Human Endometrial Epithelial Cells Research Articles

Semaglutide May Ameliorate Fibrosis and Inhibit Epithelial-Mesenchymal Transition in Intrauterine Adhesion Models.

The purpose of this study was to explore the effect of Semaglutide on intrauterine adhesions and discover new drugs for such adhesions. In this study, the cell model was simulated by TGF-β1-induced human endometrial epithelial cells, and the animal model was established through mechanical curettage and inflammatory stimulation. After co-culturing with TGF-β1 with or without different concentrations of Semaglutide for 48 h, cells were collected for RT-qPCR and Western blotting analyses. Three doses were subcutaneously injected into experimental mice once a day for two weeks, while the control group received sterile ddH2O. The serum and uterine tissues of the mice were collected. HE and Masson staining were used for the uterine histomorphological and pathological analyses. RT-qPCR and Western blotting were used for mRNA and protein expression analyses. Serum indicators were detected using ELISA kits. The results showed that Semaglutide significantly reduced the mRNA levels of fibrosis indicators ACTA2, COL1A1, and FN and inflammatory indicators TNF-α, IL-6, and NF-κB in the two models. Semaglutide improved endometrium morphology, increased the number of endometrial glands, and reduced collagen deposition in IUA mice. The results also showed that Semaglutide could inhibit vimentin, E-Cadherin, and N-Cadherin in the two models. In summary, Semaglutide can ameliorate fibrosis and inflammation of intrauterine adhesions as well as inhibit epithelial-mesenchymal transition in IUA models.

Excessive proliferating cell nuclear antigen attenuates endometrial adhesive capacity and decidualization in patients with recurrent implantation failure.

Does the expression of proliferating cell nuclear antigen (PCNA) in the endometrium regulate endometrial receptivity in patients with recurrent implantation failure (RIF)? A high abundance of PCNA attenuates endometrial adhesive capacity and decidualization in patients with RIF. Aberrant expression of PCNA has been discovered in multiple infertility-related disorders. However, the expression pattern and role of PCNA in the establishment of endometrial receptivity and endometrial decidualization in patients with RIF remain unclear. We analysed the expression of PCNA in mid-secretory endometrial tissues from 24 patients with RIF and 24 healthy women. Additionally, PCNA expression levels were measured in proliferative and mid-secretory phase endometrial tissue samples from women with regular menstrual cycles and in decidual tissue samples taken from ten women during normal early pregnancy (n = 10 per phase for each group). The function and regulatory mechanisms of PCNA in endometrial adhesive capacity and endometrial decidualization were investigated using BeWo spheroids, Ishikawa cells, and human endometrial stromal cells (HESCs). The expression of PCNA in mid-secretory endometrial tissues of patients with RIF and women with normal endometrium and in endometrial tissue at different stages of the menstrual cycle and in decidualized tissues was analysed by RT-qPCR, western blot, and immunohistochemistry staining (IHC). Furthermore, the number of BeWo spheroids directly attached to the Ishikawa cell monolayers, and the potential molecular mechanisms involved, were compared between cells overexpressing PCNA and a control group. Additionally, the effect and regulatory mechanisms of PCNA on the decidualization of HESCs in vitro were investigated. Our findings indicated that the abundance of PCNA was dramatically greater in mid-secretory endometrial tissues from patients with RIF than in those from women with healthy endometrium. The expression of PCNA increased in the proliferative phase of the menstrual cycle but decreased gradually in the mid-secretory phase and in decidual tissues. Interestingly, PCNA was expressed in both human endometrial epithelial cells (HEECs) and HESCs. In Ishikawa cells, PCNA overexpression dramatically reduced the endometrial adhesive capacity by inhibiting the expression of adhesion molecules (E-cadherin and integrin β3) and activating the FAK/paxillin signalling pathway. Furthermore, in HESCs, PCNA overexpression attenuated endometrial decidualization by activating the AKT/β-catenin signalling pathway and increasing tight junctions between cells by upregulating ZO-1 and occludin expression. In addition, PCNA-ELAVL1 interactions were confirmed by coimmunoprecipitation in decidualized HESCs. N/A. The functional analysis of PCNA was limited by the number of human endometrial tissues. A larger sample size is required to further explore the potential roles of PCNA during embryo implantation. Moreover, the present results should be taken with caution, as only a few of the embryos that were transferred in RIF patients population underwent preimplantation genetic testing for embryonic chromosome aneuploidies (PGT-A), despite embryo ploidy testing being significant in the diagnosis of unexplained RIF. High PCNA expression attenuates endometrial adhesive capacity and decidualization in patients with RIF. These findings provide new insights into the potential mechanisms underlying the occurrence of implantation failure. This work was supported by the National Natural Science Foundation of China (82101698), Shandong Provincial Natural Science Foundation (ZR2021MH012), and the Science and Technology Plan of Yantai (2023YD021 and 2022YD031). The authors have no conflicts of interest to disclose. N/A.

