Proliferation Of Endometrial Epithelial Cells Research Articles

Metformin ameliorates endometrial thickness in a rat model of thin endometrium.

Metformin, a well-established anti-diabetic drug, is also used in managing various other metabolic disorders including polycystic ovarian syndrome (PCOS). There are evidences to show that metformin improves endometrial functions in PCOS women. However, fewer studies have explored the direct effects of metformin on endometrium. Previous in vitro studies have shown that therapeutic serum concentrations of metformin enhance endometrial epithelial cell proliferation. The present study was undertaken to investigate in vivo effects of metformin on endometrial proliferation in a rat model of thin endometrium. Toward this, a rat model of thin endometrium was developed. Metformin (0.1% or 1% w/v) was administrated orally for 15 days in rats with thin endometrium. Oral metformin administration for three consecutive estrous cycles (15 days) in the thin endometrium rat model led to an increase in endometrial thickness compared to sham endometrium. Histological analysis showed a significant increase in the number of endometrial glands (P < 0.05), stromal cells (P < 0.01) and blood vessels (P < 0.01) in metformin-treated (n = 10 in each group) uterine horns compared to sham (saline-treated) uterine horns in rats. The expression of proliferating cell nuclear antigen and vascular epithelial growth factor was found to be upregulated on treatment with 1% metformin-treated group (n = 7). However, pregnancy outcomes in the rats treated with metformin remained unaltered despite the restoration of endometrial thickness. In conclusion, the study demonstrated that metformin ameliorates endometrial thickness in a rat model of thin endometrium by increasing endometrial proliferation and angiogenesis, without restoration of embryo implantation.

The effect of selenium on the proliferation of bovine endometrial epithelial cells in a lipopolysaccharide-induced damage model

BackgroundEndometritis is a common bovine postpartum disease. Rapid endometrial repair is beneficial for forming natural defense barriers and lets cows enter the next breeding cycle as soon as possible. Selenium (Se) is an essential trace element closely related to growth and development in animals. This study aims to observe the effect of Se on the proliferation of bovine endometrial epithelial cells (BEECs) induced by lipopolysaccharide (LPS) and to elucidate the possible underlying mechanism.ResultsIn this study, we developed a BEECs damage model using LPS. Flow cytometry, cell scratch test and EdU proliferation assay were used to evaluate the cell cycle, migration and proliferation. The mRNA transcriptions of growth factors were detected by quantitative reverse transcription-polymerase chain reaction. The activation of the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) and Wnt/β-catenin pathways were detected by Western blotting and immunofluorescence. The results showed that the cell viability and BCL-2/BAX protein ratio were significantly decreased, and the cell apoptosis rate was significantly increased in the LPS group. Compared with the LPS group, Se promoted cell cycle progression, increased cell migration and proliferation, and significantly increased the gene expressions of TGFB1, TGFB3 and VEGFA. Se decreased the BCL-2/BAX protein ratio, promoted β-catenin translocation from the cytoplasm to the nucleus and activated the Wnt/β-catenin and PI3K/AKT signaling pathways inhibited by LPS.ConclusionsIn conclusion, Se can attenuate LPS-induced damage to BEECs and promote cell proliferation and migration in vitro by enhancing growth factors gene expression and activating the PI3K/AKT and Wnt/β-catenin signaling pathways.

Metformin exhibits antineoplastic effects on Pten-deficient endometrial cancer by interfering with TGF-β and p38/ERK MAPK signalling

Metformin is a widespread antidiabetic agent that is commonly used as a treatment against type 2 diabetes mellitus patients. Regarding its therapeutic potential, multiple studies have concluded that Metformin exhibits antineoplastic activity on several types of cancer, including endometrial carcinoma. Although Metformin’s antineoplastic activity is well documented, its cellular and molecular anticancer mechanisms are still a matter of controversy because a plethora of anticancer mechanisms have been proposed for different cancer cell types. In this study, we addressed the cellular and molecular mechanisms of Metformin’s antineoplastic activity by using both in vitro and in vivo studies of Pten-loss driven carcinoma mouse models. In vivo, Metformin reduced endometrial neoplasia initiated by Pten-deficiency. Our in vitro studies using Pten-deficient endometrial organoids focused on both cellular and molecular levels in Metformin’s tumor suppressive action. At cellular level, we showed that Metformin is involved in not only the proliferation of endometrial epithelial cells but also their regulation via a variety of mechanisms of epithelial-to-mesenchymal transition (EMT) as well as TGF-β-induced apoptosis. At the molecular level, Metformin was shown to affect the TGF-β signalling., a widely known signal that plays a pivotal role in endometrial carcinogenesis. In this respect, Metformin restored TGF-β-induced apoptosis of Pten-deficient endometrial organoids through a p38-dependent mechanism and inhibited TGF-β-induced EMT on no-polarized endometrial epithelial cells by inhibiting ERK/MAPK signalling. These results provide new insights into the link between the cellular and molecular mechanism for Metformin’s antineoplastic activity in Pten-deficient endometrial cancers.

