Endometrial Epithelial Cells Research Articles

Assessing Postpartum Reproductive Health via Endometrial Cytology and Body Temperature in Mehsana Buffaloes

The aim of this study was to investigate the effect of body temperature and endometrial cytology on postpartum reproductive health in Mehsana buffaloes. A total of twenty-six Mehsana buffaloes were used in this study. Per-rectal and ultrasonography examinations were used to observe the postpartum reproductive function of these buffaloes and on day 60 post natural service, the pregnancy was confirmed. Based on fertile estrus, buffaloes were retrospectively divided into two groups: Group I (buffaloes conceiving before 90 days) and Group II (buffaloes conceiving after 90 days). One week before parturition and four weeks after it, the rectal temperature was taken. The cytobrush technique was used to obtain endometrial samples for cytological tests on the 21st and 30th day postpartum. During the final week of pregnancy and the first, second, third, and fourth weeks’ post-partum, the mean rectal temperature (⁰F) varied from 101.7 ± 0.07 to 102.0 ± 0.06 in Group-I and 101.6 ± 0.07 to 102.1 ± 0.05 in Group-II. During the final week pre-partum and the first week post-partum, the mean rectal temperature (⁰F) was shown to be significantly (p<0.05) lower than it was during the second, third, and fourth weeks post-partum among the groups. On the 21st and 30th day postpartum during endometrial cytology, the percentage of neutrophils was 14.31 ± 1.56 and 12.23 ± 0.93 in Group I and 17.92 ± 1.51 and 15.08 ± 0.73 in Group II, respectively. On the 30th day, there was a significant difference (p<0.05) between the groups in Group II that had a greater mean percentage of neutrophils. When endometrial cytology was performed on buffaloes at various postpartum days, the mean percentage of lymphocytes, endometrial epithelial cells and large vacuolated epithelial cells revealed non-significant changes. On the 30th day, it was discovered that the prevalence of subclinical endometritis in Group I and Group II buffaloes was 7.69 and 23.07 percent, respectively. The postpartum first behavioural estrus and first fertile estrus occurred at 62.00 ± 4.34 and 65.31±4.45 days in Group I and 77.46±11.12 and 130.08±9.26 days in Group II buffaloes, respectively. Although, the average days for first behavioural estrus did not differ significantly between the groups the fertile estrus occurred significantly (P<0.05) delayed in Group II buffaloes.

Abstract 2791: Establishment of immortalized endometrial cell lines using Sendai virus

Abstract Objective: To establish endometrial immortalized cell lines using the Sendai virus and analyze their characteristics. Methods: The study included patients with benign ovarian tumors and endometrioid carcinoma who had regular menstrual cycles. Endometrial cells were collected and separated into epithelial and non-epithelial cells using flowcytometry (FCM) (CD326 (EpCAM) antibody). Furthermore, the isolated cells were infected with Sendai virus carrying three immortalization genes (Bmi-1, hTERT, and SV40T) to create immortalized cell lines. The cells were characterized as epithelial cells by immunocytochemistry (ICC), and receptor expression was assessed by reverse transcription PCR (RT-PCR) and ICC. Non-epithelial cells from the patients with endometrial carcinoma were evaluated using ICC. Results: The immortalized cell lines were capable of more than 20 passages. Immortalized endometrial epithelial cells tested positive for anti-Keratin antibodies and negative for anti-Vimentin antibodies by ICC. Additionally, FCM detected these cells as positive for anti-EpCAM antibody, confirming that they were epithelial cells. RT-PCR confirmed mRNA expression of estrogen α-receptor and progesterone receptor after 20 passages and ICC confirmed protein expression of them after 10 passages, establishing an endometrial epithelial immortalized cell line. Non-epithelial cells isolated from patients with endometrial carcinoma tested positive for anti-α-smooth muscle actin and anti-Vimentin antibodies by ICC, indicating their characterization as cancer-associated fibroblasts. Conclusion: We successfully established an immortalized cell line of endometrial epithelial cells using Sendai virus and generated cancer-associated fibroblasts. Citation Format: Kohei Hikino, Hiroaki Komatsu, Genki Hichiwa, Kanako Kazuki, Yasuhiro Kazuki, Hiroyuki Kugoh, Masayo Okawa, Yuki Ida, Masayo Hosokawa, Mayumi Sawada, Akiko Kudoh, Shinya Sato, Fuminori Taniguchi. Establishment of immortalized endometrial cell lines using Sendai virus [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2791.

