miR-23a-3p increases endometrial receptivity via CUL3 during embryo implantation

A receptive endometrium is required in a successful embryo implantation. The ubiquitination-induced β-catenin degradation is related to the implantation failure.This study aimed to elucidate whether miR-23a-3p regulates endometrial receptivity via the modulation of β-catenin ubiquitination.The expressions of miR-23a-3p and CUL3 were detected in endometrial epithelial cells (EECs) isolated from pregnant mice and in hormone-induced EEC-like Ishikawa cells. The ubiquitination experiment was performed to explore the effect of CUL3 and miR-23a-3p on β-catenin ubiquitination level. The trophoblast attachment was detected by co-culturing JAR (choriocarcinoma cell line) spheroids with Ishikawa cell monolayers. miR-23a-3p was upregulated while CUL3 was downregulated in EECs at day 4 after pregnancy compared with day 1, as well as in hormone-induced Ishikawa cells. miR-23a-3p positively regulated the protein level of β-catenin without affecting the mRNA level. The ubiquitination and degradation of β-catenin was suppressed by miR-23a-3p, while it was promoted by CUL3. Immunoprecipitation confirmed the binding between CUL3 and β-catenin. Luciferase reporter assay confirmed the target relationship between miR-23a-3p and CUL3. The ubiquitination of β-catenin was modulated by the miR-23a-3p/CUL3 pathway. The overexpression of miR-23a-3p promoted JAR spheroid attachments in Ishikawa cells. miR-23a-3p is beneficial for the endometrial receptivity and embryo implantation, whose mechanism is partly through the modulation of CUL3/β-catenin.

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Podocalyxin promotes an impermeable epithelium and inhibits pro-implantation factors to negatively regulate endometrial receptivity

Scientific Reports ◽

10.1038/s41598-021-03425-2 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Sophea Heng ◽

Nirukshi Samarajeewa ◽

Yao Wang ◽

Sarah G. Paule ◽

James Breen ◽

...

Keyword(s):

Embryo Implantation ◽

Endometrial Receptivity ◽

Negative Regulator ◽

Epithelial Barrier ◽

Limiting Factor ◽

Ishikawa Cell ◽

Barrier Functions ◽

Functional Studies ◽

Expression Of Genes ◽

Endometrial Epithelial Cells

AbstractEmbryo implantation is a key step in establishing pregnancy and a major limiting factor in IVF. Implantation requires a receptive endometrium but the mechanisms governing receptivity are not well understood. We have recently discovered that podocalyxin (PCX or PODXL) is a key negative regulator of human endometrial receptivity. PCX is expressed in all endometrial epithelial cells in the non-receptive endometrium but selectively down-regulated in the luminal epithelium at receptivity. We have further demonstrated that this down-regulation is essential for implantation because PCX inhibits embryo attachment and penetration. However, how PCX confers this role is unknown. In this study, through RNAseq analysis of Ishikawa cell line stably overexpressing PCX, we discovered that PCX suppresses expression of genes controlling cell adhesion and communication, but increases those governing epithelial barrier functions, especially the adherens and tight junctions. Moreover, PCX suppresses multiple factors such as LIF and signaling pathways including Wnt and calcium signaling that support receptivity but stimulates anti-implantation genes such as LEFTY2. Functional studies confirmed that PCX promotes epithelial barrier functions by increasing key epithelial junction proteins such as E-cadherin and claudin 4. PCX thus promotes an anti-adhesive and impermeable epithelium while impedes pro-implantation factors to negatively control endometrial receptivity for implantation.

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

Reproductive Biology and Endocrinology ◽

10.1186/s12958-021-00879-x ◽

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.

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Characterisation of Osteopontin in an In Vitro Model of Embryo Implantation

Cells ◽

10.3390/cells8050432 ◽

2019 ◽

Vol 8 (5) ◽

pp. 432 ◽

Cited By ~ 3

Author(s):

Stéphane C Berneau ◽

Peter T Ruane ◽

Daniel R Brison ◽

Susan J Kimber ◽

Melissa Westwood ◽

...

