal/stem cells (eMSC), a cell population co-expressing the perivascular markers CD140b and CD146. Endometrial regeneration is impaired in the presence of intrauterine adhesions, leading to infertility, recurrent pregnancy loss and placental abnormalities. Several types of somatic stem cells have been used to repair the damaged endometrium in animal models, reporting successful pregnancy. However, the ability of endometrial stem cells to repair the damaged endometrium remains unknown. METHODS: Electrocoagulation was applied to the left uterine horn of NOD/SCID mice causing endometrial injury. Human eMSC or PBS was then injected into the left injured horn while the right normal horn served as controls. Mice were sacrificed at different timepoints (Day 3, 7 and 14) and the endometrial morphological changes as well as the degree of endometrial injury and repair were observed by histological staining. Gene expression of various inflammatory markers was assessed using qPCR. The functionality of the repaired endometrium was evaluated by fertility test. RESULTS: Human eMSC successfully incorporated into the injured uterine horn, which displayed significant morphological restoration. Also, endometrium in the eMSC group showed better cell proliferation and glands formation than the PBS group. Although the number of blood vessels were similar between the two groups, gene expression of VEGF-α significantly increased in the eMSC group. Moreover, eMSC had a positive impact on the regeneration of both stromal and epithelial components of the mouse endometrium, indicated by significantly higher vimentin and CK19 protein expression. Reduced endometrial fibrosis and down-regulation of fibrosis markers were also observed in the eMSC group. The eMSC group had a significantly higher gene expression of anti-inflammatory factor Il-10 and lower mRNA level of pro-inflammatory factors Ifng and Il-2, indicating the role of eMSC in regulation of inflammatory reactions. The eMSC group showed higher implantation sites than the PBS group, suggesting better endometrial receptivity with the presence of newly emerged endometrial lining. CONCLUSIONS: Our findings suggest eMSC improves regeneration of injured endometrium in mice.

Minimally invasive delivery of human umbilical cord-derived mesenchymal stem cells by an injectable hydrogel via Diels-Alder click reaction for the treatment of intrauterine adhesions.

Intrauterine adhesions (IUA) are the most common cause of uterine infertility, and conventional treatments have not consistently achieved satisfactory pregnancy rates. Stem cell therapy shows promising potential for the clinical treatment of IUA. Although various advanced biomaterials have been designed for delivering stem cells to the uterine cavity, there remain significant challenges, particularly in devising therapeutic strategies for clinical application that minimize surgical incisions and conform to the intricate structure of uterine cavity. Herein, an injectable hydrogel loaded with human umbilical cord-derived mesenchymal stem cells (UCMSCs) was synthesized via the Diels-Alder click reaction for endometrial regeneration and fertility restoration, exhibiting suitable mechanical properties, good biocompatibility, and desirable degradation properties. Notably, this hydrogel permitted minimally invasive administration and integrated seamlessly with surrounding tissue. Our study revealed that the UCMSCs-laden injectable hydrogel enhanced cell proliferation, migration, angiogenesis, and exhibited anti-fibrotic effects in vitro. The implantation of this hydrogel significantly facilitated endometrium regeneration and restored fertility in a rat endometrial damage model. Mechanistically, in vivo results indicated that the UCMSCs-laden injectable hydrogel effectively promoted macrophage recruitment and facilitated M2 phenotype polarization. Collectively, this hydrogel demonstrated efficacy in regenerating damaged endometrium, leading to the restoration of fertility. Consequently, it holds promise as a potential therapeutic strategy for endometrial damage and fertility decline arising from intrauterine adhesions. STATEMENT OF SIGNIFICANCE: Severe endometrial traumas frequently lead to intrauterine adhesions and subsequent infertility. Stem cell therapy shows promising potential for the clinical treatment of IUA; however, challenges remain, including low delivery efficiency and compromised stem cell activity during the delivery process. In this study, we fabricated an injectable hydrogel loaded with UCMSCs via the Diels-Alder click reaction, which exhibited unique bioorthogonality. The in situ-gelling hydrogels could be introduced through a minimally invasive procedure and adapt to the intricate anatomy of the uterus. The UCMSCs-laden injectable hydrogel promoted endometrial regeneration and fertility restoration in a rat endometrial damage model, efficaciously augmenting macrophage recruitment and promoting their polarization to the M2 phenotype. The administration of UCMSCs-laden injectable hydrogel presents a promising therapeutic strategy for patients with severe intrauterine adhesion.