Loss of KLF15 impairs endometrial receptivity by inhibiting EMT in endometriosis.

The impaired endometrial receptivity is a major factor contributing to infertility in patients with endometriosis (EM), but the underlying mechanism remains unclear. Our study aimed to investigate the role of Kruppel-like factor 15 (KLF15) in endometrial receptivity and its regulation in EM. We observed a significant decrease in KLF15 expression in the mid-secretory epithelial endometrial cells of EM patients compared to normal females without EM. To confirm the role of KLF15 in endometrial receptivity, we found a significantly reduced KLF15 expression and a significant decrease in embryo implantation number in the rat model via uterine horn infection with siRNA. This highlights the importance of KLF15 as a regulator receptivity. Furthermore, through ChIP-qPCR, we discovered that the progesterone receptor (PR) directly binds to KLF15 promoter regions, indicating that progesterone resistance may mediate the decrease in KLF15 expression in EM patients. Additionally, we found that the mid-secretory endometrium of EM patients exhibited impaired epithelial-mesenchymal transition (EMT). Knockdown of KLF15 upregulated E-cadherin and downregulated vimentin expression, leading to inhibited invasiveness and migration of Ishikawa cells. Overexpression KLF15 promotes EMT, invasiveness, and migration ability, and increases the attachment rate of JAR cells to Ishikawa cells. Through RNA-seq analysis, we identified TWIST2 as a downstream gene of KLF15. We confirmed that KLF15 directly binds to the promoter region of TWIST2 via ChIP-qPCR, promoting epithelial cell EMT during the establishment of endometrial receptivity. Our study reveals the involvement of KLF15 in the regulation of endometrial receptivity and its downstream effects on EMT. These findings provide valuable insights into potential therapeutic approaches for treating non-receptive endometrium in patients with EM.

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.

Secretomes from Conventional Insemination and Intra-Cytoplasmic Sperm Injection Derived Embryos Differentially Modulate Endometrial Cells In Vitro

Conventional Insemination (CI) and Intra-Cytoplasmic Sperm Injection (ICSI) are routinely used insemination methods in clinical Assisted Reproductive Technologies (ART) settings. However, the existing data on the developmental competence and implantation potential of CI and ICSI derived embryos are not unequivocal. This prospective study on 23 patients undergoing ART treatment explored whether the secretomes of CI- and ICSI-derived embryo differentially alter the expression of integrins (αv and β3 integrin) and MUCIN-1 (MUC-1) in a human endometrial epithelial cell line (Ishikawa). Immunocytochemical data demonstrated that the secretome of CI-derived top quality (GI) embryos induced higher (p < 0.05) expression of ɑv β3 compared to sibling ICSI derived G1 embryos in Ishikawa cells. Though, relative levels of the transcript for MUC-1, anti-adhesion molecule did not show a significant difference between the study groups, immunocytochemical analysis demonstrated significantly (p < 0.0001) higher expression of MUC-1 in cells treated with ICSI-derived embryo secretome, compared to that treated with CI -derived embryo secretome. These results suggest that secretomes from CI and ICSI embryos differentially modulate the endometrial cells in vitro. This hints at differences in the ability of CI- and ICSI- derived embryos to alter endometrial profile.

Effect of heat exposure on prostaglandin production and expression of COX-2, PGES, PGFS, ITGAV and LGALS15 mRNAs in endometrial epithelial cells of buffalo (Bubalus bubalis).