Keyword(s):

Secretory Pathway ◽

Embryo Implantation ◽

Giant Cells ◽

Ishikawa Cell ◽

Altered Expression ◽

Ishikawa Cells ◽

Cell Layers ◽

Endometrial Epithelium ◽

Vitro Model

At the onset of pregnancy, embryo implantation is initiated by interactions between the endometrial epithelium and the outer trophectoderm cells of the blastocyst. Osteopontin (OPN) is expressed in the endometrium and is implicated in attachment and signalling roles at the embryo–epithelium interface. We have characterised OPN in the human endometrial epithelial Ishikawa cell line using three different monoclonal antibodies, revealing at least nine distinct molecular weight forms and a novel secretory pathway localisation in the apical domain induced by cell organisation into a confluent epithelial layer. Mouse blastocysts co-cultured with Ishikawa cell layers served to model embryo apposition, attachment and initial invasion at implantation. Exogenous OPN attenuated initial, weak embryo attachment to Ishikawa cells but did not affect the attainment of stable attachment. Notably, exogenous OPN inhibited embryonic invasion of the underlying cell layer, and this corresponded with altered expression of transcription factors associated with differentiation from trophectoderm (Gata2) to invasive trophoblast giant cells (Hand1). These data demonstrate the complexity of endometrial OPN forms and suggest that OPN regulates embryonic invasion at implantation by signalling to the trophectoderm.

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Increased METTL3-mediated m6A methylation inhibits embryo implantation by repressing HOXA10 expression in recurrent implantation failure

Reproductive Biology and Endocrinology ◽

10.1186/s12958-021-00872-4 ◽

2021 ◽

Vol 19 (1) ◽

Author(s):

Pingping Xue ◽

Wenbo Zhou ◽

Wenqiang Fan ◽

Jianya Jiang ◽

Chengcai Kong ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Embryo Implantation ◽

Endometrial Receptivity ◽

Mrna Levels ◽

Implantation Failure ◽

Quantitative Real Time Pcr ◽

Recurrent Implantation Failure ◽

Ishikawa Cells ◽

Embryo Attachment

Abstract Background Recurrent implantation failure (RIF) is a major limitation of assisted reproductive technology, which is associated with impaired endometrial receptivity. Although N6-methyladenosine (m6A) has been demonstrated to be involved in various biological processes, its potential role in the endometrium of women with RIF has been poorly studied. Methods Global m6A levels and major m6A methyltransferases/demethylases mRNA levels in mid-secretory endometrium from normal and RIF women were examined by colorimetric m6A quantification strategy and quantitative real-time PCR, respectively. The effects of METTL3-mediated m6A modification on embryo attachment were evaluated by an vitro model of a confluent monolayer of Ishikawa cells co-cultured with BeWo spheroids, and the expression levels of homeo box A10 (HOXA10, a well-characterized marker of endometrial receptivity) and its downstream targets were evaluated by quantitative real-time PCR and Western blotting in METTL3-overexpressing Ishikawa cells. The molecular mechanism for METTL3 regulating HOXA10 expression was determined by methylated RNA immunoprecipitation assay and transcription inhibition assay. Results Global m6A methylation and METTL3 expression were significantly increased in the endometrial tissues from women with RIF compared with the controls. Overexpression of METTL3 in Ishikawa cells significantly decreased the ration of BeWo spheroid attachment, and inhibited HOXA10 expression with downstream decreased β3-integrin and increased empty spiracles homeobox 2 expression. METTL3 catalyzed the m6A methylation of HOXA10 mRNA and contributed to its decay with shortened half-life. Enforced expression of HOXA10 in Ishikawa cells effectively rescued the impairment of METTL3 on the embryo attachment in vitro. Conclusion Increased METTL3-mediated m6A modification represents an adverse impact on embryo implantation by inhibiting HOXA10 expression, contributing to the pathogenesis of RIF.