Novel therapeutic targets, including IGFBP3, of umbilical cord mesenchymal stem-cell-conditioned medium in intrauterine adhesion.

Mesenchymal stem cells play important roles in repairing injured endometrium. However, the molecular targets and potential mechanism of the endometrial recipient cells for stem cell therapy in intrauterine adhesion (IUA) are poorly understood. In this study, umbilical cord mesenchymal stem-cell-conditioned medium (UCMSCs-CM) produced positive effects on a Transforming growth factor beta (TGF-ß) induced IUA cell model. RNA-sequencing was performed on clinical IUA tissues, and the top 40 upregulated and top 20 downregulated mRNAs were selected and verified using high-throughput (HT) qPCR in both tissues and cell models. Based on a bioinformatic analysis of RNA-sequencing and HT-qPCR results, 11 mRNAs were uncovered to be the intervention targets of UCMSCs-CM on IUA endometrium cell models. Among them, IGFBP3 was striking as a key pathogenic gene and a potential diagnostic marker of IUA, which exhibited the area under the curve (AUC), sensitivity, specificity were 0.924, 93.1% and 80.6%, respectively in 60 endometrial tissues. The silencing of IGFBP3 exerted positive effects on the IUA cell model through partially upregulating MMP1 and KLF2. In conclusion, RNA-sequencing combined with HT qPCR based on clinical tissues and IUA cell models were used in IUA research and our results may provide some scientific ideas for the diagnosis and treatment of IUA.

Photobiomodulation therapy at 632 nm wavelength ameliorates intrauterine adhesion via activation of cAMP/PKA/CREB pathway.

Intrauterine adhesion (IUA), a major cause of uterine infertility, is pathologically characterized by endometrial fibrosis. Current treatments for IUA have poor efficacy with high recurrence rate, and restoring uterine functions is difficult. We aimed to determine the therapeutic efficacy of photobiomodulation (PBM) therapy on IUA and elucidate its underlying mechanisms. A rat IUA model was established via mechanical injury, and PBM was applied intrauterinely. The uterine structure and function were evaluated using ultrasonography, histology, and fertility tests. PBM therapy induced a thicker, more intact, and less fibrotic endometrium. PBM also partly recovered endometrial receptivity and fertility in IUA rats. A cellular fibrosis model was then established with human endometrial stromal cells (ESCs) cultured in the presence of TGF-ß1. PBM alleviated TGF-ß1-induced fibrosis and triggered cAMP/PKA/CREB signaling in ESCs. Pretreatment with the inhibitors targeting this pathway weakened PBM's protective efficacy in the IUA rats and ESCs. Therefore, we conclude that PBM improved endometrial fibrosis and fertility via activating cAMP/PKA/CREB signaling in IUA uterus. This study sheds more lights on the efficacy of PBM as a potential treatment for IUA.

Endometrial mesenchymal stromal/stem cells improve regeneration of injured endometrium in mice

BACKGROUND: The monthly regeneration of human endometrial tissue is maintained by the presence of human endometrial mesenchymal stromal/stem cells (eMSC), a cell population co-expressing the perivascular markers CD140b and CD146. Endometrial regeneration is impaired in the presence of intrauterine adhesions, leading to infertility, recurrent pregnancy loss and placental abnormalities. Several types of somatic stem cells have been used to repair the damaged endometrium in animal models, reporting successful pregnancy. However, the ability of endometrial stem cells to repair the damaged endometrium remains unknown. METHODS: Electrocoagulation was applied to the left uterine horn of NOD/SCID mice causing endometrial injury. Human eMSC or PBS was then injected into the left injured horn while the right normal horn served as controls. Mice were sacrificed at different timepoints (Day 3, 7 and 14) and the endometrial morphological changes as well as the degree of endometrial injury and repair were observed by histological staining. Gene expression of various inflammatory markers was assessed using qPCR. The functionality of the repaired endometrium was evaluated by fertility test. RESULTS: Human eMSC successfully incorporated into the injured uterine horn, which displayed significant morphological restoration. Also, endometrium in the eMSC group showed better cell proliferation and glands formation than the PBS group. Although the number of blood vessels were similar between the two groups, gene expression of VEGF-α significantly increased in the eMSC group. Moreover, eMSC had a positive impact on the regeneration of both stromal and epithelial components of the mouse endometrium, indicated by significantly higher vimentin and CK19 protein expression. Reduced endometrial fibrosis and down-regulation of fibrosis markers were also observed in the eMSC group. The eMSC group had a significantly higher gene expression of anti-inflammatory factor Il-10 and lower mRNA level of pro-inflammatory factors Ifng and Il-2, indicating the role of eMSC in regulation of inflammatory reactions. The eMSC group showed higher implantation sites than the PBS group, suggesting better endometrial receptivity with the presence of newly emerged endometrial lining. CONCLUSIONS: Our findings suggest eMSC improves regeneration of injured endometrium in mice.