Early embryonic mortality is one of the major intriguing factors of reproductive failure that causes considerable challenge to the mammalian cell biologists. Heat stress is the major factor responsible for reduced fertility in farm animals. The present study aimed to investigate the influence of heat stress on prostaglandin production and the expression of key genes, including COX-2, PGES, PGFS, ITGAV and LGALS15, in buffalo endometrial epithelial cells. Buffalo genitalia containing ovaries with corpus luteum (CL) were collected immediately post-slaughter. The stages of the estrous cycle were determined based on macroscopic observations of the ovaries. Uterine lumens of the mid-luteal phase (days 6-10 of the estrous cycle) were washed and treated with trypsin to isolate epithelial cells, which were then cultured at control temperature (38.5°C for 24h) or exposed to elevated temperatures [38.5°C for 6h, 40.5°C for 18h; Heat Stressed (HS)]. The supernatant and endometrial epithelial cells were collected at various time points (0, 3, 6, 12, and 24h) from both the control and treatment groups. Although heat stress (40.5°C) significantly (P < 0.05) increased COX-2, PGES, and PGFS transcripts in epithelial cells but it did not affect the in vitro production of PGF2α and PGE2. The expression of ITGAV and LGALS15 mRNAs in endometrial epithelial cells remained unaltered under elevated temperature conditions. It can be concluded that elevated temperature did not directly modulate prostaglandin production but, it promoted the expression of COX-2, PGES and PGFS mRNA in buffalo endometrial epithelial cells.

Meptyldinocap induces implantation failure by forcing cell cycle arrest, mitochondrial dysfunction, and endoplasmic reticulum stress in porcine trophectoderm and endometrial luminal epithelial cells

Meptyldinocap is a dinitrophenol fungicide used to control powdery mildew. Although other dinitrophenol pesticides have been found to exhibit reproductive toxicity, studies of meptyldinocaps are scarce. This study investigated the adverse effects of meptyldinocap on porcine trophectoderm (pTr) and porcine endometrial luminal epithelial (pLE) cells, which play crucial roles in implantation. We confirmed that meptyldinocap decreased cell viability, induced apoptosis, and inhibited proliferation by decreasing proliferation-related gene expression and inducing changes in the cell cycle. Furthermore, meptyldinocap treatment caused mitochondrial dysfunction, endoplasmic reticulum stress, and disruption of calcium homeostasis. Moreover, it induces alterations in mitogen-activated protein kinase signaling cascades and reduces the migration ability, leading to implantation failure. Our findings suggest that meptyldinocap reduces the cellular functions of pTr and pLE cells, which are important for the implantation process, and interferes with interactions between the two cell lines, potentially leading to implantation failure. We also propose a mechanism by which the understudied fungicide meptyldinocap exerts its cytotoxicity.

Abstract B002: ARID1A loss activates MAPK signaling via DUSP4 downregulation: a mechanistic insight in endometrial carcinoma

Abstract The intricate interplay between chromatin remodelers, transcriptional regulation, and downstream cellular processes is gradually unraveling. AT-rich interactive domain-containing protein 1A (ARID1A), a tumor suppressor and chromatin remodeler, is frequently mutated in endometrium-related malignancies including ovarian or uterine clear cell carcinoma (CCC) and endometrioid carcinoma (EMCA). How ARID1A mutation alters downstream signaling is yet to be established. Based on the TCGA proteomics datasets, MAPK and ARID1A are negatively correlated in endometrial cancer. Yet, the underlying molecular mechanism of this association remains unclear. Using ARID1A-proficient and ARID1A-deficient isogenic human endometrial epithelial cells, we explored transcriptomic changes and discovered notable downregulation of a MAPK phosphatase, dual-specificity phosphatase 4 (DUSP4), in ARID1A-deficient cells. This expression pattern was further validated in additional pairs of isogenic cell lines, ES-2, MCF10a, and HCT116. Chromatin immunoprecipitation sequencing (ChIP-seq) was utilized to assess active and repressive transcription histone marks on global and 5’ transcriptional regulatory regions of DUSP4. ChIP-seq demonstrated decreased histone acetylation marks (H3K27Ac, H3K9Ac) on DUSP4 enhancer and promoter regions of ARID1A-deficient cells, suggesting ARID1A's role in modulating chromatin accessibility and gene expression. Furthermore, a positive correlation between ARID1A and DUSP4 expression was established using a Pax8-driven conditional knockout mouse cohort and in human endometroid carcinoma tissues. Moreover, an inverse expression correlation between ARID1A and the phosphatase substrates of DUSP4, including p-p38, p-JNK, and p-ERK in the MAPK signaling pathway, was observed in this genetically engineered murine model and in the TCGA datasets of human uterine carcinomas. Interestingly, ectopic DUSP4 expression decreased cell proliferation, and MAPK pathway targeting, specifically via ERK inhibitor Ulixertinib, significantly mitigated tumor growth in vivo. In conclusion, our findings suggest ARID1A modulates DUSP4 expression by remodeling the chromatin landscape which leads to an inhibitory effect on the MAPK pathway and suppresses tumor progression. ARID1A-DUSP4-MAPK axis offers a promising avenue for developing targeted therapies against ovarian or uterine CCC and EMCA. Citation Format: Jayaprakash Mandal, Zheng-Cheng Yu, Ie-Ming Shih, Tian-Li Wang. ARID1A loss activates MAPK signaling via DUSP4 downregulation: a mechanistic insight in endometrial carcinoma [abstract]. In: Proceedings of the AACR Special Conference on Endometrial Cancer: Transforming Care through Science; 2023 Nov 16-18; Boston, Massachusetts. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(5_Suppl):Abstract nr B002.