LIF regulates the expression of miR-27a-3p and HOXA10 in bovine endometrial epithelial cells via STAT3 pathway

LIF is crucial in regulating embryo implantation, while HOXA10 is a marker gene for uterine receptivity. However, the specific mechanism of LIF regulating HOXA10 during cow embryo implantation has not been fully understood. To address this knowledge gap, the experiment involved treating bovine endometrial epithelial cells (BEECs) with LIF to investigate the relationship between LIF, miRNA, and HOXA10. The experimental findings revealed that applying LIF resulted in a substantial increase in the proliferation of endometrial epithelial cells. Moreover, the expressions of PI3K, AKT, HOXA10, CDK4, cyclinD1, and cyclinE1 were significantly elevated. Conversely, the expression of p21Cipl was significantly reduced. In the group that received a combination of LIF and a STAT3 inhibitor, the expression of PI3K/AKT remained significantly increased, but there was no significant change in the expression of HOXA10. When miRNA-27a-3p was overexpressed, it resulted in a decrease in both the RNA and protein expression of HOXA10. Conversely, inhibiting miRNA-27a-3p increased the RNA and protein expression of HOXA10. In the presence of LIF treatment, the expression of miRNA-27a-3p was reduced, while the expression of HOXA10 was increased. However, when LIF and a STAT3 inhibitor were combined, there was no significant change in the expression of miRNA-27a-3p or HOXA10. Consequently, LIF facilitated cell proliferation by activating the PI3K/AKT pathway. LIF controlled the expression of miRNA-27a-3p and HOXA10 in endometrial epithelial cells through STAT3, with miRNA-27a-3p negatively regulating the expression of HOXA10.

Impaired secretion of C-X-C motif chemokine ligand 10 by stimulation with a Toll-like receptor 4 ligand in endometrial epithelium of infertile patients with minimal-to-mild endometriosis

Successful embryo implantation requires transient, well-controlled inflammation in decidualizing cells. In mice, Toll-like receptor (TLR) 4 signaling in endometrial epithelial cells (EECs) by stimulation with factors present in seminal fluids has been shown to be a key upstream driver of a controlled inflammatory response. Clinical evidence supports that exposure of the female reproductive tract to seminal plasma promotes implantation success. We investigated the response of EECs to TLR2 (Pam3Csk4), TLR 3 (Poly I:C), and TLR4 (lipopolysaccharides [LPS]) ligands with respect to secretion of C-X-C motif chemokine ligand (CXCL) 10 (CXCL10) and interleukin-6 (IL-6) in infertile patients with minimal-to-mild endometriosis (EECs-endo) (n = 38) and those of healthy, fertile women (EECs-healthy) (n = 30). Stimulation with either Pam3Csk4, Poly I:C or LPS, significantly induced CXCL10 and IL-6 in EECs-healthy (p < 0.05). In EECs-endo, either Pam3Csk4 or Poly I:C significantly induced CXCL10 (p < 0.05), whereas no significant response was observed after stimulation with LPS. Neither LPS, Poly I:C, nor Pam3Csk4 significantly induced IL-6 secretion in EECs-endo. Secretion of CXCL10 in EECs-healthy after stimulation with LPS was significantly higher (p < 0.05) than that in EECs-endo. CXCL10 decreased cell proliferation of EECs from both groups. Activation of nuclear factor kappa light chain enhancer of activated B cells and signal transducer and activator of transcription 3 signalings was not impaired, but activation of p38 mitogen-activated protein kinases signaling by LPS stimulation was impaired in EECs-endo. The present findings suggested that an insufficient response of EECs to a TLR4 ligand may be involved in molecular mechanisms of endometriosis-associated infertility.

