Endometrial Fibrosis Research Articles

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.

Extracellular Vesicles Derived from Mesenchymal Stem Cells as Cell-Free Therapy for Intrauterine Adhesion.

Intrauterine adhesion (IUA) can occur after trauma to the basal layer of the endometrium, contributing to severe complications in females, such as infertility and amenorrhea. To date, the proposed therapeutic strategies are targeted to relieve IUA, such as hysteroscopic adhesiolysis, Foley catheter balloon, and hyaluronic acid injection have been applied in the clinic. However, these approaches showed limited effects in alleviating endometrial fibrosis and thin endometrium. Mesenchymal stem cells (MSCs) can offer the potential for endometrium regeneration owing to reduce inflammation and release growth factors. On this basis, MSCs have been proposed as promising methods to treat intrauterine adhesion. However, due to the drawbacks of cell therapy, the possible therapeutic use of extracellular vesicles released by stem cells is raising increasing interest. The paracrine effect, mediated by MSCs derived extracellular vehicles (MSC-EVs), has recently been suggested as a mechanism for their therapeutic properties. Here, we summarizes the main pathological mechanisms involved in intrauterine adhesion, the biogenesis and characteristics of extracellular vesicles, explaining how these vesicles could provide new opportunities for MSCs.

Observation on the effect of electroacupuncture on the regeneration of endometrium in rats with intrauterine adhesion

To observe the effect of electroacupuncture (EA) on the degree of endometrial fibrosis and inflammatory response in the rat model of intrauterine adhesion (IUA), so as to explore the possible mechanism of EA underlying improving IUA and promoting endometrium regeneration. Forty-five female SD rats were randomly divided into blank, model and EA groups, with 15 rats in each group. The IUA model was established by mechanical scratching combined with lipopolysaccharide infection. EA was applied to bilateral "Zigong" (EX-CA1) and "Sanyinjiao" (SP6), with acupuncture applied to "Guanyuan" (CV4) for rats in the EA group, started from the 2nd day after modeling, 15 minutes every time, once a day for 2 consecutive estrous cycles. Samples from 5 rats in each group were collected during estrus period. Changes of endometrial histopathology and number of glands were observed after HE staining. The area of endometrial fibrosis was observed and calculated after Masson staining. The positive expressions of collagen type I (Col-I) and transforming growth factor β1 (TGF-β1) proteins in endometrial tissue were detected by immunohistochemistry method. The protein expression of integrin αγβ3 in uterine tissue was detected by Western blot. The contents of interleukin (IL)-1β and tumor necrosis factor α (TNF-α) in uterine tissue were detected by ELISA. Samples from remaining 10 rats in each group were collected on the 8th day of gestation for calculation of the embryo implantation numbers of the rats. HE staining showed complete uterine tissue structure of the rats in the blank group during estrus period, with clear endometrial layer, unobstructed and regular uterine cavity, and dense glands. Destroyed endometrial layer, narrowed and adhered uterine cavity, and sparse glands of the rats were seen in the model group, which was relatively milder in the EA group. Following modeling, the number of endometrial glands, the protein expression of Integrin αγβ3, the number of implanted uterine embryos on the injured side of the model group were significantly decreased (P<0.01), while the area of endometrial fibrosis, the positive expressions of Col-I and TGF-β1 proteins, and the contents of IL-1β and TNF-α in the uterine tissue were significantly increased (P<0.01) in comparison with those in the blank group. After intervention, the number of endometrial glands, the protein expression of Integrin αγβ3, the number of implanted uterine embryos on the injured side of the EA group were significantly increased (P<0.01，P<0.05), while the area of endometrial fibrosis, the positive expressions of Col-I and TGF-β1 proteins, and the contents of IL-1β and TNF-α in the uterine tissue were significantly decreased (P<0.01,P<0.05) compared with the model group. EA can enhance endometrial receptivity, and promote endometrial regeneration, be conducive to embryo implantation in IUA model rats, which may be related to its effect in alleviating endometrial fibrosis and reducing inflammatory response.