Cecropin A Alleviates LPS-Induced Oxidative Stress and Apoptosis of Bovine Endometrial Epithelial Cells.

Dairy cows receiving a prolonged high-concentrate diet express an elevated concentration of lipopolysaccharides (LPSs) in the peripheral blood circulation, accompanied by a series of systemic inflammatory responses; however, the specific impacts of inflammation are yet to be determined. Cecropin-like antimicrobial peptides have become a research hotspot regarding antimicrobial peptides because of their excellent anti-inflammatory activities, and cecropin A is a major member of the cecropin family. To elucidate the mechanism of cecropin A as anti-inflammatory under the condition of sub-acute ruminal acidosis (SARA) in dairy cows, we induced inflammation in bEECs with LPS (10 µg/mL) and then added cecropin A (25 µM). Afterwards, we detected three categories of indexes including oxidative stress indices, inflammation-related genes, and apoptosis-related genes in bovine endometrial epithelial cells (bEECs). The results indicated that cecropin A has the ability to reduce inflammatory factors TNF-α, IL-1β, and IL-8 and inhibit the MAPK pathway to alleviate inflammation. In addition, cecropin A is able to reduce reactive oxygen species (ROS) levels and alleviates LPS-induced oxidative stress and mitochondrial dysfunction by downregulating NADPH Oxidase (NOX), and upregulating catalase (CAT), glutathione peroxidase (GPX), and superoxide dismutase (SOD). Furthermore, cecropin A demonstrates the ability to inhibit apoptosis by suppressing the mitochondrial-dependent apoptotic pathway, specifically Fas/FasL-caspase-8/-3. The observed increase in the Bcl-2/Bax ratio, a known apoptosis regulator, further supports this finding. In conclusion, our study presents novel solutions for addressing inflammatory responses associated with SARA.

Extracellular vesicle-encapsulated miR-25-3p promotes epithelial-mesenchymal transition and migration of endometrial epithelial cells by inducing macrophage polarization.

The pathogenesis of adenomyosis is closely related to the epithelial-mesenchymal transition and macrophages. MicroRNAs have been extensively investigated in relation to the epithelial-mesenchymal transition in a range of malignancies. However, there is a paucity of research on extracellular vesicles derived from the eutopic endometrium of adenomyosis and their encapsulated microRNAs. In this study, we investigated the role of microRNA-25-3p derived from extracellular vesicles in inducing macrophage polarization and promoting the epithelial-mesenchymal transition in endometrial epithelial cells of patients with adenomyosis and controls. We obtained eutopic endometrial samples and isolated extracellular vesicles from the culture supernatant of primary endometrial cells. Real-time quantitative PCR analysis demonstrated that microRNA-25-3p was highly expressed in extracellular vesicles, as well as in macrophages stimulated by extracellular vesicles from eutopic endometrium of adenomyosis; and macrophages transfected with microRNA-25-3p exhibited elevated levels of M2 markers, while displaying reduced levels of M1 markers. After co-culture with the above polarized macrophages, endometrial epithelial cells expressed higher levels of N-cadherin and Vimentin, and lower protein levels of E-cadherin and Cytokeratin 7. It was revealed that microRNA-25-3p encapsulated in extracellular vesicles from eutopic endometrial cells could induce macrophage polarization toward M2, and the polarized macrophages promote epithelial-mesenchymal transition in epithelial cells. However, in vitro experiments revealed no significant disparity in the migratory capacity of endometrial epithelial cells between the adenomyosis group and the control group. Furthermore, it was observed that microRNA-25-3p-stimulated polarized macrophages also facilitated the epithelial-mesenchymal transition and migration of endometrial epithelial cells within the control group. Thus, the significance of microRNA-25-3p-induced polarized macrophages in promoting the development of adenomyosis is unclear, and macrophage infiltration alone may be adequate for this process. We emphasize the specificity of the local eutopic endometrial microenvironment and postulate its potential significance in the pathogenesis of adenomyosis.