Synergistic effects of electroacupuncture and bone marrow mesenchymal stem cell transplantation on rat intrauterine adhesion： an observation. / 电针联合骨髓间å è´¨å¹²ç»†èƒžç§»æ¤æ²»ç–—å¤§é¼ å®«è ”ç²˜è¿žçš„ååŒæ•ˆåº”è§‚å¯Ÿ.

OBJECTIVES: To investigate the effects of electroacupuncture(EA) combined with bone marrow mesen-chymal stem cells(BMSCs) transplantation on the endometrium of rats with intrauterine adhesions(IUA), so as to explore the possible mechanisms underlying their combined therapeutic effects. METHODS: Forty adult female SD rats were randomly divided into control, model, cell, and combined groups. The IUA rat model was established using a dual injury method of mechanical scratching and lipopolysaccharide infection. After successful modeling, on days 1, 3, and 7, rats in the model group received tail vein injection of phosphate buffered solution, while rats in the cell group received tail vein injection of BMSCs suspension for BMSCs transplantation；and rats in the combined group received BMSCs transplantation combined with EA treatment (2 Hz/15 Hz, 1-2 mA), targeting the "Guanyuan"(CV4), bilateral "Zusanli"(ST36) and "Sanyinjiao"(SP6) for 20 min daily for 3 consecutive estrous cycles. After intervention, uterine tissue was collected from 5 rats in each group. Histological analysis was performed using hematoxylin and eosin staining to evaluate endometrial thickness and glandular number. Masson staining was used to assess endometrial fibrosis area. Immunohistochemistry was performed to detect the positive expressions of vascular endothelial growth factor(VEGF), proliferating cell nuclear antigen(PCNA), and estrogen receptor(ER). Western blot analysis was conducted to determine the protein expressions of homeobox A10(HoxA10) and leukemia inhibitory factor(LIF), both key regulators of endometrial receptivity. The remaining 5 rats in each group were co-housed with male rats, and the uterine function recovery was evaluated by assessing the number of embryo implantations. RESULTS: Compared with the control group, the model group showed thinning endometrium(P<0.001), decreased glandular number(P<0.001), increased endometrial fibrosis area(P<0.001), reduced positive expressions of VEGF, PCNA, ER, expressions of HoxA10 and LIF, and decreased embryo implantation number (P<0.001) on the injured side of the uterus. Compared with the model group, the combined group showed a reversal of the aforementioned indicators(P<0.001, P<0.01)；the cell group exhibited thicker endometrium(P<0.001) and reduced endometrial fibrosis area(P<0.001). Compared with the cell group, the combined group showed increased endometrial thickness(P<0.01), elevated glandular number(P<0.05), significantly decreased endometrial fibrosis area(P<0.05), enhanced positive expressions of VEGF, PCNA and ER, expressions of HoxA10 and LIF in the endometrium, and a significant increase in embryo implantation number (P<0.001, P<0.05, P<0.01) on the injured side of the uterus, indicating better results than the cell group. CONCLUSIONS: The combination of EA and BMSCs synergistically promotes the repair of damaged endometrium, improves endometrial morphology, reduces fibrosis levels, enhances vascular regeneration and matrix cell proliferation, improves endometrial receptivity, which ultimately facilitates embryo implantation.

Exploring distinct properties of endometrial stem cells through advanced single-cell analysis platforms.