CircRNA3669 promotes goat endometrial epithelial cells proliferation via miR-26a/RCN2 to activate PI3K/AKT-mTOR and MAPK pathways

The development of receptive endometrium (RE) from pre-receptive endometrium (PE) for successful embryo implantation is a complex dynamic process in which the morphology and physiological states of the endometrial epithelium undergo a series of significant changes, including cell proliferation and apoptosis. However, the molecular mechanisms are not yet fully understood. In this study, a higher circRNA3669 level was observed in PE than in RE of goats. Functional assays revealed that this overexpression promoted the proliferation of goat endometrial epithelial cells (GEECs) by activating the expression of genes related to the PI3K/AKT-mTOR and MAPK pathways, thereby inhibiting apoptosis in vitro. Furthermore, circRNA3669 functioned as a competing endogenous RNA (ceRNA) to upregulate Reticulocalbin-2 (RCN2) expression at the post-transcriptional level by interacting with and downregulating miR-26a in GEECs. In addition, RCN2, which is highly expressed in the PE of goats, was found to be regulated by β-estradiol (E2) and progesterone (P4). Our results demonstrated that RCN2 also affected the key proteins PI3K, AKT, mTOR, JNK, and P38 in the PI3K/AKT-mTOR and MAPK pathways, thereby facilitating GEECs proliferation and suppressing their apoptosis in vitro. Collectively, we constructed a new circRNA3669-miR-26a-RCN2 regulatory network in GEECs, which further provides strong evidence that circRNA could potentially play a crucial regulatory role in the development of RE in goats.

Bisphenol S Increases Cell Number and Stimulates Migration of Endometrial Epithelial Cells.

To determine whether bisphenol S (BPS), a common substitute for bisphenol A (BPA), induces cell proliferation and migration in human endometrial epithelial cells (Ishikawa) and adult mouse uterine tissues. Human endometrial Ishikawa cells were exposed to low doses of BPS (1 nM and 100 nM) for 72 hours. Cell proliferation was assessed through the viability assays MTT and CellTiter-Glo®. Wound healing assays were also used to evaluate the migration potential of the cell line. The expression of genes related to proliferation and migration was also determined. Similarly, adult mice were exposed to BPS at a dose of 30 mg/kg body weight/day for 21 days, after which, the uterus was sent for histopathologic assessment. BPS increased cell number and stimulated migration in Ishikawa cells, in association with the upregulation of estrogen receptor beta (ESR2) and vimentin (VIM). In addition, mice exposed to BPS showed a significantly higher mean number of endometrial glands within the endometrium. Overall, in vitro and in vivo results obtained in this study showed that BPS could significantly promote endometrial epithelial cell proliferation and migration, a phenotype also observed with BPA exposure. Hence, the use of BPS in BPA-free products must be reassessed, as it may pose adverse reproductive health effects to humans.

Proteomic profiles and the function of RBP4 in endometrium during embryo implantation phases in pigs

BackgroundEndometrial receptivity plays a vital role in the success of embryo implantation. However, the temporal proteomic profile of porcine endometrium during embryo implantation is still unclear.ResultsIn this study, the abundance of proteins in endometrium on days 9, 10, 11, 12, 13, 14, 15 and 18 of pregnancy (D9, 10, 11, 12, 13, 14, 15 and 18) was profiled via iTRAQ technology. The results showed that 25, 55, 103, 91, 100, 120, 149 proteins were up-regulated, and 24, 70, 169, 159, 164, 161, 198 proteins were down-regulated in porcine endometrium on D10, 11, 12, 13, 14, 15 and 18 compared with that on D9, respectively. Among these differentially abundance proteins (DAPs), Multiple Reaction Monitoring (MRM) results indicated that S100A9, S100A12, HRG and IFI6 were differentially abundance in endometrial during embryo implantation period. Bioinformatics analysis showed that the proteins differentially expressed in the 7 comparisons were involved in important processes and pathways related to immunization, endometrial remodeling, which have a vital effect on embryonic implantation.ConclusionOur results reveal that retinol binding protein 4 (RBP4) could regulate the cell proliferation, migration and apoptosis of endometrial epithelial cells and endometrial stromal cells to affect embryo implantation. This research also provides resources for studies of proteins in endometrium during early pregnancy.

METTL3 promotes proliferation of goat endometrial epithelial cells by regulating CTGF in an m6A-dependent manner.

