KRT18 regulates trophoblast cell migration and invasion which are essential for embryo implantation.

Female infertility is a worldwide concern that impacts the quality of life and well-being of affected couples. Failure of embryo implantation is a major cause of early pregnancy loss and is precisely regulated by a programmed molecular mechanism. Recent studies have shown that proper trophoblast adhesion and invasion are essential for embryo implantation. However, the potential regulatory mechanism involved in trophoblast adhesion and invasion has yet to be fully elucidated. KRT18 has been reported to play a critical role in early embryonic development, but its physiological function in embryo implantation remains unclear. In the present study, we revealed that KRT18 was highly expressed in trophoblast cells and that knockdown of KRT18 in mouse embryos inhibited embryo adhesion and implantation. In vitro experiments further showed that silencing KRT18 disturbed trophoblast migration and invasion. More importantly, we provide evidence that KRT18 directly binds to and stabilizes cell surface E-cadherin in trophoblast cells through microscale thermophoresis (MST) analysis and molecular biology experiments. In brief, our data reveal that KRT18, which is highly expressed in trophoblast cells, plays an important role in the regulation of trophoblast invasion and adhesion during embryo implantation by directly binding to E-cadherin.

Par3/integrin ß1 regulates embryo adhesion via changing endometrial luminal epithelium polarity†.

The objective is to investigate the pathophysiological significance of Par3 and integrin ß1 with regard to the functionality of the endometrial luminal epithelium (LE). Design: laboratory study; setting: university research laboratory. Analysis involved endometrial aspirates and endometrial adenocarcinoma cells (HEC-1A) and endometrial carcinoma cells (RL95-2). We first examined the expression and localization of Par3 and integrin ß1 in HEC-1A cells and RL95-2 cells. Then we knocked down Par3 and integrin ß1 in HEC-1A cells and RL95-2 cells, respectively, and found that Par3/integrin ß1 affected embryo adhesion by regulating the intercellular tight junctions' (TJs') structure and thus the polarity of the endometrial LE. These findings were also confirmed in the endometrium specimens from human and mice. The main outcome measures were the expression and localization of Par3 and integrin ß1 in the endometrial epithelial cell lines and endometrium specimens and the regulations of Par3 and integrin ß1 on TJs, polarity, and embryo adhesion. Following the knockdown of Par3 in HEC-1A cells, there was a reduction in the complexity of the TJs and cell polarity, and the adhered blastocysts number was significantly increased. However, the reduction of integrin ß1 in RL95-2 cells resulted in effects that directly opposed those following the knockdown of Par3 in HEC-1A cells. Estrogen and progesterone reduced the expression of Par3 and promoted the expression of integrin ß1 in HEC-1A cells. Par3/integrin ß1 regulates embryo adhesion by regulating intercellular TJs' structure and polarity of endometrial LE under the action of ovarian hormones.

Progesterone promotes embryo adhesion by upregulating c-Fos/c-Jun transcription factor-mediated poFUT1 expression†.

The proliferation and adhesion abilities of placental trophoblasts are critical for embryo implantation and successful pregnancy. Protein O-fucosyltransferase 1 (poFUT1) and the transcription factor c-Fos/c-Jun have been found to promote trophoblastic cell invade into the endometrium. Progesterone is critical to the regulation of embryonic implantation. However, the exact role of poFUT1 in embryo proliferation and adhesion to the endometrium, and the relationship between progesterone, c-Fos/c-Jun, and poFUT1 has not been studied in detail. In the current study, we found that the serum levels of poFUT1 and progesterone significantly was decreased in miscarriage patients compared with those in normal pregnancy women, and there is a positive correlation between the changes in progesterone and poFUT1. Employing a human embryo trophoblastic cell line (JAR), we showed that progesterone facilitated the activation of c-Fos/c-Jun. Using an electrophoretic mobility shift assay and chromatin immunoprecipitation, we confirmed that the specific transcription factor c-Fos/c-Jun regulated the poFUT1 promoter, which could enhance poFUT1 transcriptional activity, thus further increasing trophoblast cell proliferation and adhesion potential. Taking these findings together, progesterone upregulates poFUT1 expression via the specific transcription factor c-Fos/c-Jun, and then increase trophoblast cell proliferation and adhesion potential. poFUT1 and progesterone may be used together as potential markers of miscarriage, and they may be diagnostic and therapeutic targets for miscarriage.

Investigating the role of CD44 and hyaluronate in embryo-epithelial interaction using an in vitro model.