Targeting endometrial inflammation in intrauterine adhesion ameliorates endometrial fibrosis by priming MSCs to secrete C1INH

Intrauterine adhesion (IUA) is a common cause of uterine infertility and its histopathologic characteristic is endometrial fibrosis. A shortage of stem cells in the endometrial basalis has been recognized as a common cause of IUA development because approximately 90% of patients suffer from IUA after endometrial injury. In this study, we provide evidence that persistent inflammation is the main contributor to endometrial fibrosis in IUA patients. We further found that treating an IUA-like mouse model with ITI-hUC-MSCs (hUC-MSCs reprogrammed by IL-1β, TNF-α and IFN-γ) significantly decreased endometrial inflammation and fibrosis. Mechanistically, high levels of complement 1 inhibitor (C1INH) secreted by ITI-hUC-MSCs prevented inflammation from inducing profibrotic CD301+ macrophage polarization by downregulating the JAK-STAT signaling pathway. In conclusion, persistent inflammation in the endometria of IUA patients provides macrophage polarization with a profibrotic niche to promote endometrial fibrosis, and the powerful immunomodulatory effects of ITI-hUC-MSCs improve the immune microenvironment of endometrial regeneration.

MicroRNA-122-5p alleviates endometrial fibrosis via inhibiting the TGF-β/SMAD pathway in Asherman's syndrome

Research questionWhat is the effect of miR-122 on the progression and recovery of fibrosis in Asherman's syndrome? DesignEndometrial tissue was collected from 21 patients, 11 with intrauterine adhesion (IUA) and 10 without IUA. Quantitative real-time polymerase chain reaction, immunofluorescence and Western blot were applied to observe the expression of mRNAs/miRNAs and protein, respectively. The endometrial physical injury was carried out in C57BL/6 mice to create an endometrial fibrosis model, with intrauterine injection of adenovirus to compare the antifibrosis and repair function of miR-122 on endometrium. The morphology of the uterus was observed using haematoxylin and eosin staining, and fibrosis markers were detected by immunohistochemistry. ResultsmiR-122 expression was reduced in patients with IUAs, accompanied by fibrosis. MiR-122 overexpression reduced the degree of fibrosis in endometrial stromal cells. Further molecular analyses demonstrated that miR-122 inhibited fibrosis through the TGF-β/SMAD pathway by directly targeting the 3′ untranslated region of SMAD family member 3, suppressing its expression. Notably, miR-122 promoted endometrial regeneration and recovery of pregnancy capacity in a mouse endometrial injury model. ConclusionsmiR-122 is a critical regulator for repair of endometrial fibrosis and provided new insight for the clinical treatment of intrauterine adhesions.

Functionalized human umbilical cord mesenchymal stem cells and injectable HA/Gel hydrogel synergy in endometrial repair and fertility recovery

Intrauterine adhesions (IUA) caused by endometrial injury are one of the main causes of female infertility. The current treatments for endometrial injury offer limited clinical benefits and cannot improve endometrial receptivity and pregnancy outcomes. Tissue engineering and regenerative medicine are considered potential solutions to address this concern and may offer effective treatment methods for the regeneration of injured human endometrium. Herein, we prepared an injectable hydrogel based on oxidized hyaluronic acid (HA-CHO) and hydrazide-grafted gelatin (Gel-ADH). The injectable hydrogel showed satisfactory biocompatibility when mixed with human umbilical cord mesenchymal stem cells (hUCMSCs). In an endometrial injury rat model, the treatment with hUCMSCs-loaded injectable hydrogel significantly enhanced the thickness of the endometrium and increased the abundance of blood vessels and glands in the injured endometrium compared to the control group. The hUCMSCs-loaded injectable hydrogel treatment significantly reduced endometrial fibrosis, decreased the expression of the pro-inflammatory factors (IL-1β and IL-6) and increased the expression of the anti-inflammatory factor (IL-10). This treatment induced endometrial VEGF expression by activating the MEK/ERK1/2 signaling pathway. Moreover, this treatment improved endometrial receptivity to the embryo and restored the embryo implantation rate similar to the sham group (48% in the sham group vs 46% in the treatment group), and this treatment achieved pregnancy and live birth in rats with endometrial injury. In addition, we also preliminarily validated the safety of this treatment in the maternal rats and fetuses. Collectively, our study showed that the hUCMSCs-loaded injectable hydrogel hold potential as an effective treatment strategy promoting rapid recovery of endometrial injury, and this hydrogel is a promising biomaterial for regenerative medicine applications. Statement of significance1. Oxidized hyaluronic acid (HA-CHO)/hydrazide-grafted gelatin (Gel-ADH) hydrogel combined with human umbilical cord mesenchymal stem cells (hUCMSCs) are effective in improving the regeneration of endometrium in the endometrial injury rat model.2. The hUCMSCs-loaded hydrogel treatment promotes the expression of endometrial VEGF through MEK/ERK1/2 signaling pathway and regulates the balance of inflammatory factors.3. The embryo implantation and live birth rates restore to normal level in the endometrial injury rat model, and the hydrogel has no adverse effects on maternal rats, fetuses, and offspring development after the treatments.