Long-Term Maintenance of Viable Human Endometrial Epithelial Cells to Analyze Estrogen and Progestin Effects.

There are fewer investigations conducted on human primary endometrial epithelial cells (HPEECs) compared to human primary endometrial stromal cells (HPESCs). One of the main reasons is the scarcity of protocols enabling prolonged epithelial cell culture. Even though it is possible to culture HPEECs in 3D over a longer period of time, it is technically demanding. In this study, we successfully established a highly pure, stable, and long-term viable human conditionally reprogrammed endometrial epithelial cell line, designated as eCRC560. These cells stained positive for epithelial markers, estrogen and progesterone receptors, and epithelial cell-cell contacts but negative for stromal and endothelial cell markers. Estradiol (ES) reduced the abundance of ZO-1 in a time- and dose-dependent manner, in contrast to the dose-dependent increase with the progestin dienogest (DNG) when co-cultured with HPESCs. Moreover, ES significantly increased cell viability, cell migration, and invasion of the eCRC560 cells; all these effects were inhibited by pretreatment with DNG. DNG withdrawal led to a significantly disrupted monolayer of eCRC560 cells in co-culture with HPESCs, yet it markedly increased the adhesion of eCRC560 to the human mesothelial MeT-5A cells. The long-term viable eCRC560 cells are suitable for in vitro analysis of HPEECs to study the epithelial compartment of the human endometrium and endometrial pathologies.

Exosomes from uterine fluid promote capacitation of human sperm.

Extracellular vesicles (EVs) are membrane-bound vesicles containing various proteins, lipids, and nucleic acids. EVs are found in many body fluids, such as blood and urine. The release of EVs can facilitate intercellular communication through fusion with the plasma membrane or endocytosis into the recipient cell or through internalization of the contents. Recent studies have reported that EVs isolated from human endometrial epithelial cells (EECs) promote sperm fertilization ability. EVs from uterine flushing fluid more closely resemble the physiological condition of the uterus. However, it is unclear whether EVs derived directly from uterine flushing fluid have the same effect on sperm. This study aimed to research the effect of EVs from uterine flushing fluid on sperm. EVs were isolated from the uterine flushing fluid. The presence of EVs was confirmed by nanoparticle tracking analysis (NTA), Western blot, and transmission electron microscopy (TEM). EVs were incubated with human sperm for 2 h and 4 h. The effects of EVs on sperm were evaluated by analyzing acrosome reaction, sperm motility, and reactive oxygen species (ROS). The EVs fractions isolated from the uterine fluid were observed in cup-shaped vesicles of different sizes by TEM. All isolated vesicles contained similar numbers of vesicles in the expected size range (30-200 nm) by NTA. CD9 and CD63 were detected in EVs by western blot. Comparing the motility of the two groups incubated sperm motility significantly differed at 4 h. The acrosome reactions were promoted by incubating with EVs significantly. ROS were increased in sperm incubated with EVs. Our results showed EVs present in the uterine fluid. Acrosome reactions and ROS levels increased in human sperm incubated with EVs. EVs from uterine fluid can promote the capacitation of human sperm. The increased capacitation after sperm interaction with EVs suggests a possible physiological effect during the transit of the uterus.

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.

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.

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.