N6-methyladenosine (m6A), an epigenetic modification on RNAs, plays an important role in many physiological and pathological processes. However, the involvement of m6A in goat uterus during early pregnancy remains largely unknown. In this study, we found that the total m6A level was increasing in goat uterus as early pregnancy progressed. Methyltransferase-like 3 (METTL3) is a core catalytic subunit of the m6A methyltransferase. We thus determined the expression and regulation of METTL3 in goat uterus. METTL3 was highly expressed in the luminal and glandular epithelia from day 16 (D16) to D25 of pregnancy. And it could be up-regulated by estrogen and progesterone in goat uterus and primary endometrial epithelial cells (EECs). In EECs, knockdown or overexpression of METTL3 resulted in a significant decrease or increase of cell proliferation, respectively. METTL3 knockdown reduced the m6A level of not only total RNA but also connective tissue growth factor (CTGF) mRNA. Luciferase assay suggested that METTL3 might target the potential m6A sites in the 3'untranslated region (3'UTR) of CTGF mRNA. Moreover, METTL3 positively regulated CTGF expression, and CTGF knockdown significantly counteracted the promoting effect of METTL3 overexpression on EEC proliferation. Collectively, METTL3 is dynamically expressed in goat uterus and can affect EEC proliferation by regulating CTGF in an m6A-dependent manner. Our results will lay a foundation for further studying the crucial mechanism of METTL3-mediated m6A modification in goat uterus during early pregnancy.

Mechanism of human chorionic gonadotropin in endometrial receptivity via the miR‐126‐3p/PI3K/Akt/eNOS axis

Human chorionic gonadotropin (hCG) might affect endometrial receptivity, exerting integral roles in embryo implantation. This study explored the action of hCG in endometrial receptivity via the miR-126-3p/PIK3R2/PI3K/Akt/eNOS axis. The embryo implantation dysfunction (EID) mouse models were established by administrating mifepristone and human endometrial epithelial cells (EECs) were used for in vivo experiments, both followed by hCG treatment. Expression level of CD105 and protein levels of cadherin CD144 and CD146 in mice were determined by immunohistochemistry and Western blot. The levels of miR-126-3p and PIK3R2 mRNA and PIK3R2, p-PI3K p85 α, PI3K p110 α, p-Akt, Akt, p-eNOS, and eNOS protein levels were measured. Cell proliferation was evaluated by CCK-8 and EdU assays. The binding sites of miR-126-3p and PIK3R2 were predicted and verified. hCG-treated EECs were further transfected with miR-126-inhibitor for functional rescue experiments. hCG ameliorated endometrial receptivity in EID mice. Moreover, hCG promoted miR-126-3p and suppressed PIK3R2 in EID mice and EECs. miR-126-3p targeted PIK3R2. EEC proliferation was enhanced after hCG treatment but inhibited by miR-126-3p downregulation. Both in vivo and in vitro experiments validated that hCG activated the PI3K/Akt/eNOS pathway through the miR-126-3p/PIK3R2 axis. Collectively, hCG improves endometrial receptivity by activating the PI3K/Akt/eNOS pathway via regulating miR-126-3p/PIK3R2.

Detrimental actions of obesity-associated advanced glycation end-products on endometrial epithelial cell proliferation are alleviated by antioxidants

Advanced glycation end-products (AGE) are elevated in the uterine environment of obese infertile women. Can the detrimental effects of AGE on endometrial epithelial cells be mitigated with therapeutics, and recapitulated in a more physiologically relevant primary model (organoids)? Human endometrial epithelial cells (ECC-1) were exposed to AGE at concentrations physiologically representative of uterine fluid in lean or obese individuals, and three potential therapeutics: 25nmol/l receptor for AGE (RAGE) antagonist FPS-ZM1, 100μmol/l metformin, or a combination of antioxidants (10μmol/l N-acetyl-l-cysteine, 10μmol/l N-acetyl-l-carnitine and 5μmol/l α-lipoic acid). Real-time cell analysis (xCELLigence, ACEA Biosciences) determined the rate of adhesion and proliferation. The proliferation of organoid-derived cells and secretion of cytokines from organoids was characterized in the presence of AGE (n = 5). The uterine fluid of women undergoing assisted reproduction was profiled for AGE-associated inflammatory markers (n = 77). ECC-1 proliferation was reduced by AGE from obese versus lean conditions and vehicle control (P = 0.04 and P<0.001, respectively), and restored to a proliferation corresponding to lean conditions by antioxidants. AGE influenced organoid derived primary endometrial epithelial cell proliferation in a donor-dependent manner. AGE increased the organoid secretion of the proinflammatory cytokine CXCL16 (P = 0.006). Clinically, CXCL16 correlated positively to maternal body mass index (R = 0.264, P = 0.021) and intrauterine glucose concentration (R = 0.736, P<0.0001). Physiologically relevant concentrations of AGE alter endometrial epithelial cell function. Antioxidants restore the rate of proliferation of AGE-treated endometrial epithelial (ECC-1) cells. Primary endometrial epithelial cells, cultured as organoids, demonstrate altered proliferation and CXCL16 secretion in the presence of AGE equimolar with the uterine fluid from obese individuals.