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.

Decitabine effect on COL1, COL3, MMP2 and MMP9 in TGF-β1 treated mare endometrial fibroblasts

Endometrial chronic degenerative fibrosis in mares, known as endometrosis is detrimental to fertility. Endometrium type I (COL1) and III (COL3) collagen accumulation and periglandular fibrosis characterize endometrosis. Transforming growth factor β1 (TGFβ1), is a main pro-fibrotic signal for myofibroblast differentiation, promoting collagen synthesis by epigenetic mechanisms in humans. Collagen turnover is controlled by metalloproteinase-2 (MMP-2), MMP-9 and their inhibitors. Because epigenetic alterations may change fibroblast phenotype and control fibroproliferative diseases, we hypothesized that endometrial fibroblasts might be under epigenetic regulation and modulate endometrosis. Epigenetic changes can be reversed and are promising for therapy. Nevertheless, this has not been demonstrated in mares. DNA methylation, a stable epigenetic marker, can be assessed through DNA methyltransferases (DNMTs) action. This study aimed to evaluate if transcription of some genes involved in endometrosis (COL1A1, COL3A1, MMP2 and MMP9) was altered with endometrial fibrosis, and if they were epigenetically modulated, through DNA methylation. Therefore, in vitro epigenetic modulation of equine endometrial fibroblasts treated with TGF-β1 was assessed, and the epigenetic inhibitor, 5-aza-2′-deoxycytidine (5-aza-dc or decitabine), was used to impair fibrogenesis. Fibroblasts from mare endometria (n=5) were treated with TGF-β1 (10 ng/mL) for 48h, followed by decitabine (1 µM) for 48h. As controls, fibroblasts were incubated in culture medium, or with decitabine or TGF-β1, for 96h. TGF-β1 and decitabine effects on DNA methyltransferases(DNMT1, DNMT3A and DNMT3B), COL1A1, COL3A1, MMP2 and MMP9 mRNA levels (qPCR) were evaluated in fibroblasts, and collagen (EIA) and metalloproteinases MMP-2 and -9 (zymography) in conditioned medium. TGF-β1 increased DNMT3A mRNA levels (P<0.05), COL1 and COL3 mRNA (P<0.01 and P<0.001, respectively) and protein (P<0.05). It also decreased MMP2 mRNA levels and enzymatic activity (P<0.0001 and P<0.05, respectively). Decitabine diminished DNMT3A mRNA levels (P<0.001), COL1 (P<0.001) and COL3 (P<0.01) mRNA and protein (P<0.01 and P<0.05, respectively). It increased MMP2 (P<0.01) and MMP9 mRNA (P<0.001), and enzymatic activity (P<0.05 and P<0.01, respectively), in TGF-β1 challenged fibroblasts. Data show an epigenetic involvement in the pathogenesis of endometrosis, as depicted by downregulation of TGF-β1 profibrotic effect in endometrial fibroblasts treated with decitabine. Decitabine influences MMPs reflected by increasing MMP2 and 9 mRNA and enzymatic activity in TGF-β1 treated endometrial fibroblasts. Furthermore, the hypermethylation (indicated by increased DNMT3A mRNA levels) that occurred with TGF-β1 treatment, seems to inhibit the expression and activity of MMP2. Data are in agreement with previous results and suggest that endometrosis is epigenetically modulated by anti-fibrotic genes (MMP2 and MMP9) inhibition, rather than fibrotic genes activation. Further studies will allow to better understand endometrosis epigenetics modulation. UIDB/00276/2020; LA/P/0059/2020; 2022.09161.PTDC

Conditioned medium of mesenchymal stem cells: application in mares with endometrial fibrosis

