scholarly journals Versican V1 in human endometrial epithelial cells promotes BeWo spheroid adhesion in vitro

Reproduction ◽  
2019 ◽  
pp. 53-64 ◽  
Author(s):  
Yumiko Miyazaki ◽  
Akihito Horie ◽  
Hirohiko Tani ◽  
Masashi Ueda ◽  
Asuka Okunomiya ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Chondroitin Sulfate ◽  
Cell Line ◽  
Human Embryo ◽  
Embryo Implantation ◽  
Cancer Cell Line ◽  
Promoting Effect ◽  
Ishikawa Cells ◽  
Endometrial Epithelial Cells

The endometrium extracellular matrix (ECM) is essential for embryo implantation. Versican, a large chondroitin sulfate proteoglycan that binds hyaluronan and forms large ECM aggregates, can influence fundamental physiological phenomena, such as cell proliferation, adhesion and migration. The present study investigated the possible role of versican in human embryo implantation. Versican V1 expression and secretion in human endometrial epithelial cells (EECs) was most prominent in the mid-secretory phase. Versican expression in EECs significantly increased after treatment with estrogen and progesterone, but not by estrogen alone. We also established versican V1-overexpressing Ishikawa (endometrial cancer cell line) cells (ISKW-V1), versican V3-overexpressing (ISKW-V3) and control GFP-overexpressing (ISKW-GFP) Ishikawa cells. By the in vitro implantation model, the attachment ratio of BeWo (choriocarcinoma cell line) spheroids to the monolayer of ISKW-V1, but not of ISKW-V3, was found significantly enhanced compared with attachment to the ISKW-GFP monolayer. The conditioned medium derived from ISKW-V1 (V1-CM) also promoted the attachment of BeWo spheroids to the ISKW monolayer. However, this attachment-promoting effect was abolished when V1-CM was pretreated with chondroitinase ABC, which degrades chondroitin sulfate. Therefore, out of the ECM components, versican V1 may facilitate human embryo implantation.

scholarly journals Mitochondrial dysfunction induced by high estradiol concentrations in endometrial epithelial cells

2019 ◽  
Author(s):  
Chia-Hung Chou ◽  
Shee-Uan Chen ◽  
Chin-Der Chen ◽  
Chia-Tung Shun ◽  
Wen-Fen Wen ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Mitochondrial Dysfunction ◽  
Embryo Implantation ◽  
Ros Production ◽  
Vitro Fertilization ◽  
Equine Chorionic Gonadotropin ◽  
In Vivo Effects ◽  
Endometrial Epithelial Cells

Abstract Context A supraphysiological estradiol (E2) concentration after ovarian stimulation is known to result in lower embryo implantation rates in in vitro fertilization (IVF). Endometrial epithelial cells (EECs) apoptosis occurs after the stimulation with high E2 concentrations, and mitochondria play important roles in cell apoptosis. Objective To investigate the mitochondrial function in EECs after the stimulation with high E2 concentrations. Materials and Methods Human EECs were purified and cultured with different E2 concentrations (10-10, 10-9, 10-8, 10-7 M) in vitro, in which 10-7 M is supraphysiologically high. Eight-week-old female mouse endometrium was obtained 5.5 days after the injection of 1.25 IU or 20 IU equine chorionic gonadotropin (eCG), roughly during the embryo implantation window, to examine the in vivo effects of high E2 concentrations on mouse EECs. Results In vivo and in vitro experiments demonstrated decreased mitochondrial DNA contents and ATP formation after EECs were stimulated with supraphysiologically high E2 concentrations than those stimulated with a physiologic E2 concentration. Less prominent immunofluorescence mitochondrial staining, fewer mitochondria number under electron microscopy, lower JC-1 aggregate/monomer ratio, and greater reactive oxygen species (ROS) production were found after EECs were stimulated with supraphysiologically high E2 concentrations. The high E2-induced ROS production was reduced when EECs were pretreated with N-acetyl-cysteine (NAC) in vitro, but remained unchanged after the pretreatment with coenzyme Q10. Conclusion High E2 concentrations increase extra-mitochondrial ROS production in EECs and subsequently result in mitochondrial dysfunction.

scholarly journals The effects of hyaluronate-containing medium on human embryo attachment to endometrial epithelial cells in vitro