ARID1A loss activates MAPK signaling via DUSP4 downregulation

BackgroundARID1A, a tumor suppressor gene encoding BAF250, a protein participating in chromatin remodeling, is frequently mutated in endometrium-related malignancies, including ovarian or uterine clear cell carcinoma (CCC) and endometrioid carcinoma (EMCA). However, how ARID1A mutations alter downstream signaling to promote tumor development is yet to be established.MethodsWe used RNA-sequencing (RNA-seq) to explore transcriptomic changes in isogenic human endometrial epithelial cells after deleting ARID1A. Chromatin immunoprecipitation sequencing (ChIP-seq) was employed to assess the active or repressive histone marks on DUSP4 promoter and regulatory regions. We validated our findings using genetically engineered murine endometroid carcinoma models, human endometroid carcinoma tissues, and in silico approaches.ResultsRNA-seq revealed the downregulation of the MAPK phosphatase dual-specificity phosphatase 4 (DUSP4) in ARID1A-deficient cells. ChIP-seq demonstrated decreased histone acetylation marks (H3K27Ac, H3K9Ac) on DUSP4 regulatory regions as one of the causes for DUSP4 downregulation in ARID1A-deficient cells. Ectopic DUSP4 expression decreased cell proliferation, and pharmacologically inhibiting the MAPK pathway significantly mitigated tumor formation in vivo.ConclusionsOur findings suggest that ARID1A protein transcriptionally modulates DUSP4 expression by remodeling chromatin, subsequently inactivating the MAPK pathway, leading to tumor suppression. The ARID1A-DUSP4-MAPK axis may be further considered for developing targeted therapies against ARID1A-mutated cancers.

Maternal and embryonic signals cause functional differentiation of luminal epithelial cells and receptivity establishment.

Embryo implantation requires temporospatial maternal-embryonic dialog. Using single-cell RNA sequencing for the uterus from 2.5 to 4.5days post-coitum (DPC) and bulk sequencing for the corresponding embryos of 3.5 and 4.0 DPC pregnant mice, we found that estrogen-responsive luminal epithelial cells (EECs) functionally differentiated into adhesive epithelial cells (AECs) and supporting epithelial cells (SECs), promoted by progesterone. Along with maternal signals, embryonic Pdgfa and Efna3/4 signaling activated AECs and SECs, respectively, enhancing the attachment of embryos to the endometrium and furthering embryo development. This differentiation process was largely conserved between humans and mice. Notably, the developmental defects of SOX9-positive human endometrial epithelial cells (similar to mouse EEC) were related to thin endometrium, whereas functional defects of SEC-similar unciliated epithelial cells were related to recurrent implantation failure (RIF). Our findings provide insights into endometrial luminal epithelial cell development directed by maternal and embryonic signaling, which is crucial for endometrial receptivity.

TNFAIP3 interacting protein 2 relieves lipopolysaccharide (LPS)-induced inflammatory injury in endometritis by inhibiting NF-kappaB activation.

Endometritis seriously affects the health of women, and it is important to identify new targets for its treatment. This study aimed to explore the role of TNFAIP3 interacting protein 2 (TNIP2) in endometritis through human endometrial epithelial cells (hEECs) stimulated by lipopolysaccharide (LPS). hEECs were induced with LPS to build a cellular model of endometritis. Cell growth and apoptosis were detected by cell counting kit-8 and flow cytometry. The TNIP2 mRNA and protein levels were measured using reverse transcription quantitative polymerase chain reaction (RT-qPCR) and western blot analysis, respectively. The caspase3 activity was calculated using a Caspase3 activity kit. Interleukin (IL)-1β, IL-6, and tumor necrosis factor-alpha (TNF-α) levels were determined by enzyme-linked-immunosorbent-assay. The reactive oxygen species (ROS), lactate dehydrogenase (LDH), catalase (CAT), and superoxide dismutase (SOD) levels were determined using the corresponding kits. Nuclear factor-kappaB (NF-κB) pathway was determined by western blot assay. TNIP2 was downregulated in the LPS-induced endometritis cell model. Cell viability was reduced, apoptosis was enhanced, and IL-6, IL-1β, and TNF-α levels increased in LPS-induced hEECs. Additionally, LDH activity and ROS concentration were upregulated, whereas CAT and SOD activities were downregulated in LPS-induced hEECs. These results were reversed by TNIP2 overexpression. Moreover, the results hinted that NF-κB was involved in the effects of TNIP2 on the LPS-induced endometritis cell model. TNIP2 alleviated endometritis by inhibiting the NF-κB pathway, suggesting a potential therapeutic target for endometritis.

Mechanism of E2F1 in the proliferation, migration, and invasion of endometrial carcinoma cells via the regulation of BMI1 transcription.