The use of stem cells to promote tissue regeneration has increased in recent years mainly due to their role in growth, remodeling, and repair processes, increasing the number of progenitor cells in the affected tissue. Conditioned medium, a by-product containing metabolites, growth factors, and extracellular matrix proteins is secreted by cultured stem cells. This conditioned medium has been used as a cell-free therapy in regenerative medicine. The objective of the present study was to evaluate the effect of mesenchymal stem cell conditioned medium on endometrial fibrosis in mares through histopathological evaluation. The selected mares had an average age of 18.61 ± 5.68 years and a history of no foaling in the last two breeding seasons. They were grouped according to the degree of uterine fibrosis described by Kenney in 1978: Grade II (n=4), Grade III (n=2). All mares underwent evaluation of vulvar conformation, complete gynecological examination, and ultrasonographic evaluation of the reproductive tract, as well as uterine cytological, bacteriological, and histopathological examinations. The conditioned medium was made from the culture of stem cells derived from the adipose tissue of the lumbarregion of a young horse. During diestrus, using an artificial insemination pipette, 20 mL of conditioned medium was infused into the uterus of each mare, divided into 10 mL per uterine horn. After applying the medium to a horn, 5 mL of sterile 0.9% NaCl solution was administered to ensure that the conditioned medium was infused. Twenty-one days after treatment with the conditioned medium, the six mares were submitted to a new collection of uterine biopsies for histopathological evaluation. In this second evaluation, the same changes characteristic of endometrosis were observed in the histopathology, such as nests with glandular dilation and the presence of areas of fibrosis. These findings suggest that, although no improvement was observed in the histopathological characteristics of endometrial biopsies, the use of the conditioned medium of stem cells did not induce an inflammatory reaction following its intrauterine use, suggesting its safety for further studies in equine medicine. In conclusion, the application of 20 mL of conditioned medium from stem cells in the uterine lumen of six mares with endometrosis grades II and III did not induce endometrial regeneration in the evaluated period.

Endometrial fibrosis in mares: Picrosirius Red and Collagen-2 and 3

In mares, endometrial fibrosis results from abnormal collagen deposition in the stroma and around the endometrial glands. The pathogenesis of endometrial fibrosis remains unknown, but the different types of collagens are a fundamental part of the process. The Picrosirius Red (PSR) staining is widely studied and necessary for collagen fiber and tissue remodeling. The present study aimed to determine the collagen deposition and the gene expression of collagen-2(COL-2) and collagen-3(COL-3) in the endometrium of mares with different degrees of fibrosis. Cyclic mares (n = 34) with a mean age of 14 ± 6.4 years old were used. A uterine biopsy from each mare was obtained at diestrus (D5–D10). Mares were grouped according to the degree of uterine fibrosis described by Kenney (1978): Grade I (n = 12), Grade II (n = 12), and Grade III (n = 10). Collagen in the equine endometrium was morphometrically quantified under PSR staining. The morphometric quantification of the images was performed from each sample after digital microphotographs were taken at 200x magnification, processed, and evaluated using the ImageJ 1.52a software. COL-2 and COL-3 expression was performed using qPCR. Grades were considered independent factors. Collagen-stained area and transcript expression were considered dependent factors. Data were analyzed with ANOVA andt-test. A Pearson correlation was performed between age and collagen deposition. The percentage of tissue collagen area stained with PSR revealed a significant increase between all groups in collagen deposition according to the degree of fibrosis: Grade I (11.7 ± 1.4%), Grade II (17.8 ± 1.3%), and Grade III (24.1 ± 1.9%). Age and the percentage of collagen deposited in the endometrial tissue correlated (r = 0.5) (P = 0.015). Grade II samples presented higher expression of COL-2 transcripts compared to Grade I (P = 0.026) and Grade III (P = 0.003). However, COL-3 did not show differences between groups. This study agrees with other studies that indicate that the worsening of degenerative changes coincides with advanced mare age. Sample evaluations emphasizing degenerative changes rely on an experienced evaluator, while the PSR, followed by computerized analysis, presents quantitative data. In conclusion, the increase in PSR analysis in Grade II and Grade III mares compared to Grade I demonstrates that this technique could play an important role in routine analyses for the detection, quantification, and dynamics of collagen in specific tissues. The study revealed that COL-2 might be involved in the progression and differentiation of healthy endometrium into an endometrium with severe fibrosis.

Are degenerative changes in a single endometrial biopsy representative for the endometrial state?