The endometrium is a dynamic tissue that undergoes cyclic changes in response to ovarian hormones during the menstrual cycle. These changes are crucial for pregnancy establishment and maintenance. Endometrial stem cells play a pivotal role in endometrial regeneration and repair by differentiating into various cell types within the endometrium. However, their involvement in endometrial disorders such as endometriosis, infertility, and endometrial cancer is still not fully understood yet. Traditional bulk sequencing methods have limitations in capturing heterogeneity and complexity of endometrial stem cell populations. To overcome these limitations, recent single-cell analysis techniques, including single-cell RNA sequencing (scRNA-Seq), single-cell ATAC sequencing (scATAC-Seq), and spatial transcriptomics, have emerged as valuable tools for studying endometrial stem cells. In this review, although there are still many technical limitations that require improvement, we will summarize the current state-of-the-art single-cell analysis techniques for endometrial stem cells and explore their relevance to related diseases. We will discuss studies utilizing various single-cell analysis platforms to identify and characterize distinct endometrial stem cell populations and investigate their dynamic changes in gene expression and epigenetic patterns during menstrual cycle and differentiation processes. These techniques enable the identification of rare cell populations, capture heterogeneity of cell populations within the endometrium, and provide potential targets for more effective therapies.

Telocyte-Derived Exosomes Provide an Important Source of Wnts That Inhibits Fibrosis and Supports Regeneration and Repair of Endometrium.

Intrauterine adhesions (IUAs) often occurred after common obstetrical and gynecological procedures or infections in women of reproductive age. It was characterized by the formation of endometrial fibrosis and prevention of endometrial regeneration, usually with devastating fertility consequences and poor treatment outcomes so far. Telocytes (TCs), as a novel interstitial cell type, present in female uterus with in vitro therapeutic potential in decidualization-defective gynecologic diseases. This study aims to further investigate the role of TC-derived Wnt ligands carried by exosomes (Exo) in reversal of fibrosis and enhancement of regeneration repair in endometrium. IUA cellular and animal models were established from endometrial stromal cells (ESCs) and mice, followed with treatment of TC-conditioned medium (TCM) or TC-derived Exo. In cellular model, fibrosis markers (collagen type 1 alpha 1 [COL1A1], fibronectin [FN], and α-smooth muscle actin [α-SMA]), angiogenesis (vascular endothelial growth factor [VEGF]), and pathway protein (ß-catenin) were determined by quantitative reverse transcription polymerase chain reaction (qRT-PCR), Western blotting (WB), and immunofluorescence. Results showed that, TCs (either TCM or TC-derived Exo) provide a source of Wnts that inhibit cellular fibrosis, as evidenced by significantly elevated VEGF and ß-catenin with decreased fibrotic markers, whereas TCs lost salvage on fibrosis after being blocked with Wnt/ß-catenin inhibitors (XAV939 or ETC-159). Further in mouse model, regeneration repair (endometrial thickness, number of glands, and fibrosis area ratio), fibrosis markers (fibronectin [FN]), mesenchymal-epithelial transition (MET) (E-cadherin, N-cadherin), and angiogenesis (VEGF, microvessel density [MVD]) were studied by hematoxylin-eosin (HE), Masson staining, and immunohistochemistry. Results demonstrated that TC-Exo treatment effectively promotes regeneration repair of endometrium by relieving fibrosis, enhancing MET, and angiogenesis. These results confirmed new evidence for therapeutic perspective of TC-derived Exo in IUAs.

The humanin analogue (HNG) alleviates intrauterine adhesions by inhibiting endometrial epithelial cells ferroptosis: a rat model-based study.