RGMb expression in goat uterine tissues: possible role of RGMb in the proliferation and apoptosis of endometrial epithelial cells.

Context Bone morphogenetic proteins (BMPs) play an important role in the uteri. Repulsive guidance molecule b (RGMb; a.k.a. Dragon) has been confirmed as the coreceptor of BMPs to function through drosophila mothers against decapentaplegic protein (Smads) and mitogen-activated protein kinases (MAPK) pathways. We hypothesise that RGMb regulates the uterine function through the Smads and MAPK pathways. Aims This study aimed to investigate the expression of RGMb in goat uteri and the potential role of RGMb in the endometrial epithelial cells (EECs). Methods The localisation of RGMb in goat uterine tissues was detected by immunohistochemistry (IHC), EECs were isolated and transfected with siRNA to investigate the role of RGMb in proliferation, and apoptosis. The expression levels of Smads and MAPK members was measured by western blot (WB) and real-time PCR (RT-PCR). Key results IHC showed that RGMb was localised in goat endometrial luminal cells, glandular epithelial cells, and circular muscle fibres, but not in stromal cells. RT-PCR results showed that treatment with RGMb siRNA suppressed the expressions of proliferation-related genes cyclin D1 (CCND1 , P =0.0291), cyclin-dependent kinase 2 (CDK2 P =0.0107), and proliferating cell nuclear antigen (PCNA, P =0.0508), leading to the reduced viability of EECs (P =0.0010). WB results showed that the expression ratio of cleaved-caspase 3/caspase 3 (P =0.0013) was markedly increased after RGMb siRNA transfection. Likewise, the level of phospho-extracellular signal-regulated kinase 1/2 (p-ERK1/2, P =0.0068) and p-Smad1/5/8 (P =0.0011) decreased significantly, while there were no appreciable differences in the level of p-P38 MAPK expression (P >0.05). Conclusions RGMb might participate in the regulation of cell proliferation and apoptosis through Smads and ERK signalling pathways in goat EECs. Implications RGMb is involved in regulating the proliferation and apoptosis in goat endometrial epithelial cells.

CRC Therapy Identifies Indian Hedgehog Signaling in Mouse Endometrial Epithelial Cells and Inhibition of Ihh-KLF9 as a Novel Strategy for Treating IUA.

Intrauterine adhesion (IUA) causes menstrual disturbance and infertility. There is no effective treatment available for moderate to severe IUA cases. Stem cell-based therapy has been investigated for treating IUA but is limited in clinical applications due to issues including the precise induction of differentiation, tumorigenesis, and unclear molecular mechanisms. In our recent study, we isolated and expanded the long-term cultures of conditional reprogrammed (CR) mouse endometrial epithelial cells. Treating IUA mice with these CR cells (CRCs) restored the morphology and structure of the endometrium and significantly improved the pregnancy rate. In this study, our data with high-throughput sequencing, CRISPR knockout Ihh-/-CRCs, and transplantation identified for the first time that the Indian hedgehog (Ihh) gene plays a critical role in the regulation of endometrial epithelial cell proliferation. We also found that aberrant activated Ihh-krüppel-like factor 9 (KLF9) signaling contributes to the inhibition of normal progesterone receptor (PR) function in IUA mice. Thus, we hypothesized that inhibition of the Ihh-KLF9 pathway may be a novel strategy to treat IUA. Our data demonstrated that treatment with the hedgehog signaling inhibitor Vismodegib restored the morphology, structure, and microenvironment of the endometrium, and greatly improved the pregnancy rate in IUA mice. This study suggests a promising application of hedgehog inhibitors as a targeted drug in the IUA clinic.

Therapeutic metformin concentrations positively regulate proliferation in endometrial epithelial cells (EECs) via mTOR activation and augmented mitochondrial strength.