Equine endometrial fibrosis is diagnosed by microscopy of an endometrial biopsy. It is however uncertain how representative a single biopsy is for the state of the entire endometrium. While previous studies have compared samples from multiple sites, further clarification is needed on specific degenerative changes that may vary in severity between sites. Histopathology was compared, with an emphasis on degenerative changes, in two full thickness specimens from the ventral base of the left and right uterine horns, collected post slaughter from cyclic mares (n=82). Hematoxylin and eosin-stained sections were blinded evaluated by the same evaluator for layers of periglandular fibroblasts (mild[0-3], moderate[4-10], severe[>10]), glandular nests per view (mild[0-2], moderate[3-4], severe[>4]), dilated glands per view (mild[0-3], moderate[4-6], severe[>6]), degree of glandular dilation (mild, moderate, severe), excessive lymphatic vessels (absent, present), and lymphatic lacunae (absent, present). In addition, each section was scored according to a modified Kenney and Doig classification. Statistical analysis included a multiple logistic regression model for fraction of mares with disagreement in Kenney and Doig category, a multiple generalized linear regression model for fraction of parameters with disagreement, a multiple logistic regression model for fraction of mares with disagreement within each parameter, and equivalence tests for agreement of a cumulative score ofdegenerative parameters. Possible effect of age, breed and estrous cycle stage on disagreement between paired uterine horns were included in the analysis. In 49% of mares, specimens from paired uterine horns were assigned the same Kenney and Doig category, and in 46% of mares, the specimens were assigned either of two adjacent categories. In 5% of mares, the specimens varied with two categories. Disagreement in Kenney and Doig category between paired uterine horns increased with each year of age (OR=1.1, p=0.012). Analysis of degenerative parameters showed a disagreement in at least two parameters in 39% of mares, and the number of parameters with disagreement increased with age (HR=1.04, p=0.005). Disagreement within each parameter ranged from 10% of mares (glandular nests), to 31% (periglandular fibroblasts) and 38% (excessive lymphatic vessels). Disagreement increased with age for parameters involving glands (OR=1.14-1.16, p<0.04). When tested for equivalence, the cumulative scores for paired uterine horns did not differ for mares younger than 20 years (p<0.001). In conclusion, although cumulative scores of degenerative changes were equal for paired samples in mares under 20 years of age, the changes were not uniformly distributed. This suggests that conclusions based on a single biopsy, particularly in mares over 20 years of age, should be interpreted with caution, and evaluation of two biopsies is recommended.

Epigenetic regulation of collagen in the mare's placenta: putative relationship with maternal age

Placental plasticity influences fetal development, pregnancy success, and foal growth. Older fertile mares deliver heavier foals and placentas, with increased collagen deposition. In humans, epigenetics regulates placental development and function. Epigenetics, through DNA methylation, also modulates endometrial fibrosis. As such, we hypothesized that as mares get older, collagen deposition in the placenta might be epigenetically modulated. This study assessed if transcription of collagen genes in the equine placenta was epigenetically modulated through DNA methyltransferases (DNMTs), and affected by aging. Immediately after foaling, samples from different chorioallantoic areas (gravid horn, non-gravid horn, and body) were collected from young (n=10; 4-6 years) and older mares (n=10; 12-18 years). Gene transcription of collagen type 1 (COL1A1), collagen type 3 (COL3A1), and DNA methyltransferases (DNMT1, DNMT3A and DNMT3B) was determined (qRT-PCR). Unpaired T-test and Mann-Whitney test were used with statistical significance set a p <0.05. In older mares, COL1A1 mRNA levels were higher in the gravid than in the non-gravid horn (p<0.05), and also upregulated in comparison to young mares’ placentas (p<0.05). In young mares, these transcripts were increased in the placenta body in comparison to the gravid (p<0.05) and non-gravid horns (p<0.01). In older mares, COL3A1 transcriptionwas higher in gravid and non-gravid horns, with respect to the same placenta regions in young mares (p<0.05). In older mares, DNMT1 mRNA expression in the body was lower than in younger mares (p<0.05). For all placentas, DNMT1 transcripts in the non-gravid horn were lowest, compared to the gravid horn (p<0.01) and body (p<0.001). In young mares, DNMT1 mRNA expression in the body was also higher than in the gravid horn (p<0.05). With mares aging, there was a downregulation of DNMT3A transcripts in gravid and non-gravid placenta horns, compared to young mares’ placentas (p< 0.05). In older mares, its levels were lowest in the gravid horn, compared to the non-gravid horn (p<0.05) and body (p<0.01). Similarly, in older mares’ placentas, DNMT3B transcripts were downregulated in the gravid horn (p<0.05) and placenta body (p<0.001), compared to young mares. DNMT3B mRNA levels were lowest in the non-gravid horn, both in young mares, in comparison to placenta body (p<0.01) and in older mares, with respect to the gravid horn and body (p<0.01). Considering mares aging, hypomethylation indicated by decreased DNMTs transcripts, in parallel with upregulation of collagen gene expression in gravid and non-gravid horns, may suggest epigenetic involvement on placental plasticity. However, further research is needed to unravel the intricacies of epigenetic modulation of collagen in equine placentas.