Endometrial carcinoma (EC) is the most prevalent gynecological cancer. Transcription factor (TF) regulates a large number of downstream target genes and is a key determinant of all physiological activities, including cell proliferation, differentiation, apoptosis, and cell cycle. The transcription factor E2F1 shows prominent roles in EC. BMI1 is a member of Polycomb suppressor Complex 1 (PRC1) and has been shown to be associated with EC invasiveness. It is currently unclear whether E2F1 can participate in the proliferation, migration, and invasion processes of EC cells by regulating BMI1 transcription. We investigated whether E2F1 could participate in the proliferation, migration, and invasion processes of EC cells by regulating BMI1 transcription, in order to further clarify the pathogenesis and etiology of EC, and provide reference for identifying potential therapeutic targets and developing effective prevention and treatment strategies for this disease. Human endometrial epithelial cells (hEECs) and human EC cell lines were selected. E2F1 expression was assessed by Western blot. E2F1 was silenced in AN3CA or overexpressed in HEC-1 by transfections, or E2F1 was silenced and BMI1 was overexpressed in AN3CA by cotransfection. Cell proliferation, migration, and invasion were detected by MTT, wound healing, and Transwell assays. The binding sites between E2F1 and BMI1 promoters were predicted through JASPAR website, and the targeted binding was verified by dual-luciferase report and ChIP assays. E2F1 was up-regulated in human EC cell lines, with its expression highest in AN3CA, and lowest in HEC-1. AN3CA invasion, migration, and proliferation were repressed by E2F1 knockdown, while those of HEC-1 cells were promoted by E2F1 overexpression. E2F1 overexpression increased the activity of wild type BMI1 reporter vector promoter, while this promotion was weakened after mutation of the predicted binding site in the BMI1 promoter. In the precipitated E2F1, BMI1 promoter site level was higher than that of IgG immunoprecipitant. BMI1 silencing suppressed AN3CA cell growth. BMI1 overexpression partially abrogated E2F1 silencing-inhibited EC cell growth. E2F1 promoted EC cell proliferation, invasion, and migration by promoting the transcription of BMI1.

Organoid co-culture model of the human endometrium in a fully synthetic extracellular matrix enables the study of epithelial-stromal crosstalk

The human endometrium undergoes recurring cycles of growth, differentiation, and breakdown in response to sex hormones. Dysregulation of epithelial-stromal communication during hormone-mediated signaling may be linked to myriad gynecological disorders for which treatments remain inadequate. Here, we describe a completely defined, synthetic extracellular matrix that enables co-culture of human endometrial epithelial and stromal cells in a manner that captures healthy and disease states across a simulated menstrual cycle. We parsed cycle-dependent endometrial integrin expression and matrix composition to define candidate cell-matrix interaction cues for inclusion in a polyethylene glycol (PEG)-based hydrogel crosslinked with matrix metalloproteinase-labile peptides. We semi-empirically screened a parameter space of biophysical and molecular features representative of the endometrium to define compositions suitable for hormone-driven expansion and differentiation of epithelial organoids, stromal cells, and co-cultures of the two cell types. Each cell type exhibited characteristic morphological and molecular responses to hormone changes when co-encapsulated in hydrogels tuned to a stiffness regime similar to the native tissue and functionalized with a collagen-derived adhesion peptide (GFOGER) and a fibronectin-derived peptide (PHSRN-K-RGD). Analysis of cell-cell crosstalk during interleukin 1B (IL1B)-induced inflammation revealed dysregulation of epithelial proliferation mediated by stromal cells. Altogether, we demonstrate the development of a fully synthetic matrix to sustain the dynamic changes of the endometrial microenvironment and support its applications to understand menstrual health and endometriotic diseases. This work was supported by The John and Karine Begg Foundation, the Manton Foundation, and NIH U01 (EB029132).

Human blastocysts uptake extracellular vesicles secreted by endometrial cells containing miRNAs related to implantation.