Implantation failure is an important impediment to increasing success rates in assisted reproductive technologies. Knowledge of the cascade of morphological and molecular events at implantation remains limited. Cell surface CD44 and hyaluronate (HA) have been reported in the uterus, but a role in intercellular interaction at implantation remains to be evaluated. Mouse embryos were co-cultured with human Ishikawa endometrial epithelial monolayers over 2 days. Attachment was tenuous during the first 24 h, after which it became stable, leading to breaching of the monolayer. The effects of enzymatically reducing the density of HA, or introducing a function-blocking antibody to CD44, were monitored during progression from weak to stable embryonic attachment. Hyaluronidase-mediated removal of surface HA from the epithelial cells enhanced the speed of attachment, while a similar treatment of embryos had no effect. The antibody to CD44 caused retardation of initial attachment. These results suggest that CD44-HA binding could be employed by embryos during initial docking, but the persistence of HA in epithelial cells might be detrimental to later stages of implantation by retarding attainment of stable attachment.

Hsa-miR-30d, secreted by the human endometrium, is taken up by the pre-implantation embryo and might modify its transcriptome.

During embryo implantation, the blastocyst interacts with and regulates the endometrium, and endometrial fluid secreted by the endometrial epithelium nurtures the embryo. Here, we propose that maternal microRNAs (miRNAs) might act as transcriptomic modifier of the pre-implantation embryo. Microarray profiling revealed that six of 27 specific, maternal miRNAs were differentially expressed in the human endometrial epithelium during the window of implantation--a brief phase of endometrial receptivity to the blastocyst--and were released into the endometrial fluid. Further investigation revealed that hsa-miR-30d, the expression levels of which were most significantly upregulated, was secreted as an exosome-associated molecule. Exosome-associated and free hsa-miR-30d was internalized by mouse embryos via the trophectoderm, resulting in an indirect overexpression of genes encoding for certain molecules involved in the murine embryonic adhesion phenomenon--Itgb3, Itga7 and Cdh5. Indeed, this finding was supported by evidence in vitro: treating murine embryos with miR-30d resulted in a notable increase in embryo adhesion. Our results suggest a model in which maternal endometrial miRNAs act as transcriptomic modifiers of the pre-implantation embryo.

Activating transcription factor 3 promotes embryo attachment via up-regulation of leukemia inhibitory factor in vitro.

BACKGROUND: A receptive endometrium is essential for maternal-embryonic molecular communication during implantation. However, the specific molecular regulatory mechanisms of the endometrial capacity remain poorly understood. Here, we examined activating transcription factor 3 (ATF3) expression in human endometria and the functional effect of ATF3 on embryo attachment in vitro. METHODS: Immunohistochemistry (IHC) was used to assess the ATF3 expression patterns in human endometria. Quantitative real-time PCR (qRT-PCR), western blotting and immunofluorescence (IF) studies were applied to explore ATF3 expression in Ishikawa cells upon estrogen (E2) and medroxyprogesterone acetate (MPA) treatment. qRT-PCR and western blotting were performed to inspect LIF (leukemia inhibitory factor) expression after enhancement or inhibition of ATF3, and a luciferase reporter assay and ChIP-PCR were used to confirm the regulatory mechanism of ATF3 to LIF. Endometrial epithelial capacity was assessed by an in vitro model of attachment of BeWo spheroids to Ishikawa cells. Western blotting was performed to compare the expression of ATF3 in endometrial samples of recurrent implantation failure (RIF) patients with that in samples from fertile women (FER) who had undergone no less than one successful embryo transplantation. RESULTS: ATF3 was located in human endometrial epithelial cells and stromal cells and was significantly induced by E2 and MPA in Ishikawa cells. Adenovirus-mediated overexpression of ATF3 in Ishikawa cells activated LIF promoter activity and enhanced its expression. Accordingly, the stimulation of BeWo spheroid adhesion promoted by ATF3 was inhibited by pretreatment with a specific antibody against LIF via the antibody-blocking assay. Moreover, ATF3 was aberrantly decreased in the endometria of RIF patients. CONCLUSIONS: Our findings suggest that ATF3 plays a significant role in regulating human endometrial receptivity and embryo attachment in vitro via up-regulation of leukemia inhibitory factor. TRIAL REGISTRATION: Construction and management of the Nanjing multi-center biobank. No. 2013-081-01 . Registered 10 Dec. 2013.

Activation of matrix metalloproteinase-26 by HOXA10 promotes embryo adhesion in vitro.