Modeling embryo-endometrial interface recapitulating human embryo implantation.

The initiation of human pregnancy is marked by the implantation of an embryo into the uterine environment; however, the underlying mechanisms remain largely elusive. To address this knowledge gap, we developed hormone-responsive endometrial organoids (EMO), termed apical-out (AO)-EMO, which emulate the in vivo architecture of endometrial tissue. The AO-EMO comprise an exposed apical epithelium surface, dense stromal cells, and a self-formed endothelial network. When cocultured with human embryonic stem cell-derived blastoids, the three-dimensional feto-maternal assembloid system recapitulates critical implantation stages, including apposition, adhesion, and invasion. Endometrial epithelial cells were subsequently disrupted by syncytial cells, which invade and fuse with endometrial stromal cells. We validated this fusion of syncytiotrophoblasts and stromal cells using human blastocysts. Our model provides a foundation for investigating embryo implantation and feto-maternal interactions, offering valuable insights for advancing reproductive medicine.

Effect of estradiol after bacterial infection on the Wnt/β-catenin pathway in bovine endometrium epithelial cells and organoids

Endometritis is a disease caused by a postpartum bacterial infection with a poor prognosis that primarily affects dairy cows. Three-dimensional organoids have been used as a model for endometritis, because they exhibit a structure comparable to that of the endometrium, demonstrating both expansibility and hormone responsiveness. These characteristics render them an ideal platform for in vitro investigations of endometrial diseases. Estradiol (E2) is an endogenous steroid hormone with demonstrated anti-inflammatory properties, and the objective of this study was to determine the mechanism by which E2 modulates the inflammatory response and the Wnt signal transduction pathway in bovine endometrial epithelial cells and organoids following E. coli infection. We present the techniques for isolating and culturing primary bovine endometrial epithelial cells (BEECs), and producing endometrial organoids. For the experiments, the endometrial epithelial cells and organoids were infected with E. coli for 1 h, followed by incubation with E2 for 12 h. The mRNA and protein expressions of the inflammation-related genes, IL-1β, IL-6, TLR4, and NF-κB, as well as the Wnt pathway-related genes, Wnt4, β-catenin, c-Myc, and CyclinD1, were assessed using real-time quantitative-PCR and western blotting, respectively. The CCK8 viable cell counting assay was utilized to determine the optimal concentration of the Wnt inhibitor, IWR-1. The mRNA and protein expression of Wnt pathway-related genes was assessed following IWR-1 treatment, while the expression levels of proliferation-associated genes (Ki67, PCNA) and barrier repair genes (occludin, claudin, and Zo-1) in BEECs and organoids were evaluated after E2 treatment. The results of this study show that mRNA expression of the inflammatory genes, IL-1β, TLR4, and NF-κB (P < 0.05) decreased in BEECs following E2 treatment compared to the E. coli group. The protein expression of the IL-1β, IL-6, TLR4 and NF-κB genes was also inhibited (P < 0.05). Similar results were observed in tests on the organoids. Our findings demonstrate that E2 significantly upregulates the expression of Wnt-related genes, including β-catenin and c-Myc, while concurrently downregulating the expression of GSK3β (P < 0.05). Next, we treated E. coli-infected BEECs and organoids with the Wnt inhibitor, IWR-1. Compared with E. coli and E. coli + E2, the expression of mRNA and protein from Wnt 4, β-catenin, and CyclinD1 in E. coli + E2 and E. coli + IWR-1 was down-regulated (P < 0.05). The expression of the proliferation genes, Ki67, PCNA, and the tight junction genes, occludin, claudin1, and Zo-1, in organoids was significantly higher than that in BEECs (P < 0.05). In summary, we found strong potential for E2 mitigation of the E. coli-induced inflammatory response in BEECs and organoids, through activation of the Wnt pathway. In addition, the proliferation and repair capacity of organoids was much higher than that of BEECs.