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182 CALBINDIN-D28k IS PREDOMINANTLY EXPRESSED AND REGULATED BY ESTROGEN IN HUMAN ENDOMETRIUM DURING MENSTRUAL CYCLE

Reproduction Fertility and Development ◽

10.1071/rdv23n1ab182 ◽

2011 ◽

Vol 23 (1) ◽

pp. 192

Author(s):

K.-C. Choi ◽

H. Yang ◽

E.-B. Jeung

Keyword(s):

Menstrual Cycle ◽

Calcium Binding ◽

Potential Role ◽

Embryo Implantation ◽

Endometrial Receptivity ◽

Human Endometrium ◽

Ishikawa Cell ◽

Proliferative Phase ◽

Calbindin D28k ◽

The Brain

The human endometrium resists embryo implantation except during the window of receptivity. A change in endometrial gene expression is required for the development of receptivity. Uterine calbindin-D28k (CaBP-28k) has been shown to be involved in the regulation of endometrial receptivity by intracellular Ca2+. Nowadays, this protein has been mainly linked to the brain, kidneys, and pancreas, but potential role(s) of CaBP-28k remain to be clarified in the uterus of humans during the menstrual cycle. Thus, we demonstrated in this study that the expression of CaBP-28k in the human endometrium in more divided in the menstrual phases. During the menstrual cycle of humans, uterine expression levels of CaBP-28k mRNA and protein increased at the proliferative phase and fluctuated in these tissues, compared with other phases. We assessed the effects of the sex-steroid hormones E2 and P4 on the expression of CaBP-28k in the Ishikawa cell line. A significant increase in the expression of CaBP-9k mRNA was observed at the concentration of 17β-oestradiol (E2; 10–9 to 10–7 M). In addition, spatial expression of CaBP-28k was detected by immunohistochemistry. CaBP-28k is abundantly localised in the cytoplasm of the luminal and glandular epithelial cells during the menstrual cycle. Taken together, these results indicate that CaBP-28k, a uterine calcium-binding protein, is abundantly expressed in the human uterus, suggesting that uterine expression of CaBP-28k may be involved in reproductive functions during the menstrual cycle of humans.

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Uterine SOX17: a key player in human endometrial receptivity and embryo implantation

Scientific Reports ◽

10.1038/s41598-019-51751-3 ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 2

Author(s):

Sophie Kinnear ◽

Lois A. Salamonsen ◽

Mathias Francois ◽

Vincent Harley ◽

Jemma Evans

Keyword(s):

Epithelial Cells ◽

Clinical Investigation ◽

Embryo Implantation ◽

Adhesive Contact ◽

Endometrial Receptivity ◽

Large Numbers ◽

Yin And Yang ◽

Hormonal Milieu ◽

Endometrial Epithelial Cells

Abstract The yin and yang of female fertility is a complicated issue; large numbers of women/couples desire fertility and seek assisted reproduction intervention to achieve conception, while others seek to prevent pregnancy. Understanding specific molecules which control endometrial-embryo interactions is essential for both facilitating and preventing pregnancy. SOX17 has recently emerged as an important transcription factor involved in endometrial receptivity and embryo implantation. However, studies to date have examined mouse models of pregnancy which do not necessarily translate to the human. Demonstration of a role for ‘implantation factors’ in a human system is critical to provide a rationale for in depth clinical investigation and targeting of such factors. We demonstrate that SOX17is present within the receptive human endometrium and is up-regulated within human endometrial epithelial cells by combined estrogen & progesterone, the hormonal milieu during the receptive window. SOX17 localizes to the point of adhesive contact between human endometrial epithelial cells and a human ‘embryo mimic’ model (trophectodermal spheroid). Targeting SOX17 in endometrial epithelial cells using CRISPR/Cas9 knockdown or a SOX-F family inhibitor, MCC177, significantly inhibited adhesion of an trophectodermal spheroids to the epithelial cells thereby preventing ‘implantation’. These data confirm the important role of endometrial SOX17 in human endometrial receptivity and embryo implantation.