Metformin, an antidiabetic drug, has recently been repositioned in the treatment of several nondiabetic disorders including reproductive disorders such as polycystic ovarian syndrome (PCOS), where it improves endometrial functions. In vitro studies employing supratherapeutic concentrations (5-20 mM) of metformin, have reported anti-proliferative effects on endometrial epithelial and stromal cells. However, animal and human studies have revealed that therapeutic serum concentrations of metformin range between 20-70 µM. In the present study, the effect of therapeutic concentrations of metformin was studied on endometrial epithelial cells (EECs). Therapeutic concentrations of metformin induced proliferation in Ishikawa and HEC-1A cells. The proliferation of EECs was found mTOR dependent. Interestingly, therapeutic metformin concentrations were not able to activate the classical AMPK signaling. On the contrary, supratherapeutic metformin concentration (10 mM) inhibited mTOR and activated AMPK signaling. Microarray analysis of metformin-treated HEC-1A cells revealed dose-dependent differential effects on biological pathways associated with translation, ribosomal RNA processing, mitochondrial translation and cell proliferation. Therapeutic concentrations of metformin upregulated mitochondrial number as demonstrated by increased MitotrackerTM Red staining and enhanced succinate dehydrogenase (SDHD) expression; however higher concentration (10 mM) abrogated the same. Our results suggest that therapeutic concentrations of metformin augment mitochondrial strength and induce mTOR dependent endometrial cell proliferation.

Protective Role of Bone Marrow Mesenchymal Stem Cells (BMSCs) Expressing miR-124 in Endometritis

Endometritis is correlated to repeated implantation failures. MicroRNA (miRNA) participates in several inflammatory diseases and miR-124 is involved in many diseases. Bone marrow stromal cells (BMSCs) are closely connected to the regulation of inflammation. Human endometrial epithelial cells (HEECs) were cultured in vitro , assigned into control group, LPS group and BMSC group, and miR-124 overexpressing BMSCs were constructed and co-cultured with HEECs followed by analysis of HMGB1 and NF- κ B expression by Western Blot, and the proliferation and apoptosis of HEECs. In LPS group, HEECs proliferation and miR-124 decreased, apoptosis and HMGB1 increased ( P &lt; 0.05). After co-culture with BMSCs, it can promote HEECs proliferation, inhibit apoptosis, increase miR-124, and decrease HMGB1, NF- κ b and the secretion of inflammatory factors ( P &lt; 0.05) with more significant changes in the high miR-124 expression group. miR-124 in endometritis endometrial epithelial cells is downregulated. In conclusion, BMSCs with high expression of miR-124 can inhibit inflammation and regulate endometrial epithelial cell apoptosis by regulating HMGB1 and NF- κ B, thereby promoting endometrial epithelial cells proliferation and delaying endometritis progression.

Human Umbilical Cord Mesenchymal Stem Cell-Derived Conditioned Medium Promotes Human Endometrial Cell Proliferation through Wnt/β-Catenin Signaling.

Purpose Mesenchymal stem cells (MSCs) and their derivant are among the promising treatments for intrauterine adhesion (IUA); they have been reported to repair the endometrial injury by proliferating endometrial cells. However, the signal pathways involved are not clear. This study investigated the role of human umbilical cord mesenchymal stem cell-derived conditioned medium (hUCMSC-CM) in relieving IUA to find out whether Wnt/β-catenin signaling was involved, and if so, to determine the possible ligands. Methods After endometrial epithelial cells (EECs) were treated with hUCMSC-CM, their proliferation and migration were measured by the CCK8 assay and the scratch assay. The activation of Wnt/β-catenin signaling was measured by Western blots, fluorescent staining, and T-cell factor/lymphoid enhancer factor (TCF/LEF) luciferase. A Wnt inhibitor (XAV393) was used to inhibit the proliferation effect of hUCMSC-CM in EECs. Wnt5a expression in hUCMSC was measured by Western blots and fluorescent staining, and Wnt5a in hUCMSC-CM was detected by enzyme-linked immunosorbent assay (ELISA), to further clarify the mechanism. Results As shown by the CCK8 assay, hUCMSC-CM promoted proliferation and migration of EECs. The expression of β-catenin, c-myc, and cyclin D1 increased in EECs after being treated with hUCMSC-CM. Moreover, hUCMSC-CM was found to promote β-catenin delivery into nuclei by Western blot and fluorescent staining; meanwhile, the inhibitor (XAV393) could restrain this process and inhibit the effect of hUCMSC-CM on EEC proliferation. Wnt5a was detected in hUCMSCs and hUCMSC-CM, which might be a potential therapeutic target. Conclusion This study demonstrated that hUCMSC-CM promoted human endometrial cell proliferation through Wnt/β-catenin signaling, and Wnt5a might be a potential activator. This would be one of the activating signal pathways in the MSC-related treatment of IUA.