Absence of Heme Oxygenase-1 Affects Trophoblastic Spheroid Implantation and Provokes Dysregulation of Stress and Angiogenesis Gene Expression in the Uterus.

The enzyme heme oxygenase-1 (HO-1) is pivotal in reproductive processes, particularly in placental and vascular development. This study investigated the role of HO-1 and its byproduct, carbon monoxide (CO), in trophoblastic spheroid implantation. In order to deepen our understanding of the role of HO-1 during implantation, we conducted in vivo experiments on virgin and pregnant mice, aiming to unravel the cellular and molecular mechanisms. Using siRNA, HO-1 was knocked down in JEG-3 and BeWo cells and trophoblastic spheroids were generated with or without CO treatment. Adhesion assays were performed after transferring the spheroids to RL-95 endometrial epithelial cell layers. Additionally, angiogenesis, stress, and toxicity RT2-Profiler™ PCR SuperArray and PCR analyses were performed in uterine murine samples. HO-1 knockdown by siRNA impeded implantation in the 3D culture model, but this effect could be reversed by CO. Uteruses from virgin Hmox1-/- females exhibited altered expression of angiogenesis and stress markers. Furthermore, there was a distinct expression pattern of cytokines and chemokines in uteruses from gestation day 14 in Hmox1-/- females compared to Hmox1+/+ females. This study strongly supports the essential role of HO-1 during implantation. Moreover, CO appears to have the potential to compensate for the lack of HO-1 during the spheroid attachment process. The absence of HO-1 results in dysregulation of angiogenesis and stress-related genes in the uterus, possibly contributing to implantation failure.

Unveiling the protective effects of BMSCs/anti-miR-124-3p exosomes on LPS-induced endometrial injury.

Researchers have reported that miR-124-3p is highly expressed in patients with chronic endometritis. However, the underlying mechanism of miR-124-3p in the development of endometritis remains unclear. This study constructed an in vitro endometrial cell injury model by treating HEECs with 2 μg/mL LPS for 48 h. Then, 1 mg/kg LPS was injected into both sides of the mouse uterus to construct an in vivo endometrial injury model. The expression of miR-124-3p in human endometrial epithelial cells (HEECs) was assessed using RT‒qPCR. Exosomes were separated from bone marrow-derived mesenchymal stem cells (BMSCs) and cocultured with HEECs. A dual-luciferase reporter assay was performed to confirm the relationship between miR-124-3p and DUSP6. The results indicated that LPS inhibited HEEC viability in a time- and dose-dependent manner. The miR-124-3p inhibitor reversed the LPS-induced apoptosis and inhibition of HEEC viability. In addition, miR-124-3p could be transferred from BMSCs to HEECs by exosomes. Exosomes were derived from BMSCs treated with an NC inhibitor (BMSCs/NC Exo) or miR-124-3p inhibitor (BMSCs/anti-miR-124-3p Exo). In addition, BMSCs/anti-miR-124-3p Exo abolished the LPS-induced inhibition of HEEC viability and proliferation by inducing HEEC apoptosis. Moreover, BMSCs/anti-miR-124-3p Exo alleviated the LPS-induced inflammation of HEECs by upregulating DUSP6 and downregulating p-p65 and p-ERK. Furthermore, in an LPS-induced in vivo endometrial injury model, BMSCs/anti-miR-124-3p Exo increased the expression level of DUSP6 and decreased the expression levels of p-p65 and p-ERK. BMSCs/anti-miR-124-3p Exo protected against LPS-induced endometrial damage in vitro and in vivo by upregulating DUSP6 and downregulating p-p65 and p-ERK1/2. This study showed that BMSCs/anti-miR-124-3p Exo might be a potential alternative for the treatment of endometritis.