Immunohistochemical expression of myeloperoxidase in the equine endometrium suffering from endometrosis

Myeloperoxidase (MPO) is a pro-oxidant enzyme with potent bactericidal activity playing an important role in the innate immune response against microbial pathogens. MPO is a marker of neutrophil activation, but it is also expressed by epithelial cells. In the endometrium of mares in physiological conditions, MPO is immunohistochemically expressed in a hormonal-dependent fashion with maximal expression in mid and basal glands during estrus (Parrilla Hernández et al. Animals. 2023;13(3):375). This suggests that MPO, as a part of the uterine mucosal immune system, contributes to the prevention of equine endometrial contamination and inflammation. Thus, an alteration of the MPO expression may have an impact on uterine immune defense. A deviation in the endometrial expression pattern of some other proteins has already been associated with endometrosis (Hoffmann et al. Theriogenology. 2009;71:264-274) and it has been proposed as one of the reasons for the reduced fertility in mares suffering of this condition. Therefore, the aim of this study was to investigate the immunohistochemical expression pattern of MPO in the estrous equine endometrium affected by endometrosis. Endometrial biopsies (n=7) obtained from mares in estrus with histological detection of three or less neutrophils per field(400x) and presenting a moderate endometrial fibrosis were studied. Immunohistochemical analysis using MPO-specific horse antibody was performed. The uterine glandular epithelium within fibrotic foci mostly presented a non-steroidal dependent immunostaining pattern reaction. In comparison to unaffected glands, most glandular epithelial cells surrounded by periglandular fibrosis exhibited no or decreased MPO immunostaining. No differences were observed between single glands or nests. These results are in accordance with previous studies for other endometrial expressed proteins. This decrease of MPO expression in the presence of periglandular fibrosis may be produced by a lack of paracrine communication between stromal and epithelial cells, which is necessary for the regulation of uterine secretory proteins by sex hormones (Pierro et al. Biology of reproduction. 2001;64(3):831-838). Although further investigation with different degrees of endometrosis is necessary, the reduced expression of MPO compared to that expected in estrus we observed in this study suggests that endometrosis may lead to disturbance in the uterine immune environment, which in turn may predispose to an increase of susceptibility to uterine infections and endometritis in mares.

Contribution of TGFß and of a cocktail of TGFß and the pro-inflammatory cytokines IL-1, IL- 6 and TNFα to the establishment of a fibrotic phenotype in endometrial fibroblasts of mares

Endometrosis in mares is characterized by inflammation, with continuous activity mainly of the profibrotic cytokine TGFβ. Other proinflammatory cytokines (IL-1, IL-6 and TNF-α) might impact on fibrosis. This aberrant signaling favors conversion of fibroblasts into myofibroblasts, exacerbating the expression of a-SMA, an imbalance of MMPs/TIMPs and uncontrolled deposition of extracellular matrix components. Also, miRNA deregulation impacts on gene expression controlling fibrosis. MicroRNA might be engulfed in extracellular vesicles (EV) secreted by fibrotic cells and induce the pathogenic phenotype in neighboring cells. We challenged endometrial fibroblasts in vitro with either TGFβ alone, combined with other cytokines (TGFβ + IL1 + IL6 + TNF -α; 10ng/ml each cytokine) or vehicle (DMEM, 1% FCS) for 24h. At time points zero, 24, 48 and 72h, cellular proteins were isolated for Western blot analysis and mRNA was converted to cDNA for RT-qPCR. Culture medium was also collected and EV separated by ultracentrifugation, quantified using NTA and Western blotting and their cargo offibrotic-related miRNAs was evaluated using the DDCT qPCR method; this was expressed in Fold Change (FC) with the vehicle group as the reference. Statistical analysis included ANOVA and Tukey posthoc, significance was set at p<0.05. Profibrotic genes aSMA, Col3A1/Col1a1, MMP9/TIMP1, MMP2/TIMP2 were upregulated in the treated groups, as well as phosphorylation of SMAD2 protein. Profibrotic miRNAs (miR17, 21 and 433) were upregulated, while anti-fibrotic miRNAs (miR26, 29a/b, 145,378 and 488) were downregulated in the treatment groups, but not in the vehicle control. TGFβ is sufficient for converting fibroblast to myofibroblast; however, the combined used of selected pro-inflammatory cytokines exacerbates the fibrotic phenotype. These findings may help to unravel the role of inflammation in the early stages of endometrial fibrosis in mares and also to develop cellular models in which to test anti-fibrotic drugs or therapeutic approaches. Funding: FONDECYT 1210349 and VRID 219.153.027-INV.