Circ_0061140 Facilitates Adenomyosis Progression by Upregulating LIN28B in vitro

Objectives: The aim of our study was to explore the role of circular RNA_0061140 (circ_0061140) in adenomyosis progression and its associated mechanism. Design: We first analyzed the expression pattern of circ_0061140 in endometrial tissues of adenomyosis patients (n = 27) and uterine fibroid patients (n = 15). Loss-of-function experiments were conducted to analyze the biological roles of circ_0061140 in regulating the viability, apoptosis, proliferation, migration, and invasion of endometrial epithelial cells. The downstream microRNA (miRNA)/messenger RNA (mRNA) axis of circ_0061140 was predicted by bioinformatics tool Starbase, and its working mechanism was verified by rescue experiments. Methods: Cell viability, apoptosis, proliferation, invasion, and migration were assessed by cell counting kit-8 assay, flow cytometry analysis, 5-ethynyl-2′-deoxyuridine assay, transwell assay, and scratch test. The binding relationship between miR-141-3p and circ_0061140 or lin-28 homolog B (LIN28B) was verified by dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay. Results: Circ_0061140 expression was upregulated in adenomyosis patients. Circ_0061140 knockdown suppressed the viability, proliferation, invasion, and migration and triggered the apoptosis of endometrial epithelial cells. Circ_0061140 served as a miRNA sponge for miR-141-3p, and miR-141-3p silencing partly reversed circ_0061140 knockdown-induced effects in endometrial epithelial cells. miR-141-3p directly interacted with LIN28B mRNA. LIN28B overexpression partly diminished miR-141-3p overexpression-mediated influences in endometrial epithelial cells. Circ_0061140 knockdown downregulated LIN28B expression by elevating miR-141-3p level in endometrial epithelial cells. Limitations: The functional verification of circ_0061140/miR-141-3p/LIN28B axis was merely conducted in vitro. Conclusion: Circ_0061140 contributed to adenomyosis progression by binding to miR-141-3p to induce LIN28B expression in vitro.

Bone marrow mesenchymal stem cell-derived extracellular vesicles facilitate endometrial injury repair by carrying the E3 ubiquitin ligase WWP1.

Bone marrow mesenchymal stem cells-derived extracellular vesicles (BMSC-EVs) relieve endometrial injury. This study aimed to elucidate the BMSC-EV mechanism in alleviating endometrial injury. Endometrial injury model in vivo was induced using 95% ethanol, and endometrial epithelial cells (EECs) treated with mifepristone were applied as an endometrial injury model in vitro. After BMSCs and BMSC-EVs were isolated and identified, the BMSC-EV function was evaluated by hematoxylin-eosin and Masson staining, immunohistochemistry, quantitative real-time PCR, Cell Counting Kit-8 assay, flow cytometry, enzyme-linked immunosorbent assay, and Transwell and tubule formation assays. The BMSC-EV mechanism was assessed using Western blot, ubiquitination, and cycloheximide-chase assays. After isolation and identification, BMSC-EVs were effective in endometrial injury repair in vivo and facilitated EEC proliferation and repressed cell apoptosis in vitro; the EEC supernatants accelerated human umbilical vein endothelial cell proliferation, migration, and invasion and facilitated angiogenesis after endometrial injury in vitro. For the BMSC-EV mechanism, E3 ubiquitin ligase WWP1 in BMSC-EVs mediated the ubiquitination of peroxisome proliferator-activated receptor gamma (PPARγ), thus relieving the PPARγ inhibition on vascular endothelial growth factor expression. Furthermore, the WWP1 in BMSC-EVs alleviated endometrial injury in vitro and in vivo. BMSC-EVs facilitated endometrial injury repair by carrying WWP1.

P-413 Non-autologous human platelet lysate stimulates in vitro proliferation of primary human endometrial cells from patients with recurrent implantation failure