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Effects of DEHP on endometrial receptivity and embryo implantation in pregnant mice

Journal of Hazardous Materials ◽

10.1016/j.jhazmat.2012.09.038 ◽

2012 ◽

Vol 241-242 ◽

pp. 231-240 ◽

Cited By ~ 31

Author(s):

Rui Li ◽

Chao Yu ◽

Rufei Gao ◽

Xueqing Liu ◽

Jing Lu ◽

...

Keyword(s):

Embryo Implantation ◽

Endometrial Receptivity ◽

Pregnant Mice

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GRIM-19, a gene associated with retinoid-interferon-induced mortality, affects endometrial receptivity and embryo implantation

Reproduction Fertility and Development ◽

10.1071/rd16104 ◽

2017 ◽

Vol 29 (7) ◽

pp. 1447 ◽

Cited By ~ 2

Author(s):

Yang Yang ◽

Yanyan Sun ◽

Laiyang Cheng ◽

Anna Li ◽

Yanjun Shen ◽

...

Keyword(s):

Immune Tolerance ◽

Embryo Implantation ◽

Pregnant Mouse ◽

Endometrial Receptivity ◽

Mrna Levels ◽

Protein Levels ◽

Tnf Α ◽

Pregnant Mice ◽

Adhesion Rate

GRIM-19 is associated with apoptosis, abnormal proliferation, immune tolerance and malignant transformation, and it also plays an important role in early embryonic development. Although the homologous deletion of GRIM-19 causes embryonic lethality in mice, the precise role of GRIM-19 in embryo implantation has not been elucidated. Here we show that GRIM-19 plays an important role in endometrial receptivity and embryo implantation. Day 1 to Day 6 pregnant mouse uteri were collected. Immunohistochemistry studies revealed the presence of GRIM-19 on the luminal epithelium and glandular epithelium throughout the implantation period in pregnant mice. The protein and mRNA levels of GRIM-19 were markedly decreased on Day 4 of pregnancy in pregnant mice, but there was no change in GRIM-19 levels in a group of pseudopregnant mice. Overexpression of GRIM-19 decreased the adhesion rate of RL95–2–BeWo co-cultured spheroids and increased apoptosis. Furthermore, STAT3 and IL-11 mRNA and protein levels were reduced by overexpressing GRIM-19, but protein and mRNA levels of TNF-α were increased. These findings indicate the involvement of GRIM-19 in the embryo implantation process by regulating adhesion, apoptosis and immune tolerance.

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hsa_circ_001946 elevates HOXA10 expression and promotes the development of endometrial receptivity via sponging miR-135b

Diagnostic Pathology ◽

10.1186/s13000-021-01104-4 ◽

2021 ◽

Vol 16 (1) ◽

Author(s):

Fang Zhao ◽

Yihong Guo ◽

Zhanrong Shi ◽

Menglan Wu ◽

Yuzhen Lv ◽

...

Keyword(s):