IFNT-induced IRF1 enhances bovine endometrial receptivity by transactivating LIFR

Interferon-τ (IFN-τ) participates in the establishment of endometrial receptivity in ruminants. However, the precise mechanisms by which IFN-τ establishes bovine endometrial receptivity remain largely unknown. Interferon regulatory factor 1 (IRF1) is a classical interferon-stimulated gene (ISG) induced by type I interferon, including IFN-τ. Leukemia inhibitory factor receptor (LIFR) is a transmembrane receptor for leukemia inhibitory factor (LIF), which is a key factor in regulating embryo implantation in mammals. This study aimed to investigate the roles of IRF1 and LIFR in the regulation of bovine endometrial receptivity by IFN-τ. In vivo, we found IRF1 and LIFR were upregulated in the bovine endometrial luminal epithelium on Day 18 of pregnancy compared to Day 18 of the estrous cycle. In vitro, IFN-τ could upregulate IRF1, LIFR, and endometrial receptivity markers (LIF, HOXA10, ITGAV, and ITGB3) expression, downregulate E-cadherin expression and reduce the quantity of microvilli of bovine endometrial epithelial cells (bEECs). Overexpression of IRF1 had similar effects to IFN-τ on endometrial receptivity, and interference of LIFR could block these effects, suggesting the positive effects of IRF1 on endometrial receptivity were mediated by LIFR. Dual luciferase reporter assay verified that IRF1 could transactivate LIFR transcription by binding to its promoter. In conclusion, IFN-τ can induce IRF1 expression in bovine endometrial epithelial cells, and IRF1 upregulates LIFR expression by binding to LIFR promoter, contributing to the enhancement of bovine endometrial receptivity.

Transcriptomic analysis of bovine endometrial epithelial cells in response to interferon tau and hormone stimulation.

The embryonic loss during early stage of gestation is one of the major causes of infertility for domestic ruminants, causing huge economic losses to pasture. Maternal recognition of pregnancy and implantation are the crucial process for determining the successful establishment and development of pregnancy in cattle. The research on molecular mechanisms of pregnancy recognition will facilitate illustrating the complex process of pregnancy establishment and help to improve pregnancy outcomes. In this study, we performed transcriptomic analysis of primary bovine endometrial epithelial cells (BEND) with or without IFNT and hormones intervention through RNA sequencing. We eventually identified 608 differentially expressed genes (DEGs) including 409 up-regulated genes and 199 down-regulated genes in IFNT and hormones-treated group compared with control group. Gene Ontology (GO) enrichment analysis demonstrated that the majority of DEGs were implicated in immune system process, response to external stimulus, response to cytokine, regulation of response to stress. Results from KEGG analysis showed a significant enrichment of NOD-like receptor signaling pathway, antigen processing and presentation, necroptosis, oxidative phosphorylation, RIG-I-like receptor signaling pathway. Additionally, a set of promising candidate genes, including (USP18, STAT1, PSMB8, IFIH1, MX2, IFI44, DHX58, CASP8, DRAM1, CXCR4), were characterized by constructing an integrated interaction network. Specifically, the mRNA expression of HOXA11, PTGS1 and PTGS2 were remarkably suppressed by silencing DRAM1 under IFNT and hormone administration, thus speculating that DRAM1 might play a crucial role in early pregnancy by regulating endometrial function. The results of this study depicted a relatively comprehensive transcriptional profiles of BEND in response to IFNT and hormones, which contributes to a better understanding of gene interaction network and underlying regulatory mechanisms in endometrium of ruminants during early pregnancy.

Autophagy activation alleviates the LPS-induced inflammatory response in endometrial epithelial cells in dairy cows.