Abstract Study question Does patient diagnosis impact induction of primary human endometrial cell proliferation by commercial non-autologous human platelet lysate (HPL)? Summary answer HPL stimulates in vitro proliferation of both primary endometrial epithelial cells (EECs) and stromal cells (ESCs) from patients with and without recurrent implantation failure (RIF). What is known already Inadequate endometrial receptivity and thickness are major causes for RIF. Our preliminary results suggested that non-autologous HPL stimulates in vitro proliferation of primary human EECs and ESCs isolated from patients with a history of RIF, with and without a thin endometrium (TE). In addition, using an in vitro model of embryo attachment, we reported that 48-hour treatment with HPL significantly augments the attachment of trophoblast spheroids (generated with HTR-8/SVneo cells) to EECs, suggesting an increase in endometrial receptivity following treatment. This suggests that HPL may standardize future clinical treatments for a TE and endometrial origins of RIF. Study design, size, duration Endometrial tissue was collected from five patients without RIF (control) and eighteen RIF patients at the CReATe Fertility Centre, Toronto, Canada. The eighteen RIF patients were further classified into three groups (N = 6 each) based on endometrial phenotype: 1) proliferative phase RIF only (without a TE), 2) secretory phase RIF only (without a TE), and 3) secretory phase RIF+TE (with a TE). Primary EECs and ESCs were enzymatically isolated and cultured separately. Participants/materials, setting, methods Primary EECs and ESCs were serum-starved with serum-free culture media (SFM) for 24 hours and then treated for 48 hours with the following treatment media: SFM (negative control), or SFM supplemented with 1% HPL for EECs, or 10% HPL for ESCs. Cell viability and proliferation were assessed using the metabolic assay PrestoBlue reagent and immunocytochemistry to quantify cells actively expressing the nuclear proliferation marker Ki67. Main results and the role of chance The metabolic assay demonstrated that 48-hour treatment with non-autologous HPL stimulates a similar significant increase in EEC viability and proliferation for all patient groups. EECs from patients without RIF (control) had the highest fold increase (1.49-fold, P &amp;lt;0.001), followed by proliferative phase RIF only (1.41-fold, P &amp;lt;0.001), secretory phase RIF+TE (1.39-fold, P &amp;lt;0.001), and secretory phase RIF only (1.24-fold, P &amp;lt;0.05). For ESCs, HPL stimulated a significant 2.69-fold increase in cell viability and proliferation for the secretory phase RIF+TE patient group (P &amp;lt;0.01). Pairwise comparison of the ratio of actively proliferating (Ki67+) cells between SFM and HPL treatment revealed that EECs isolated from control patients had the highest and most significant increase by 27.3% (P &amp;lt;0.05) after HPL treatment. Although not statistically significant, EECs isolated from all RIF patients displayed a similar trend of increased Ki67+ cells after HPL treatment. Whereas HPL treatment significantly increased the ratio of Ki67+ cells for ESCs isolated from both the proliferative phase (by 23.9%, P &amp;lt;0.05) and secretory phase (by 29.9%, P &amp;lt;0.05) endometrium of RIF only patients. Limitations, reasons for caution Although our data suggests that HPL treatment significantly stimulates primary endometrial cell proliferation in vitro, our sample size per group is small (N = 5-6). A larger sample size and future randomized controlled trials are needed to determine the efficacy of HPL as a treatment for a TE and/or RIF. Wider implications of the findings Our study provides the groundwork to improve clinical treatment of a TE and endometrial origins of RIF. We anticipate that in addition to stimulating cell proliferation, HPL will also induce a broad transcriptional response towards improved endometrial receptivity. We will next focus on characterizing the transcriptomic profile following HPL treatment. Trial registration number not applicable

Circ_0004712 promotes endometriotic epithelial cell proliferation, migration and invasion by regulating miR-488-3p/ROCK1 axis in vitro

Recent evidence indicates that circular RNAs (circRNAs) play crucial regulatory roles in the pathogenesis and development of endometriosis. Circ_0004712 was found to be differentially expressed in endometriosis. However, the detailed function and mechanism of circ_0004712 in endometriosis are still unclear. Quantitative real-time polymerase chain reaction and Western blot were used for the detection of circ_0004712, miR-488-3p and ROCK1 (Rho Associated Coiled-Coil Containing Protein Kinase 1) levels. In vitro experiments in endometrial endothelial cells were performed by cell counting kit-8, EdU, transwell, wound healing assays, and flow cytometry, respectively. The molecular mechanism of circ_0004712 function was investigated using bioinformatics target predication, dual-luciferase reporter, and RNA immunoprecipitation (RIP) assays. The expression of circ_0004712 was higher in endometriotic endometrial tissues and epithelial cells. Knockdown of circ_0004712 suppressed cell proliferation, migration, invasion, EMT process and induced apoptosis in ectopic endometrial epithelial cells in vitro. Mechanistically, circ_0004712 acted as a ceRNA to sponge miR-488-3p, thus elevating the expression of ROCK1, which was confirmed to be a target of miR-488-3p. Rescue experiments suggested that miR-488-3p inhibition reversed the inhibitory effects of circ_0004712 silencing on cell growth and metastasis. Moreover, miR-488-3p restoration restrained the proliferation and metastasis in ectopic endometrial epithelial cells, which were attenuated by ROCK1 overexpression. Circ_0004712 knockdown suppressed the proliferation and metastasis of ectopic endometrial epithelial cells via miR-488-3p/ROCK1 axis in vitro, suggesting a new insight into the pathogenesis of endometriosis.