Are the extracellular vesicles (EVs) secreted by the maternal endometrium uptaken by human embryos and is their miRNA cargo involved in implantation and embryo development? Data suggest that EVs secreted by human endometrial epithelial cells are internalized by human blastocysts, and transport miRNAs to modulate biological processes related to implantation events and early embryo development. Successful implantation is dependent on coordination between maternal endometrium and embryo, and EVs role in the required cell-to-cell crosstalk has recently been established. In this regard, our group previously showed that protein cargo of EVs secreted by primary human endometrial epithelial cells (pHEECs) is implicated in biological processes related to endometrial receptivity, embryo implantation, and early embryo development. However, little is known about the regulation of these biological processes through EVs secreted by the endometrium at a transcriptomic level. A prospective descriptive study was performed. Endometrial biopsies were collected from healthy oocyte donors with confirmed fertility on the day of oocyte retrieval, 36 h after the LH surge. pHEECs were isolated from endometrial biopsies (n = 8 in each pool) and cultured in vitro. Subsequently, conditioned medium was collected and EVs were isolated and characterized. Uptake of EVs by human blastocysts and miRNA cargo of these EVs (n = 3 pools) was analyzed. EVs were isolated from the conditioned culture media using ultracentrifugation, and characterization was performed using western blotting, nanoparticle tracking analysis, and transmission electron microscopy. EVs were fluorescently labeled with Bodipy-TR ceramide, and their uptake by human blastocysts was analyzed using confocal microscopy. Analysis of the miRNA cargo of EVs was performed using miRNA sequencing, target genes of the most expressed miRNA were annotated, and functional enrichment analysis was performed. EVs measured 100-300 nm in diameter, a concentration of 1.78 × 1011 ± 4.12 × 1010 (SD) particles/ml and expressed intraluminal protein markers Heat shock protein 70 (HSP70) and Tumor Susceptibility Gene 101 (TSG101), in addition to CD9 and CD81 transmembrane proteins. Human blastocysts efficiently internalized fluorescent EVs within 1-2 h, and more pronounced internalization was observed in the hatched pole of the embryos. miRNA-seq analysis featured 149 annotated miRNAs, of which 37 were deemed most relevant. The latter had 6592 reported gene targets, that in turn, have functional implications in several processes related to embryo development, oxygen metabolism, cell cycle, cell differentiation, apoptosis, metabolism, cellular organization, and gene expression. Among the relevant miRNAs contained in these EVs, we highlight hsa-miR-92a-3p, hsa-let-7b-5p, hsa-miR-30a-5p, hsa-miR-24-3p, hsa-miR-21-5p, and hsa-let-7a-5p as master regulators of the biological processes. This is an in vitro study in which conditions of endometrial cell culture could not mimic the intrauterine environment. This study defines potential biomarkers of endometrial receptivity and embryo competence that could be useful diagnostic and therapeutic targets for implantation success, as well as open insight further investigations to elucidate the molecular mechanisms implicated in a successful implantation. This study was supported by the Spanish Ministry of Education through FPU awarded to M.S.-B. (FPU18/03735), the Health Institute Carlos III awarded to E.J.-B. (FI19/00110) and awarded to H.F. by the Miguel Servet Program 'Fondo Social Europeo «El FSE invierte en tu futuro»' (CP20/00120), and Generalitat Valenciana through VALi+d Programme awarded to M.C.C.-G. (ACIF/2019/139). The authors have no conflicts of interest to disclose. N/A.

O-232 Extracellular vesicles secreted by the maternal endometrium functionally regulate processes related to embryo development and implantation in human blastocysts