Cell Cycle ◽

Cell Proliferation ◽

Western Blotting ◽

Embryo Implantation ◽

Endometrial Receptivity ◽

Luciferase Reporter ◽

Implantation Failure ◽

Recurrent Implantation Failure ◽

Tissue Samples ◽

Qrt Pcr

Abstract Background Impaired endometrial receptivity is a major reason for embryo implantation failure. There’s a paucity of information regarding the role of circRNAs on endometrial receptivity. Here, we investigated the function of hsa_circ_001946 on endometrial receptivity and its mechanisms. Methods A total of 50 women composing 25 with recurrent implantation failure and 25 who conceived after their implantation were recruited in this study. Expression of hsa_circ_001946, miR-135b, and HOXA10 was evaluated by quantitative RT-PCR (qRT-PCR) in biopsied endometrial tissue samples. The levels of HOXA10, and cell cycle markers (CCNB1, CDK1, and CCND1) were determined by IHC and western blotting assays. Binding relationship among miR-135b, hsa_circ_001946 and HOXA10 were confirmed by dual luciferase reporter assays and western blotting. MTT assays and cell cycle assays by FACS were employed to evaluate the proliferation and cell cycle of cells. T-HESCs were cultured with 1 µM medroxyprogesterone acetate (MPA) and 0.5 mM 8-bromoadenosine 3’:5’-cyclic monophosphate (8-Br-cAMP) to induce decidualization. The mechanisms and functions of hsa_circ_001946 on decidualization were further assessed by qRT-PCR evaluating the expression of hsa_circ_001946, miR-135b, HOXA10 and decidual markers (PRL and IGFBP1) in T-HESCs. Results Endometrial tissues from patients with recurrent implantation failure had lower hsa_circ_001946 expression, higher miR-135b expression, and lower HOXA10 expression. Hsa_circ_001946 promoted HOXA10 expression by sponging miR-135b in T-HESCs. Overexpression of hsa_circ_001946 restored cell proliferation and cell cycle that were disrupted by miR-135b overexpression in T-HESCs. Decidualized T-HESCs had higher hsa_circ_001946 expression, lower miR-135b expression, and higher HOXA10 expression. Overexpression of hsa_circ_001946 reversed the expression of decidual markers (PRL and IGFBP1) that were suppressed by miR-135b overexpression in T-HESCs. Conclusions In conclusion, our findings suggest that hsa_circ_001946 promotes cell proliferation and cell cycle process and increases expression of decidualization markers to enhance endometrial receptivity progression via sponging miR-135b and elevating HOXA10.

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Serum estradiol levels in controlled ovarian stimulation directly affect the endometrium

Journal of Molecular Endocrinology ◽

10.1530/jme-17-0036 ◽

2017 ◽

Vol 59 (2) ◽

pp. 105-119 ◽

Cited By ~ 19

Author(s):

Kamran Ullah ◽

Tanzil Ur Rahman ◽

Hai-Tao Pan ◽

Meng-Xi Guo ◽

Xin-Yan Dong ◽

...

Keyword(s):

Embryo Implantation ◽

Controlled Ovarian Hyperstimulation ◽

Endometrial Receptivity ◽

Protein Profiling ◽

Differentially Expressed ◽

Differentially Expressed Proteins ◽

Serum Estradiol ◽

Endometrial Cells ◽

Ishikawa Cells ◽

Estradiol Levels

Previous studies have shown that increasing estradiol concentrations had a toxic effect on the embryo and were deleterious to embryo adhesion. In this study, we evaluated the physiological impact of estradiol concentrations on endometrial cells to reveal that serum estradiol levels probably targeted the endometrium in controlled ovarian hyperstimulation (COH) protocols. An attachment model of human choriocarcinoma (JAr) cell spheroids to receptive-phase endometrial epithelial cells and Ishikawa cells treated with different estradiol (10−9 M or 10−7 M) concentrations was developed. Differentially expressed protein profiling of the Ishikawa cells was performed by proteomic analysis. Estradiol at 10−7 M demonstrated a high attachment rate of JAr spheroids to the endometrial cell monolayers. Using iTRAQ coupled with LC–MS/MS, we identified 45 differentially expressed proteins containing 43 significantly upregulated and 2 downregulated proteins in Ishikawa cells treated with 10−7 M estradiol. Differential expression of C3, plasminogen and kininogen-1 by Western blot confirmed the proteomic results. C3, plasminogen and kininogen-1 localization in human receptive endometrial luminal epithelium highlighted the key proteins as possible targets for endometrial receptivity and interception. Ingenuity pathway analysis of differentially expressed proteins exhibited a variety of signaling pathways, including LXR/RXR activation pathway and acute-phase response signaling and upstream regulators (TNF, IL6, Hmgn3 and miR-140-3p) associated with endometrial receptivity. The observed estrogenic effect on differential proteome dynamics in Ishikawa cells indicates that the human endometrium is the probable target for serum estradiol levels in COH cycles. The findings are also important for future functional studies with the identified proteins that may influence embryo implantation.

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