2020 ◽  
Vol 2020 (2) ◽  
Author(s):  
Peter T Ruane ◽  
Chelsea J Buck ◽  
Phoebe A Babbington ◽  
Wedad Aboussahoud ◽  
Stéphane C Berneau ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Embryo Transfer ◽  
Human Embryo ◽  
Research Network ◽  
Human Embryos ◽  
Ishikawa Cell ◽  
Practitioner Training ◽  
Cell Layers ◽  
Endometrial Epithelial Cells

Abstract STUDY QUESTION Does embryo transfer medium containing hyaluronate (HA) promote the attachment phase of human embryo implantation? SUMMARY ANSWER HA-containing medium does not promote human blastocyst attachment to endometrial epithelial cells in vitro. WHAT IS KNOWN ALREADY Embryo transfer media containing high concentrations of HA are being used to increase implantation and live birth rates in IVF treatment, although the mechanism of action is unknown. STUDY DESIGN, SIZE, DURATION Expression of HA-interacting genes in frozen-thawed oocytes/embryos was assessed by microarray analysis (n = 21). Fresh and frozen human blastocysts (n = 98) were co-cultured with human endometrial epithelial Ishikawa cell layers. Blastocyst attachment and the effects of a widely used HA-containing medium were measured. PARTICIPANTS/MATERIALS, SETTING, METHODS Human embryos surplus to treatment requirements were donated with informed consent from several ART centres. Blastocyst-stage embryos were transferred at day 6 to confluent Ishikawa cell layers; some blastocysts were artificially hatched. Blastocyst attachment was monitored from 1 to 48 h, and the effects of blastocyst pre-treatment for 10 min with HA-containing medium were determined. MAIN RESULTS AND THE ROLE OF CHANCE Human embryos expressed the HA receptor genes CD44 and HMMR, hyaluronan synthase genes HAS1–3, and hyaluronidase genes HYAL1–3, at all stages of preimplantation development. Attachment of partially hatched blastocysts to Ishikawa cells at 24 and 48 h was related to trophectoderm grade (P = 0.0004 and 0.007, respectively, n = 34). Blastocysts of varying clinical grades that had been artificially hatched were all attached within 48 h (n = 21). Treatment of artificially hatched blastocysts with HA-containing medium did not significantly affect attachment at early (1–6 h) or late (24 and 48 h) time points, compared with control blastocysts (n = 43). LIMITATIONS, REASONS FOR CAUTION Using an adenocarcinoma-derived cell line to model embryo-endometrium attachment may not fully recapitulate in vivo interactions. The high levels of blastocyst attachment seen with this in vitro model may limit the sensitivity with which the effects of HA can be observed. WIDER IMPLICATIONS OF THE FINDINGS Morphological trophectoderm grade can be correlated with blastocyst attachment in vitro. HA-containing medium may increase pregnancy rates by mechanisms other than promoting blastocyst attachment to endometrium. STUDY FUNDING/COMPETING INTEREST(S) This work was funded by a grant from the Wellbeing of Women, the NIHR Local Comprehensive Research Network and NIHR Manchester Clinical Research Facility, the Department of Health Scientist Practitioner Training Scheme, and the Ministry of Higher Education, The State of Libya. None of the authors has any conflict of interest to declare.

scholarly journals Proteomic analysis of extracellular vesicles secreted by primary human epithelial endometrial cells reveals key proteins related to embryo implantation

2022 ◽  
Vol 20 (1) ◽  
Author(s):  
Marina Segura-Benítez ◽  
María Cristina Carbajo-García ◽  
Ana Corachán ◽  
Amparo Faus ◽  
Antonio Pellicer ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Proteomic Analysis ◽  
Embryo Implantation ◽  
Endometrial Receptivity ◽  
Biological Processes ◽  
Endometrial Cells ◽  
Isolation Methods ◽  
Ishikawa Cells ◽  
Endometrial Epithelial Cells ◽  
Implantation Success