Abstract Study question What is the response of human embryos in a transcriptomic level to the uptake of extracellular vesicles (EVs) secreted by the human endometrium? Summary answer EVs secreted by the maternal endometrium induce a transcriptomic response in human embryos that modulates molecular mechanisms related to embryo development and implantation. What is known already Communication between the maternal endometrium and the embryo is essential for a successful implantation, and EV role in this cross-talk has been recently established. Results of our previous experiments showed that endometrial EVs carry miRNAs and proteins related to endometrial receptivity, embryo implantation process and early embryo development, and that they can be uptaken by human blastocysts. However, there are no studies which address the transcriptomic response of human embryos to the uptake of these EVs, which would demonstrate the functional relevance of this communication system. Study design, size, duration Endometrial biopsies were collected from oocyte donors (n = 20) with confirmed fertility, on oocyte retrieval day, and primary human endometrial epithelial cells (pHEECs) were isolated and in vitro cultured. Hormonal treatment was added to mimic secretory phase of menstrual cycle. Conditioned medium was collected and EVs were isolated and characterized. Day 5 human blastocysts (n = 24) were divided into two groups (n = 12/group) and cultured in vitro with or without these EVs. RNA-sequencing of these embryos was performed. Participants/materials, setting, methods Conditioned media from pHEECs was pooled into 4 replicates (n = 5 biopsies/replicate) and EVs were isolated by ultracentrifugation. Characterization was performed by western blot, nanoparticle tracking analysis and transmission electron microscopy (TEM). Human blastocysts were devitrified and cultured in vitro for 24h with or without EVs previously isolated. RNA-sequencing analysis was performed, and DESeq2 was used to identify differentially expressed genes (FDR&amp;lt;0.05). QIAGEN Ingenuity Pathway Analysis (IPA) was used to perform the functional enrichment analysis. Main results and the role of chance EVs presented a size range within 100-300nm, and expression of EV protein markers HSP70, TSG101, CD9 and CD81. EV morphology was corroborated by TEM. RNA-sequencing identified 26610 genes, being significant 519 upregulated and 395 downregulated in EV-embryos compared with control. Among top20 most significant upregulated genes, CSF1R (log2FoldChange [logFC]=3.36) has been associated with formation of blastocyst cavity, and PLOD2 (logFC=2) deficiency leads to early embryonic lethality. Genes related to antioxidant defense, as PDK1 (logFC=2.82), or vesicle trafficking, as SEC24D (logFC=2.17), were also upregulated. Regarding top20 downregulated genes, DAB2IP (logFC=-1.19) negative regulation was found beneficial in early pregnancy, and DUSP6 (logFC=-2.07) was associated with cell differentiation and pluripotency. Functional analysis demonstrated positive regulation of biological processes related to embryonic development, cellular invasion and migration, cell cycle, cellular organization, and cell viability in EV-embryos. Contrary, there was a negative regulation of processes related to cell death, apoptosis, and DNA fragmentation. Some pathways upregulated in EV-embryos were NANOG role in embryonic pluripotency (z-score [z]=1.89), unfolded protein response (z = 3.05), involved in cellular homeostasis, VEGF signaling (z = 1.67), that regulates trophoblast adaptation to hypoxia, and integrin signaling (z = 3.36), which regulates trophoblast adhesion and differentiation; PTEN signaling (z=-1.73) was inhibited in EV-embryos, suppression of which prevents apoptosis. Limitations, reasons for caution This is an in vitro study in which conditions of endometrial cell culture could not mimic the intrauterine environment. Wider implications of the findings EVs secreted by the maternal endometrium and uptaken by human blastocysts regulate processes related to embryo development and implantation, suggesting the functional relevance of this endometrial-embryo crosstalk during the implantation process. This study opens further research insight to define potential targets of implantation success and embryo competence. Trial registration number Not applicable

Mechanism of HCG regulating epithelial–mesenchymal transition progression in endometrial cells through the FoxO1/miR223-5p/Wnt5α pathway

Does human chorionic gonadotrophin (HCG) influence endometrial receptivity and epithelial-mesenchymal transition (EMT) via the FoxO1/miR223-5p/Wnt5α pathway? This study aimed to establish the co-culture system of human embryonic trophoblast cell line (HTR-8-Svneo) cells and human endometrial epithelial cell line (HEEC) cells. The expression of Wnt5α protein and EMT-related proteins in HTR-8-Svneo and HEEC cells treated in a gradient-dependent manner using HCG and exosome inhibitor GW4869 were detected in the co-culture system. In the HTR-8-Svneo/HEEC co-culture system, miR223-5p in HEEC cells increased significantly with induction of HTR-8-Svneo cells by 100 IU/ml HCG for 48 h (P = 0.046), and Wnt5α protein decreased significantly in HEEC cells (P = 0.021). Pretreatment of HTR-8-Svneo cells with GW4869, and knockdown of FoxO1 in HTR-8-Svneo cells, significantly inhibited the above effects of HCG on miR223-5p and Wnt5α expression in HEEC cells in the HTR-8-Svneo/HEEC co-culture system. HTR-8-Svneo cells induced with 100 IU/ml HCG for 48 h significantly enhanced the logarithmic phase proliferation activity of HEEC cells in the co-culture system (P < 0.001), while knockdown of FoxO1 in HTR-8-Svneo cells and inhibition of miR223-5p in HEEC cells suppressed proliferation of HEEC cells in the HTR-8-Svneo/HEEC co-culture system (P < 0.001). HCG exposure induces HTR-8-Svneo cells to up-regulate miR223-5p expression, which enters HEEC cells in the co-culture system through the exosomal pathway, and inhibits Wnt5α expression and the progress of EMT.