STUDY QUESTION: Does a humanin analogue (HNG) have a therapeutic effect on intrauterine adhesions (IUAs) caused by uterine cavity surgery in a rat model? SUMMARY ANSWER: HNG supplementation attenuated the development of endometrial fibrosis and IUAs, improved fertility, and contributed to the regulation of endometrial fibrosis by inhibiting endometrial ferroptosis in rats with IUAs. WHAT IS KNOWN ALREADY: IUAs, which are characterized by endometrial fibrosis, are a common cause of female infertility. Humanin (rattin in rats) is a mitochondrial-derived peptide that is widely expressed in multiple tissues. S14G-humanin (HNG) is an HNG that has been reported to have a protective effect against myocardial fibrosis. STUDY DESIGN, SIZE, DURATION: Endometrial tissues from three patients with IUAs and three controls were tested for humanin expression. Two animal models were used to evaluate the modelling effect of IUAs and the preventive effect of HNG against IUAs. In the first model, 40 rats were equally randomized to control and Day 7, 14, and 21 groups to establish the IUA model. In the second model, 66 rats were equally randomized to the control, IUA, and IUA + humanin analogue (HNG) groups. Erastin was used to induce ferroptosis in the Ishikawa cell line. PARTICIPANTS/MATERIALS, SETTING, METHODS: The endometrium was scraped with a surgical spatula, combined with lipopolysaccharide treatment, to establish the rat model of IUAs. Rats were intraperitoneally injected with 5 mg/kg/day HNG for 21 consecutive days beginning from the day of operation to evaluate the therapeutic effect on IUAs. Haematoxylin-eosin and Masson's trichrome staining were used to assess endometrial morphology and evaluate fibrosis. Ferroptosis-related markers, namely nuclear factor E2-related factor 2 (Nrf2), acyl-CoA synthetase long-chain family member 4 (ACSL4), haeme oxygenase-1 (HO-1), solute carrier family 7 member 11 (SLC7A11), glutathione peroxidase 4 (GPX4), and ferritin, were measured by immunohistochemistry and western blotting to determine whether ferroptosis was involved in the development of IUAs and to assess the attenuative effect of HNG on ferroptosis. Additionally, the female rats were mated with male rats with normal fertility to assess fertility. MAIN RESULTS AND THE ROLE OF CHANCE: Humanin was widely expressed in endometrial cells, including epithelial and stromal cells, in both humans and rats. Humanin expression levels were downregulated in the endometria of patients and rats with IUAs relative to the endometria of controls. Endometrial thickness and the number of glands were significantly decreased on Day 7, 14, and 21 after endometrial scraping when compared with the controls (all P < 0.05), whereas the fibrotic area was significantly increased (P < 0.05). Among the tested ferroptosis markers, the expression levels of Nrf2, SLC7A11, and GPX4 were significantly downregulated and those of ACSL4, HO-1, and ferritin were significantly upregulated after endometrial scraping relative to their expression levels in controls (all P < 0.05). The mating rates in the control, IUA, and IUA + HNG groups were 100% (10/10), 40% (4/10), and 80% (8/10), respectively. The number of embryos in rats with IUAs (mean ± SD: 1.6 ± 2.1) was significantly less than the number in the controls (11.8 ± 1.5). HNG supplementation significantly attenuated this decrease in the number of implanted embryos (6.3 ± 4.5) (P < 0.01). Further results showed that HNG significantly attenuated the altered expression levels of proteins involved in ferroptosis in the endometria of rats with IUAs. Moreover, in vitro experiments showed that HNG significantly attenuated the erastin-induced decrease in the viability of the Ishikawa cell line and also attenuated the increase in reactive oxygen species production and the downregulation of GPX4. LARGE SCALE DATA: None. LIMITATIONS, REASONS FOR CAUTION: The findings of this study showed that HNG inhibited ferroptosis and reduced fibrosis in a rat model of IUAs. However, we could not establish a causal relationship between ferroptosis and the development of IUAs. WIDER IMPLICATIONS OF THE FINDINGS: HNG may be effective at alleviating fibrosis during the development of IUAs, and the inhibition of ferroptosis is a promising new strategy for IUA therapy. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by the National Natural Science Foundation of China (No. 82171647); the '1000 Talent Plan' of Yunnan Province (No. RLQN20200001); and the Basic Research Project of the Yunnan Province-Outstanding Youth Foundation (No. 202101AW070018). The authors declare no competing financial interests.

A molecular staging model for accurately dating the endometrial biopsy.

Natural variability in menstrual cycle length, coupled with rapid changes in endometrial gene expression, makes it difficult to accurately define and compare different stages of the endometrial cycle. Here we develop and validate a method for precisely determining endometrial cycle stage based on global gene expression. Our 'molecular staging model' reveals significant and remarkably synchronised daily changes in expression for over 3400 endometrial genes throughout the cycle, with the most dramatic changes occurring during the secretory phase. Our study significantly extends existing data on the endometrial transcriptome, and for the first time enables identification of differentially expressed endometrial genes with increasing age and different ethnicities. It also allows reinterpretation of all endometrial RNA-seq and array data that has been published to date. Our molecular staging model will significantly advance understanding of endometrial-related disorders that affect nearly all women at some stage of their lives, such as heavy menstrual bleeding, endometriosis, adenomyosis, and recurrent implantation failure.