Abstract Background Successful implantation is dependent on coordination between maternal endometrium and embryo, and the role of EVs in the required cross-talk cell-to-cell has been recently established. In this regard, it has been reported that EVs secreted by the maternal endometrium can be internalized by human trophoblastic cells transferring their contents and enhancing their adhesive and invasive capacity. This is the first study to comprehensively evaluate three EV isolation methods on human endometrial epithelial cells in culture and to describe the proteomic content of EVs secreted by pHEECs from fertile women. Methods Ishikawa cells and pHEECs were in vitro cultured and hormonally treated; subsequently, conditioned medium was collected and EVs isolated. Ishikawa cells were used for the comparison of EVs isolation methods ultracentrifugation, ExoQuick-TC and Norgen Cell Culture Media Exosome Purification Kit (n = 3 replicates/isolation method). pHEECs were isolated from endometrial biopsies (n = 8/replicate; 3 replicates) collected from healthy oocyte donors with confirmed fertility, and protein content of EVs isolated by the most efficient methodology was analysed using liquid chromatography–tandem mass spectrometry. EV concentration and size were analyzed by nanoparticle tracking analysis, EV morphology visualized by transmission electron microscopy and protein marker expression was determined by Western blotting. Results Ultracentrifugation was the most efficient methodology for EV isolation from medium of endometrial epithelial cells. EVs secreted by pHEECs and isolated by ultracentrifugation were heterogeneous in size and expressed EV protein markers HSP70, TSG101, CD9, and CD81. Proteomic analysis identified 218 proteins contained in these EVs enriched in biological processes involved in embryo implantation, including cell adhesion, differentiation, communication, migration, extracellular matrix organization, vasculature development, and reproductive processes. From these proteins, 82 were selected based on their functional relevance in implantation success as possible implantation biomarkers. Conclusions EV protein cargos are implicated in biological processes related to endometrial receptivity, embryo implantation, and early embryo development, supporting the concept of a communication system between the embryo and the maternal endometrium via EVs. Identified proteins may define new biomarkers of endometrial receptivity and implantation success.

scholarly journals GnRH antagonist alters the migration of endometrial epithelial cells by reducing CKB

Reproduction ◽  
2020 ◽  
Vol 159 (6) ◽  
pp. 733-743 ◽  
Author(s):  
Qian Chen ◽  
Yong Fan ◽  
Xiaowei Zhou ◽  
Zheng Yan ◽  
Yanping Kuang ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Molecular Mechanisms ◽  
Implantation Rate ◽  
Endometrial Receptivity ◽  
Ishikawa Cells ◽  
Atp Generation ◽  
And Migration ◽  
Insight Into ◽  
Endometrial Epithelial Cells

Some studies have demonstrated that the implantation rate of fresh transfer cycles is lower in the gonadotropin-releasing hormone antagonist (GnRH-ant) protocol than in the GnRH agonist (GnRH-a) protocol during in vitro fertilization (IVF). This effect may be related to endometrial receptivity. However, the mechanisms are unclear. Here, endometrial tissues obtained from the mid-secretory phase of patients treated with GnRH-a or GnRH-ant protocols and from patients on their natural cycle were assessed. Endometrial expression of B-type creatine kinase (CKB), which plays important roles in the implantation phase, was significantly reduced in the GnRH-ant group. At the same time, expression of the endometrial receptivity marker HOXA10 was considerably reduced in the GnRH-ant group. GnRH-ant exposure in endometrial epithelial cells (EECs) in vitro decreased CKB expression and ATP generation and blocked polymerization of actin. Furthermore, in vitro GnRH-ant-exposed Ishikawa cells showed enhanced F-actin depolymerization, and these effects were rescued by CKB overexpression. Similar effects were observed after CKB knockdown, and these effects were rescued by CKB overexpression. Moreover, cell migration was decreased in CKB-knockdown Ishikawa cells compared with that in control cells, and this effect was also rescued by CKB overexpression. Overall, these findings showed that GnRH-ant affected CKB expression in EECs, resulting in cytoskeletal damage and migration failure. These results provide insight into the roles and molecular mechanisms of GnRH-ant treatment in the endometrium.

TKI258, a novel, multitargeted tyrosine kinase inhibitor for the treatment of breast cancer

2009 ◽  
Vol 27 (15_suppl) ◽  
pp. e14625-e14625
Author(s):  
H. Liu ◽  
C. O. Schulz ◽  
A. C. Regierer ◽  
A. Dieing ◽  
K. Possinger ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Cell Line ◽  
Cancer Cell ◽  
Breast Cancer Cell ◽  
Breast Cancer Cell Line ◽  
Cancer Cell Line ◽  
Tumor Stroma ◽  
Promoting Effect ◽  
Cancer Stroma