Antioxidant Supplementation Alleviates Mercury-Induced Cytotoxicity and Restores the Implantation-Related Functions of Primary Human Endometrial Cells.

Mercury (Hg) cytotoxicity, which is largely mediated through oxidative stress (OS), can be relieved with antioxidants. Thus, we aimed to study the effects of Hg alone or in combination with 5 nM N-Acetyl-L-cysteine (NAC) on the primary endometrial cells' viability and function. Primary human endometrial epithelial cells (hEnEC) and stromal cells (hEnSC) were isolated from 44 endometrial biopsies obtained from healthy donors. The viability of treated endometrial and JEG-3 trophoblast cells was evaluated via tetrazolium salt metabolism. Cell death and DNA integrity were quantified following annexin V and TUNEL staining, while the reactive oxygen species (ROS) levels were quantified following DCFDA staining. Decidualization was assessed through secreted prolactin and the insulin-like growth factor-binding protein 1 (IGFBP1) in cultured media. JEG-3 spheroids were co-cultured with the hEnEC and decidual hEnSC to assess trophoblast adhesion and outgrowth on the decidual stroma, respectively. Hg compromised cell viability and amplified ROS production in trophoblast and endometrial cells and exacerbated cell death and DNA damage in trophoblast cells, impairing trophoblast adhesion and outgrowth. NAC supplementation significantly restored cell viability, trophoblast adhesion, and outgrowth. As these effects were accompanied by the significant decline in ROS production, our findings originally describe how implantation-related endometrial cell functions are restored in Hg-treated primary human endometrial co-cultures by antioxidant supplementation.

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.

Protease secretions by the invading blastocyst induce calcium oscillations in endometrial epithelial cells via the protease-activated receptor 2

BackgroundEarly embryo implantation is a complex phenomenon characterized by the presence of an implantation-competent blastocyst and a receptive endometrium. Embryo development and endometrial receptivity must be synchronized and an adequate two-way dialogue between them is necessary for maternal recognition and implantation. Proteases have been described as blastocyst-secreted proteins involved in the hatching process and early implantation events. These enzymes stimulate intracellular calcium signaling pathways in endometrial epithelial cells (EEC). However, the exact molecular players underlying protease-induced calcium signaling, the subsequent downstream signaling pathways and the biological impact of its activation remain elusive.MethodsTo identify gene expression of the receptors and ion channels of interest in human and mouse endometrial epithelial cells, RNA sequencing, RT-qPCR and in situ hybridization experiments were conducted. Calcium microfluorimetric experiments were performed to study their functional expression.ResultsWe showed that trypsin evoked intracellular calcium oscillations in EEC of mouse and human, and identified the protease-activated receptor 2 (PAR2) as the molecular entity initiating protease-induced calcium responses in EEC. In addition, this study unraveled the molecular players involved in the downstream signaling of PAR2 by showing that depletion and re-filling of intracellular calcium stores occurs via PLC, IP3R and the STIM1/Orai1 complex. Finally, in vitro experiments in the presence of a specific PAR2 agonist evoked an upregulation of the ‘Window of implantation’ markers in human endometrial epithelial cells.ConclusionsThese findings provide new insights into the blastocyst-derived protease signaling and allocate a key role for PAR2 as maternal sensor for signals released by the developing blastocyst.Graphical

Abstract 4877: Mutant or overexpressed SF3B1 facilitates endometrial cancer tumorigenesis