Endometritis is a common disease that affects dairy cow reproduction. Autophagy plays a vital role in cellular homeostasis and modulates inflammation by regulating interactions with innate immune signaling pathways. However, little is known about the regulatory relationship between autophagy and inflammation in bovine endometrial epithelial cells (BEECs). Thus, we aimed to determine the role of autophagy in the inflammatory response in BEECs. In the present study, the expression levels of proinflammatory cytokines were measured by quantitative real-time polymerase chain reaction. Changes in the nuclear factor-κB (NF-κB) pathway and autophagy were determined using immunoblotting and immunocytochemistry. The induction of autophagosome formation was visualized by transmission electron microscopy. Our results demonstrated that autophagy activation was inhibited in LPS-treated BEECs, while activation of the NF-κB pathway and the mRNA expression of IL-6, IL-8, and TNF-α were increased. Furthermore, blocking autophagy with the inhibitor chloroquine increased NF-κB signaling pathway activation and proinflammatory factor expression in LPS-treated BEECs. Conversely, activation of autophagy with the agonist rapamycin inhibited the NF-κB signaling pathway and downregulated proinflammatory factors. These data indicated that LPS-induced inflammation was related to the inhibition of autophagy in BEECs. Thus, the activation of autophagy may represent a novel therapeutic strategy for eliminating inflammation in BEECs.

Role of immune cells in the establishment of implantation and maintenance of pregnancy and immunomodulatory therapies for patients with repeated implantation failure and recurrent pregnancy loss.

Immune cells play an important role in the establishment of pregnancy, and abnormalities in the immune system can cause implantation failure and miscarriage. Previous papers have been summarized and the role of immune cells in reproduction is reviewed. The immune environment in the uterus changes drastically from before implantation to after pregnancy to maintain pregnancy. In allogeneic pregnancies, immature dendritic cells (DCs) that induce immune tolerance from outside the uterus flow into the uterus, and mature DCs that remain in the uterus express programmed cell death ligand 2, which suppresses the immune response. Macrophages are classified into M1-macrophages, which induce inflammation, and M2-macrophages, which suppress inflammation; M1-macrophages are required for luteinization, and M2-macrophages induce the differentiation of endometrial epithelial cells to enable implantation. Regulatory T cells, which suppress rejection, are essential for the implantation and maintenance of allogeneic pregnancies. Implantation failure and fetal loss are associated with decreased numbers or qualitative abnormalities of DCs, macrophages, and regulatory T cells. The clinical usefulness of immunomodulatory therapies in patients with repeated implantation failure and recurrent pregnancy loss has been reported. The provision of individualized medical care in cases of implantation failure or miscarriage may improve clinical outcomes.

Zearalenone Promotes Uterine Hypertrophy through AMPK/mTOR Mediated Autophagy.

Zearalenone (ZEN), a non-steroidal Fusarium graminearum with an estrogen effect, can cause damage to the gastrointestinal tract, immune organs, liver, and reproductive system. Further analysis of the mechanism of ZEN has become an important scientific issue. We have established in vivo and in vitro models of ZEN intervention, used AMPK/mTOR as a targeted pathway for ZEN reproductive toxicity, and explored the molecular mechanism by which ZEN may induce uterine hypertrophy in weaned piglets. Our study strongly suggested that ZEN can activate the phosphorylation of AMPK in uterine endometrial epithelium cells, affect the phosphorylation level of mTOR through TSC2 and Rheb, induce autophagy, upregulate the expression of proliferative genes PCNA and BCL2, downregulate the expression of apoptotic gene BAX, promote uterine endometrial epithelium cells proliferation, and ultimately lead to thickening of the endometrial and myometrium, increased density of uterine glands, and induce uterine hypertrophy.

Upregulated α-actinin-1 impairs endometrial epithelial cell adhesion by downregulating NEBL in recurrent implantation failure

Poor endometrial receptivity results in embryo implantation failure. Acquisition of endometrial receptivity involves substantial structural alterations in the cytoskeleton and plasma membrane of epithelial cells, which facilitate embryo adhesion. However, the underlying molecular mechanism remains largely unknown. In this study, we identified that α-actinin-1 (ACTN1) was significantly downregulated in the mid-secretory phase of the endometrium compared with other phases; however, ACTN1 significantly increased in women with recurrent implantation failure (RIF). In Ishikawa and human endometrial epithelial cells (HEECs), ACTN1 overexpression significantly decreased NEBL levels, enhanced F-actin fiber levels, and caused a notable impairment in blastocyst adhesion, which mimicked the process of embryo adhesion. However, NEBL overexpression notably restored adhesion. Moreover, NEBL expression was reduced in patients with RIF compared with that in controls. Finally, our data showed that ACTN1 upregulation impaired endometrial receptivity in women with RIF, possibly by regulating NEBL expression and subsequent cell-adhesion capability.