e14625 TKI258 (4-amino-5-fluor-3-[5-(4-metylpiperazin-1-yl)-1H-benzimidazol-2-yl]quinolin-2(1H)-one, formerly known as CHIR258) is an ATP-competitive inhibitor with activities against class III or IV receptor kinases, which include FGFR, VEGFR, PDGFR, FLT3, and KIT. It has been demonstrated to possess strong anti-tumor and anti-angiogenetic activities in different tumor models, and, therefore, this compound is currently being clinically assessed for the treatment of diverse malignancies. In this study, we chose the breast cancer cell line MDA-MB-231, a cell line with high invasive capacities, as an in vitro model to analyze the effect and functional mechanism of TKI258 on the breast cancer invasiveness. Treatment of MDA-MB-231 cells with TKI258 resulted in reduced invasive capacities in a dose-dependent manner. In association with this effect, we observed that TKI258 down-regulated the phosphorylation of ERK1/2 and STAT3 and inhibited the VEGF production in the cell supernatants. Most interestingly, we found TKI258 had influence on the inflammatory chemokines CCL5 and CCL2 level if MDA-MB-231 cells were co-cultured with breast cancer stroma cells. We found that CCL5/CCL2 mRNA level in MDA-MB-231 cells, in stroma cells, or in co-culture of MDA-MB-231/breast cancer stroma was strongly inhibited by TKI258 as detected with real-time PCR. Parallel to this result, the dramatically elevated CCL2/CCL5 level in the media supernatants from co-cultured MDA-MB-231/stroma cells was reduced by TKI258 effectively. Furthermore, we demonstrated that the invasion-promoting effect of the tumor stroma cells was antagonized by TKI258 significantly. CCL5 stimulated invasion of MDA-MB-231 cells could be partially abrogated by TKI58 and/or by CCL5-neutralizing antibody. Therefore, it is most likely that the inhibitory effect of TKI258 on invasion of MDA-MB-231 cells in the presence of stroma cells is achieved, at least in part, by antagonizing the invasion-promoting effect of CCL5. Overall, our data show that TKI258 inhibited invasive capacities of aggressive breast cancer cell line MDA-MB-231, either in the absence or presence of tumor stroma cells in vitro. No significant financial relationships to disclose.

P–379 Human platelet lysate improves trophoblast spheroid attachment to primary endometrial epithelial cells from patients with recurrent implantation failure

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
T T T N Nguyen ◽  
Y S S Kwok ◽  
S Russell ◽  
C Librach
Keyword(s):  
Epithelial Cells ◽  
Cell Line ◽  
Fluorescent Microscopy ◽  
Endometrial Receptivity ◽  
Platelet Lysate ◽  
Implantation Failure ◽  
Trophoblast Cells ◽  
Recurrent Implantation Failure ◽  
Endometrial Epithelial Cells