The Therapeutic Potential of Multipotent Mesenchymal Stromal Cell-Derived Extracellular Vesicles in Endometrial Regeneration.

Disruption of endometrial regeneration, fibrosis formation, and intrauterine adhesions underlie the development of "thin" endometrium and/or Asherman's syndrome (AS) and are a common cause of infertility and a high risk for adverse obstetric outcomes. The methods used (surgical adhesiolysis, anti-adhesive agents, and hormonal therapy) do not allow restoration of the regenerative properties of the endometrium. The experience gained today with cell therapy using multipotent mesenchymal stromal cells (MMSCs) proves their high regenerative and proliferative properties in tissue damage. Their contribution to regenerative processes is still poorly understood. One of these mechanisms is based on the paracrine effects of MMSCs associated with the stimulation of cells of the microenvironment by secreting extracellular vesicles (EVs) into the extracellular space. EVs, whose source is MMSCs, are able to stimulate progenitor cells and stem cells in damaged tissues and exert cytoprotective, antiapoptotic, and angiogenic effects. This review described the regulatory mechanisms of endometrial regeneration, pathological conditions associated with a decrease in endometrial regeneration, and it presented the available data from studies on the effect of MMSCs and their EVs on endometrial repair processes, and the involvement of EVs in human reproductive processes at the level of implantation and embryogenesis.

Exosomes from Human Umbilical Cord Mesenchymal Stem Cells Facilitates Injured Endometrial Restoring in Early Repair Period through miR-202-3p Mediating Formation of ECM.

Endometrial damage repair disorder is the main reason of intrauterine adhesions (IUA) and thin endometrium (TA), which is caused by curettage or infection. Exosomal miRNAs derived from human umbilical cord mesenchymal stem cells (hucMSCs) were reported to play an important role in damage repair disorder, including endometrial fibrosis. In this study, we aimed to investigate the role of hucMSCs-derived exosomal microRNA-202-3p (miR-202-3p) in endometrial damage repair. We established rat endometrial injury model according to curettage to mimic women curettage abortion operation. The miRNA array analysis indicated that miR-202-3p was increased and matrix metallopeptidase 11 (MMP11) was decreased in the exosomes-treated rat uterine tissues. Bioinformatics analysis suggested that MMP11 is the target gene of miR-202-3p. We observed that the mRNA and protein of MMP11 were significantly decreased in exosome treatment group on day 3, and the components of extracellular matrix (ECM) COL1A1, COL3A1, COLVI and fibronectin (FN) protein were increased. And we found that when the injured human stromal cells were treated with miR-202-3p overexpression exosomes, the COLVI and FN were also upregulated in protein and mRNA expression level. For the first time MMP11 was proved to be the target gene of miR-202-3p by dual luciferase reporter system. At last, we found the state of stromal cells was better in miR-202-3p overexpression exosomes group compared to exosomes group, and miR-202-3p overexpression exosomes markedly upregulated the FN and collagen on day 3 after endometrial injury. We thought that miR-202-3p overexpression exosomes promoted endometrial repair by regulating ECM remodeling in early repair of damaged endometrium. Taken together, these experimental findings may provide a theoretical basis for understanding endometrial repair and an insight into the clinical treatment for IUA. Human umbilical cord mesenchymal stem cells exosomal miR-202-3p could regulate the expression of MMP11 and promote the accumulation of extracellular matrix, such as COL1A1, COL3A1, COLVI, FN, in the early repair period of endometrial injury.

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-β1. PBM alleviated TGF-β1-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.

Effect of stem cell conditional medium-loading adhesive hydrogel on TGF-β1-induced endometrial stromal cell fibrosis.