Melatonin-pretreated human umbilical cord mesenchymal stem cells improved endometrium regeneration and fertility recovery through macrophage immunomodulation in rats with intrauterine adhesions†.

Intrauterine adhesions (IUA) are a common gynecological problem. Stem cell therapy has been widely used in the treatment of IUA. However, due to the complex and harsh microenvironment of the uterine cavity, the effectiveness of such therapy is greatly inhibited. This study aimed to investigate whether melatonin pretreatment enhances the efficacy of human umbilical cord mesenchymal stem cells (HucMSCs) in IUA treatment in rats. First, we explored the effect of melatonin on the biological activity of HucMSCs in vitro through a macrophage co-culture system, Cell Counting Kit 8 (CCK-8), 5-Ethynyl-2'-deoxyuridine (EdU), flow cytometry, immunofluorescence staining, and qRT-PCR. Subsequently, we established the IUA rat model and tracked the distribution of HucMSCs in this model. In addition, we observed the number of M1 and M2 macrophages through immunofluorescence staining and detected the levels of inflammatory cytokines. Four weeks after cell transplantation, HE, Masson, and immunohistochemical staining were performed. In vitro experiments showed that melatonin pretreatment of HucMSCs promoted proliferation, reduced apoptosis, up-regulated the stemness gene, and regulated macrophage polarization. In vivo, melatonin pretreatment caused more HucMSCs to remain in the uterine cavity. Melatonin-pretreated HucMSCs recruited more macrophages, regulated macrophage polarization, and reduced inflammation. Melatonin-pretreated HucMSCs relieved fibrosis, increased endometrium thickness, and up-regulated CD34, vimentin, proliferating cell nuclear antigen (PCNA), and alpha small muscle antigen (α-SMA) expression. Fertility tests showed that melatonin-pretreated HucMSCs increased the number of embryos. In summary, pretreatment with melatonin was beneficial for HucMSC treatment because it enhanced the cell's ability to recruit macrophages and regulate macrophage polarization, which led to the regeneration of the endometrium and improved pregnancy outcomes.

Targeting CD301+ macrophages inhibits endometrial fibrosis and improves pregnancy outcome.

Macrophages are a key and heterogeneous cell population involved in endometrial repair and regeneration during the menstrual cycle, but their role in the development of intrauterine adhesion (IUA) and sequential endometrial fibrosis remains unclear. Here, we reported that CD301+ macrophages were significantly increased and showed their most active interaction with profibrotic cells in the endometria of IUA patients compared with the normal endometria by single-cell RNA sequencing, bulk RNA sequencing, and experimental verification. Increasing CD301+ macrophages promoted the differentiation of endometrial stromal cells into myofibroblasts and resulted in extracellular matrix accumulation, which destroyed the physiological architecture of endometrial tissue, drove endometrial fibrosis, and ultimately led to female infertility or adverse pregnancy outcomes. Mechanistically, CD301+ macrophages secreted GAS6 to activate the AXL/NF-κB pathway, upregulating the profibrotic protein synthesis. Targeted deletion of CD301+ macrophages or inhibition of AXL by Bemcentinib blunted the pathology and improved the outcomes of pregnancy in mice, supporting the therapeutic potential of targeting CD301+ macrophages for treating endometrial fibrosis.

TT-10 may elevate YAP and repair mouse uterine damage resulting from the inhibition effect of ibuprofen on COX2-PGE2 and YAP.