Abstract Study question Could non-autologous platelet lysate (PL) increase attachment of HTR–8 spheroids in vitro to primary endometrial epithelial cells (EECs) from patients with recurrent implantation failure (RIF)? Summary answer Increased quantity of HTR–8 spheroids attached to primary EECs, isolated from patients with RIF, suggests in vitro treatment with non-autologous PL could improve endometrial receptivity. What is known already Inadequate endometrial receptivity and thickness are major causes for RIF. Recent studies suggest that platelet-rich plasma (PRP) may improve pregnancy outcomes for RIF and/or thin endometrium (TE) patients. Our previous results show that a commercially sourced and non-autologous human PRP/PL (HPL) promotes EC proliferation in vitro, suggesting that HPL may help to standardize future clinical treatments. In addition to EC proliferation, HPL treatment may improve embryo attachment to primary EECs isolated from patients with a history of RIF. In vitro attachment assays with trophoblast spheroids (embryo model) could help elucidate the effect of HPL on endometrial receptivity in RIF patients. Study design, size, duration Endometrial tissue was collected from nine RIF patients at the CReATe Fertility Centre, Toronto, Canada (Veritas REB#16580): five with (RIF+TE) and four without a TE (RIF only). Primary EECs were enzymatically isolated and treated with serum-free culture media (SFM) or 1% HPL in SFM for 48 hours before performing the attachment assay. Trophoblast cells (HTR–8/SVneo) were grown in suspension on a rocker to form 70–100 uM spheroids over 24 hours before use in the assay. Participants/materials, setting, methods Spheroids were fluorescently labelled with calcein-AM for 30 minutes and size-selected to capture spheroids similar in size to a human blastocyst. Spheroids were seeded on top of EEC monolayers and calcein fluorescence was immediately measured by a spectrophotometer. Following the 1-hour incubation, unattached spheroids were aspirated, and fluorescence was measured again. Spheroids were also individually quantified by fluorescent microscopy and ImageJ™ software. The percentage of spheroid attachment was calculated for calcein fluorescence and ImageJ™ quantification. Main results and the role of chance The HTR–8/SVneo cell line, derived from human first-trimester extravillous trophoblast cells (EVT), has been shown to be a suitable cell line to assess adhesion and invasion in vitro. Trophoblast spheroids generated from this cell line visually resembled a blastocyst and maintained expression of the EVT and implantation biomarkers: GATA3, ITGA5, and LIF. Primary EECs, treated for 48 hours with SFM supplemented with 1% commercially sourced and non-autologous HPL, overall exhibited increased attachment to HTR–8 spheroids. The percentage of spheroid attachment, as measured by fluorescence alone, significantly increase from 47.98% to 64.27% (P < 0.01) of seeded spheroids in RIF+TE EEC cultures, and from 48.12% to 85.77% (P < 0.001) of seeded spheroids in RIF only EEC cultures. Quantification by fluorescent microscopy and ImageJ™ software for individual calcein-stained spheroids, revealed a significant increase in spheroid attachment, from 57.52% to 86.5% (P < 0.01) in RIF+TE EEC cultures, and from 42.58% to 68.90% (P < 0.01) in RIF only EEC cultures. Limitations, reasons for caution Although there was a positive correlation between calcein fluorescence and spheroid quantity, quantification by fluorescence alone may be unreliable due to the variable numbers of cells in each spheroid. Our data suggest a more precise increase in attachment is detected when quantified by fluorescent microscopy and ImageJ™ software. Wider implications of the findings: We report a method for functional assessment of endometrial receptivity in vitro. HPL appears to promote implantation in RIF patients in a model of embryo attachment. We predict that the observed increase in attachment is due to increased endometrial receptivity gene expression, which will be our next investigative avenue. Trial registration number N/A

Regulation of tight junctions by sex hormones in normal human endometrial epithelial cells and uterus cancer cell line Sawano

2013 ◽  
Vol 354 (2) ◽  
pp. 481-494 ◽  
Author(s):  
Masayuki Someya ◽  
Takashi Kojima ◽  
Marie Ogawa ◽  
Takafumi Ninomiya ◽  
Kazuaki Nomura ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Cell Line ◽  
Tight Junctions ◽  
Sex Hormones ◽  
Cancer Cell ◽  
Cancer Cell Line ◽  
Normal Human ◽  
Endometrial Epithelial Cells

New Platinum (II) Ternary Complexes of Formamidine and Pyrophosphate: Synthesis, Characterization and DFT Calculations and In vitro Cytotoxicity

2020 ◽  
Vol 23 (7) ◽  
pp. 611-623
Author(s):  
Ahmed A. Soliman ◽  
Fawzy A. Attaby ◽  
Othman I. Alajrawy ◽  
Azza A.A. Abou-hussein ◽  
Wolfgang Linert
Keyword(s):  
Cell Line ◽  
Cancer Cell ◽  
Theoretical Calculations ◽  
Thermal Analyses ◽  
Cancer Cell Line ◽  
Cellular Growth ◽  
Planar Geometry ◽  
Square Planar ◽  
Vis Spectroscopy

Aim and Objective: Platinum (II) and platinum (IV) of pyrophosphate complexes have been prepared and characterized to discover their potential as antitumor drugs. This study was conducted to prepare and characterize new ternary platinum (II) complexes with formamidine and pyrophosphate as an antitumor candidate. Materials and Methods: The complexes have been characterized by mass, infrared, UV-Vis. spectroscopy, elemental analysis, magnetic susceptibility, thermal analyses, and theoretical calculations. They have been tested for their cytotoxicity, which was carried out using the fastcolorimetric assay for cellular growth and survival against MCF-7 (breast cancer cell line), HCT- 116 (colon carcinoma cell line), and HepG-2 (hepatocellular cancer cell line). Results: All complexes are diamagnetic, and the electronic spectral data displayed the bands due to square planar Pt(II) complexes. The optimized complexes structures (1-4) indicated a distorted square planar geometry where O-Pt-O and N-Pt-N bond angles were 82.04°-96.44°, respectively. Conclusion: The complexes showed noticeable cytotoxicity and are considered as promising antitumor candidates for further applications.

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