Abstract Aberrant expression or mutations in core splicing factors including U2AF1, SRSF2, ZRSR2, and SF3B1 found to drive tumorigenesis of various malignant tumors e.g., myelodysplastic syndromes, chronic lymphocytic leukemia, uveal melanoma, and breast cancer. Importantly, the amplifications and mutations in SF3B1, the pioneer splicing factor, are reportedly far more common than those in other splicing factors. However, the functional relevance and underlying mechanisms of dysregulated SF3B1 in endometrial cancer are not investigated. Endometrial cancer is the fourth most common cancer in women in the United States and there will be about 65,950 new endometrial cancer cases in the current year alone with 12,550 estimated cancer deaths in the United States. In this study, we thus investigated the role of SF3B1 in endometrial adenocarcinoma. We found SF3B1 is one of the most frequently amplified or mutated splicing factors in endometrial cancer. Specifically, analysis of The Cancer Genome Atlas (TCGA) uterine pan-cancer dataset revealed that up to 9% of endometrial cancers have SF3B1 mutations or amplification. Additionally, we showed that the SF3B1 protein is overexpressed in human as well as mouse endometrial tumor samples. Further analysis found elevated SF3B1 protein in multiple endometrial cancer cell lines even in the absence of SF3B1 mutations. However, we did not observe any altered expression at the transcript level. Further, we identified a correlation between the over-expressed SF3B1 levels and the tumorigenicity/invasive potential of cancer cell lines. For instance, endometrial cancer cell lines (Ishikawa, AN3CA, KLE) with over-expressed SF3B1 levels were much more tumorigenic and invasive than those with low endogenous SF3B1 levels (normal human endometrial epithelial cells, RL-95-2 cells). Moreover, cell viability assays revealed that Ishikawa, AN3CA, and KLE cells were more susceptible to the loss of SF3B1 or treatment with the SF3B1 inhibitor Pladienolide-B (PLAD-B) than RL-95-2 cells. To further ensure that overexpressed or mutated SF3B1 drives endometrial tumorigenesis, we transiently transfected an empty vector or a vector expressing FLAG-tagged wild-type SF3B1 or vector with mutated SF3B1 construct R957Q into RL-95-2 cells which have minimal endogenous SF3B1. Analysis of proliferation assays revealed that cells overexpressing SF3B1 proliferated significantly more than cells expressing empty vector. Intriguingly, we observed that RL-95-2 cells over-expressing R957Q mutants were even more proliferative than the cells expressing wild-type SF3B1. Together, this data suggest that SF3B1 over-expression and mutation play a crucial oncogenic role in endometrial cancer, and these findings may support the development of SF3B1 inhibitors to treat this disease. Citation Format: Pooja Popli, Xinchao Deng, Premal Thaker, Ramakrishna Kommagani. Mutant or overexpressed SF3B1 facilitates endometrial cancer tumorigenesis. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4877.

Downregulation of SEPTIN11 inhibits endometrial epithelial cell adhesive function in patients with elevated peripheral blood natural killer cell counts

Research questionWhat is the underlying mechanism of IVF and embryo transfer (IVF-ET) failure in patients with elevated peripheral blood natural killer cell (pNK) counts? DesignPatients undergoing IVF-ET cycles for tubal obstruction or pelvic adhesion (n = 486) were assigned to three groups: high (CD56+CD16+pNK >30% [n = 49]); medium (15< CD56+CD16+pNK ≤30% [n = 211]); and normal pNK groups (5≤ CD56+CD16+pNK ≤15% [n = 226]). Their general condition, previous pregnancy history and IVF outcomes were compared. Uterine fluid and endometrial tissue from patients in the high and normal pNK groups were collected during the mid-secretory phase and studied to elucidate the molecular mechanism underlying impaired endometrial receptivity. ResultsThe highest incidence of IVF-ET cycles (P < 0.0001) and biochemical pregnancy losses (P < 0.0001), and lowest implantation and clinical pregnancy rates (both P < 0.0001), were observed in patients with pNK over 30%. No significant difference was found in the number of previous miscarriages and rate of spontaneous miscarriage in IVF outcomes. Lower Septin11 (SEPT11) expression in the uterine fluid and endometrial epithelial cells (EEC), and higher endometrial IFN-γ, was observed in patients with high pNK. Ishikawa cell and human endometrial epithelial cell (HEEC) adhesion was inhibited after SEPT11 knock-down. Elevated IFN-γ decreased the SEPT11 protein levels in Ishikawa cells and HEECs. ConclusionsCD56+CD16+pNK above 30% may be a threshold for adverse IVF-ET outcomes. Low SEPT11 expression in EEC inhibits cell adhesion, which may cause impaired endometrial receptivity in patients with elevated pNK. The level of SEPT11 in mid-secretory uterine fluid could serve as a non-invasive marker to assess endometrial receptivity in these patients.