Ibuprofen (IBU) is an emerging environmental contaminant that, in high doses, can damage reproductive organs in humans and other mammals. Recently, its effects on the uterus have been investigated. It is known that the COX2-PGE2 pathway and Yes-associated protein (YAP) are involved in female reproductive organ development and form a COX2-PGE2-EP2-Gas-ß-catenin-YAP-COX2 positive feedback loop, in addition, TT-10, a pharmacological product, has been found to increase YAP. In this study, IBU was orally administrated to female mice for 7 d at doses of 0, 50, 100, and 200 mg/kg·bw/day (control, low, medium, and high doses, respectively). In addition, 0, 50, 100, and 200 µmol/L IBU was added in vitro to cultured uterine cells for 7 d at control, low, medium, and high doses, respectively; then, 0, 5, 10, and 20 µmol/L TT-10 were given to the in vitro uterine culture containing 100 µmol/L IBU to observe the effect of YAP activation. The results showed that medium and high doses of IBU inhibited the COX2-PGE2 pathway, decreasing YAP and increasing pYAP, leading to reduced circPVT1, elevated miR-149, and increased apoptosis, ultimately damaging the uterus. Conversely, 10 µmol/L TT-10 maximally enhanced YAP, which regulated COX2-PGE2 pathway activation, increased circPVT1, and decreased miR-149, and promoted cell proliferation, preventing uterine damage. This suggests that IBU may cause uterine damage by inhibiting the COX2-PGE2 pathway and YAP, and that appropriate doses of TT-10 may repair this damage by elevating YAP and stimulating COX2 via the feedback loop.

MenSCs Transplantation Improve the Viability of Injured Endometrial Cells Through Activating PI3K/Akt Pathway.

Endometrial injury is one of the leading causes of female infertility and is caused by intrauterine surgery, endometrial infection, repeated abortion, or genital tuberculosis. Currently, there is little effective treatment to restore the fertility of patients with severe intrauterine adhesions and thin endometrium. Recent studies have confirmed the promising therapeutic effects of mesenchymal stem cell transplantation on various diseases with definite tissue injury. The aim of this study is to investigate the improvements of menstrual blood-derived endometrial stem cells (MenSCs) transplantation on functional restoration in the endometrium of mouse model. Therefore, ethanol-induced endometrial injury mouse models were randomly divided into two groups: the PBS-treated group, and the MenSCs-treated group. As expected, the endometrial thickness and gland number in the endometrium of MenSCs-treated mice were significantly improved compared to those of PBS-treated mice (P < 0.05), and fibrosis levels were significantly reduced (P < 0.05). Subsequent results revealed that MenSCs treatment significantly promoted angiogenesis in the injured endometrium. Simultaneously, MenSCs enhance the proliferation and antiapoptotic capacity of endometrial cells, which is likely contributed by activating the PI3K/Akt signaling pathway. Further tests also confirmed the chemotaxis of GFP-labeled MenSCs towards the injured uterus. Consequently, MenSCs treatment significantly improved the pregnant mice and the number of embryos in pregnant mice. This study confirmed the superior improvements of MenSCs transplantation on the injured endometrium and uncovered the potential therapeutic mechanism, which provides a promising alternative for patients with serious endometrial injury.

Ferroptosis contributes to endometrial fibrosis in intrauterine adhesions.

Intrauterine adhesions (IUA), characterized by endometrial fibrosis, is a challenging clinical issue in reproductive medicine. We previously demonstrated that epithelial-mesenchymal transition (EMT) and fibrosis of endometrial stromal cells (HESCs) played a vital role in the development of IUA, but the precise pathogenesis remains elucidated. Ferroptosis has now been recognized as a unique form of oxidative cell death, but whether it is involved in endometrial fibrosis remains unknown. In the present study, we performed an RNA-seq of the endometria from 4 severe IUA patients and 4 normal controls. Enrichment analysis and protein-protein interactions (PPIs) network analysis of differentially expressed genes (DEGs) were conducted. Immunohistochemistry was used to assess ferroptosis levels and cellular localization. The potential role of ferroptosis for IUA was investigated by in vitro and in vivo experiments. Here, we demonstrated that ferroptosis load is increased in IUA endometria. In vitro experiments showed that erastin-induced ferroptosis promoted EMT and fibrosis in endometrial epithelial cells (P < 0.05), but did not lead to pro-fibrotic differentiation in endometrial stromal cells (HESCs). Cell co-culture experiments showed that erastin-stimulated epithelial cell supernatants promoted fibrosis in HESCs (P < 0.05). In vivo experiments suggested that elevation of ferroptosis level in mice by erastin led to mild endometrial EMT and fibrosis. Meanwhile, the ferroptosis inhibitor Fer-1 significantly ameliorated endometrial fibrosis in a dual-injury IUA murine model. Overall, our findings revealed that ferroptosis may serve as a potential therapeutic target for endometrial fibrosis in IUA.