Successful embryonic implantation requires an effective maternal-embryonic molecular dialogue. However, the detailed mechanisms of epithelial-embryo adhesion remain poorly understood. Here, we report that matrix metalloproteinase-26 (MMP-26) is a novel downstream target gene of homeobox a 10 (HOXA10) in human endometrial cells. HOXA10 binds directly to a conserved TTAT unit (-442 to -439) located within the 5' regulatory region of the MMP-26 gene and regulates the expression and secretion of MMP-26 in a concentration-dependent manner. Moreover, the adenovirus-mediated overexpression of MMP-26 in Ishikawa cells markedly increased BeWo spheroid adhesion. An antibody blocking assay further demonstrated that the promotion of BeWo spheroid adhesion by HOXA10 and MMP-26 was significantly inhibited by pre-treatment with a specific antibody against MMP-26. These results demonstrate that the HOXA10-mediated expression of MMP-26 promotes embryo adhesion during the process of embryonic implantation.

The Role of Extracellular Vesicles in Embryo Implantation.

Extracellular vesicles (EVs) are membrane-coating nanoparticles derived from cells. The effect of cell-to-cell communication mediated by EVs has been investigated in different fields of physio-logical as well as pathological process in recent years. Reproduction, regarded as a definitive characteristic of organisms, has been a focus in both animal and medical sciences. It is well agreed that implantation is a critical event during early pregnancy in viviparous animals, and a proper implantation is essential for the establishment and maintenance of normal pregnancy. However, successful implantation requires the synchronized development of both the uterus and the embryo, therefore, in which well communication and opportune regulation are necessary. This review focuses on the progression of studies that reveal the role of EVs in early pregnancy, especially during implantation. Based on current evidence, EVs are produced and exist in the environment for implantation. It has been proved that EVs of different origins such as endometrium and embryo, have positive influences on embryo implantation. With their cargos of proteins and nucleic acids (especially microRNAs), EVs exert their effects including information transportation, immune stimulation and regulation of gene expression.

Connective tissue growth factor gene expression in goat endometrium during estrous cycle and early pregnancy.

Embryo implantation is crucial for a successful pregnancy. Although many essential molecular modulators and pathways have been identified, the precise mechanisms of the process in goat remain largely unknown. CCN2 is a connective tissue growth factor participating in many biological processes; however, its presence or function in goat uterus has not yet been reported. In this study, we determined the expression and regulation of CCN2 in goat uterus. CCN2 was not detected by in situ hybridization at ED0 (Day 0 of the estrous cycle), but at ED6 (metestrus), ED12 (dioestrus), and ED16 (proestrus), with high signals in luminal epithelium, superficial glands, and caruncula matrix. During early pregnancy, CCN2 was also detected in these locations on D0 and D6 (pre-receptive uterus). The signals significantly increased on D16 and D19 (receptive uterus), and remained at high levels on D25 and D30. Similarly, the RT-qPCR assays showed that the mRNA level of CCN2 was relatively low on D0 and D6, increased on D16, peaked on D19, and kept high thereafter. Moreover, CCN2 was up-regulated not only in ovariectomized ewes subcutaneously injected with 17ß-estradiol and progesterone (separately or together), but also in cultured goat uterine epithelial cells treated with the two hormones or interferon tau (IFNτ). In conclusion, CCN2 expression may be induced by 17ß-estradiol, progesterone, and IFNτ in the luminal epithelium of goat receptive uterus, suggesting that CCN2 is involved in goat embryo adhesion during early pregnancy.

Nur77 promotes embryo adhesion by transcriptionally regulating HOXA10 expression.

HOXA10 is an important regulator of embryo adhesion. Reduced expression may contribute to embryo implantation failure. The expression of Nur77 and HOXA10 has been shown to be reduced in the endometrium of patients with recurrent implantation failure. In this study, we found that Nur77 was directly bound to the HOXA10 promoter and promoted HOXA10 protein expression in a dose-dependent manner in Ishikawa cells. Furthermore, the promoting effect of Nur77 on embryo adhesion was significantly blocked when endogenous HOXA10 expression was knocked down in Ishikawa cells. Moreover, the Nur77 agonist 6-MP was also confirmed to promote embryo adhesion. This study is the first to show that Nur77 promotes embryo adhesion by transcriptionally regulating HOXA10 expression. Nur77 and 6-MP may provide a basis to develop a new treatment for patients who suffer from embryo adhesion dysfunction.Abbreviations: HB-EGF: heparin-binding epidermal growth factor-like growth factor; HOXA10: homeobox A10; NGFI-B: nerve growth factor-induced gene B; RIF: recurrent implantation failure; RPL: recurrent pregnancy loss; 6-MP: 6-mercaptopurine; IGFBP-1: insulin-like growth factor binding protein-1; LIF: leukemia inhibitory factor; IL-1ß: Interleukin - 1ß; CRISPR/Cas9: Clustered Regularly Interspaced Short Palindromic Repeats; AF-1: activation function-1 domain; HIF-1α: hypoxia-inducible factor 1α; CREB: cAMP response element binding.