Introduction: Uterine adhesion (IUA) is a severe complication that results from uterine operations or uterine infections. Hysteroscopy is considered the gold standard for the diagnosis and treatment of uterine adhesions. Yet, this invasive procedure leads to re-adhesions after hysteroscopic treatment. Hydrogels loading functional additives (e.g., placental mesenchymal stem cells (PC-MSCs)) that can act as physical barriers and promote endometrium regeneration are a good solution. However, traditional hydrogels lack tissue adhesion which makes them unstable under a rapid turnover of the uterus, and PC-MSCs have biosafety risks when used as functional additives. Methods: In this study, we coupled an adhesive hydrogel with a PC-MSCs conditioned medium (CM) to form a hybrid of gel and functional additives (CM/Gel-MA). Results and Discussion: Our experiments show that CM/Gel-MA enhances the activity of endometrial stromal cells (ESCs), promotes cell proliferation, and reduces the expression of α-SMA, collagen I, CTGF, E-cadherin, and IL-6, which helps to reduce the inflammatory response and inhibit fibrosis. We conclude that CM/Gel-MA can more potentially prevent IUA by combining the physical barriers from adhesive hydrogel and functional promotion from CM.

Oroxylin A relieves intrauterine adhesion in mice through inhibiting macrophage pyroptosis via SIRT3-SOD2-ROS pathway.

Intrauterine adhesion (IUA) is manifested by endometrial fibrosis and inflammation, which seriously affects female reproductive health. Macrophages are mainly inflammatory cells and have been reported to participate in the fibrosis of IUA. Oroxylin A (OA), a kind of flavonoid compounds, was showed to possess the inhibitory effects on inflammation and fibrosis. However, the role of OA in IUA remains unclear. In the present study, we found that OA effectively alleviated the level of inflammation and uterine fibrosis in IUA mice. OA also decreased the macrophage pyroptosis which increased in uteri of IUA mice. Pyroptosis is a programmed cell death accompanied by an inflammatory response. Moreover, OA repressed the mediators of pyroptosis including the expression of NOD-like receptor family pyrin domain containing 3 (NLRP3), caspase-1 and Gasdermin D (GSDMD) and the release of IL-1β, IL-18 and cleaved-caspase-1 in J774A.1 cells induced by LPS/ATP in vitro. Mechanistically, the alleviation of OA on uterine fibrosis is achieved by inhibiting macrophage pyroptosis via SIRT3-SOD2-ROS pathway. Our data indicate that OA may serve as an effective agent for the treatment of the endometrial fibrosis with IUA.

Single-Cell Profiling Uncovers the Roles of Endometrial Fibrosis and Microenvironmental Changes in Adenomyosis.

Adenomyosis (AM) is a common benign uterine disorder that has deleterious effects on women's health. However, the pathogenesis of AM is not clearly understood. We aimed to investigate the pathophysiological changes and molecular mechanism in AM. Single-cell RNA sequencing (scRNA-seq) was employed to construct a transcriptomic atlas of various cell subsets from the ectopic endometrium (EC) and eutopic endometrium (EM) of one AM patient and evaluate differential expression. The Cell Ranger software pipeline (version 4.0.0) was applied to conduct sample demultiplexing, barcode processing and mapping reads to the reference genome (human GRCh38). Different cell types were classified with markers with the "FindAllMarkers" function, and differential gene expression analysis was performed with Seurat software in R. The findings were confirmed by Reverse Transcription Real-Time PCR using samples from three AM patients. We identified nine cell types: endothelial cells, epithelial cells, myoepithelial cells, smooth muscle cells, fibroblasts, lymphocytes, mast cells, macrophages and unknown cells. A number of differentially expressed genes, including CLO4A1, MMP1, TPM2 and CXCL8, were identified from all cell types. Functional enrichment showed that aberrant gene expression in fibroblasts and immune cells was related to fibrosis-associated terms, such as extracellular matrix dysregulation, focal adhesion and the PI3K-Akt signaling pathway. We also identified fibroblast subtypes and determined a potential developmental trajectory related to AM. In addition, we identified increased cell-cell communication patterns in EC, highlighting the imbalanced microenvironment in AM progression. Our results support the theory of endometrial-myometrial interface disruption for AM, and repeated tissue injury and repair could lead to increased fibrosis in the endometrium. Therefore, the present study reveals the association between fibrosis, the microenvironment, and AM pathogenesis. This study provides insight into the molecular mechanisms regulating AM progression.