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EHD1 impairs decidualization by regulating the Wnt4/?-catenin signaling pathway in recurrent implantation failure.

ABSTRACT: BACKGROUND:Recurrent implantation failure (RIF) remains a critical and challenging problem in assisted reproductive technology mainly due to impaired decidualization. The endocytic and transcytotic activity in the endometrium are crucial for decidualization. The most representative endocytic gene is the C-terminal Eps15 homology domain-containing 1 (EHD1), but whether EHD1-mediated endocytic function is responsible for embryo implantation during decidualization remains unclear. METHODS:A transcriptomic analysis was performed to evaluate the differentially expressed genes between the fertile control and RIF group. The expression and location of EHD1 in endometrial tissues were further examined by IHC, qRT-PCR and Western blotting. The transduction of an EHD1 recombinant adenovirus into human endometrial stromal cells was performed to investigate relevant decidualization marker genes. Additionally, a microarray analysis following the adenovirus-mediated overexpression of EHD1 was conducted to identify EHD1-related changes in HESCs, and the potential molecular mechanisms were further confirmed through immunofluorescence and coimmunoprecipitation analyses. FINDINGS:An RNA-seq analysis demonstrated that EHD1 expression was significantly higher in the mid-secretory endometrium of the RIF group than in that of the fertile control group. The analysis of the menstrual cycle showed that expression of EHD1 increased in the mid-proliferative phase and showed a gradual decrease in the mid-secretory and decidual phases. Furthermore, EHD1 overexpression impaired decidualization by suppressing the expression of prolactin and insulin-like growth factor binding protein-1 and the formation of the cytoskeleton. The mechanistic analysis revealed the EHD1 regulated LRP5/6 protein function through the endocytic pathway, and subsequently suppressed the Wnt4/?-catenin pathway during decidualization. In addition, a Wnt4 agonist improved an impaired decidualization process. INTERPRETATION:Regulation of the EHD1-Wnt4 pathway might serve as a promising therapeutic strategy for improving endometrial receptivity in RIF women.

SUBMITTER: Zhou Q

PROVIDER: S-EPMC6921214 | biostudies-literature | 2019 Dec

REPOSITORIES: biostudies-literature

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EHD1 impairs decidualization by regulating the Wnt4/β-catenin signaling pathway in recurrent implantation failure.

Zhou Quan Q Yan Guijun G Ding Lijun L Liu Jingyu J Yu Xiaoying X Kong Shuangbo S Zhang Mei M Wang Zhilong Z Liu Yang Y Jiang Yue Y Kong Na N Sun Jianxin J Sun Haixiang H

EBioMedicine 20191106

<h4>Background</h4>Recurrent implantation failure (RIF) remains a critical and challenging problem in assisted reproductive technology mainly due to impaired decidualization. The endocytic and transcytotic activity in the endometrium are crucial for decidualization. The most representative endocytic gene is the C-terminal Eps15 homology domain-containing 1 (EHD1), but whether EHD1-mediated endocytic function is responsible for embryo implantation during decidualization remains unclear.<h4>Method ...[more]

PMID: 31707150

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Project description:BackgroundThe precise pathogenesis of poor endometrial receptivity in recurrent implantation failure (RIF) remains unclear. This study was aimed at exploring the effects of different CD44 isoforms in the mid-secretory phase endometrium on endometrial receptivity in women with RIF.MethodsMid-secretory phase endometrial tissue samples were obtained from the following two groups of women who had undergone IVF: (a) 24 patients with RIF and (b) 18 patients with infertility due to tubal obstruction, who had achieved a successful clinical pregnancy after the first embryo transfer in IVF (control group). Identification of differentially expressed CD44 isoforms in endometrial tissues was assessed using immunohistochemistry, qPCR, and western blotting. Effects of overexpression and knockdown of CD44v3 on proliferation and decidualization of immortalized human endometrial stromal cells (T-HESCs) and primary HESCs were investigated by qPCR and western blot analysis. A heterologous coculture system of embryo implantation was constructed to mimic the process of trophoblast invasion during implantation.ResultsThe expression of CD44v3 was significantly higher in the mid-secretory phase of endometrial stromal cells than in the proliferation phase, but was notably lower in RIF patients. Knockdown of CD44v3 significantly downregulated cell proliferation both in T-HESCs and HESCs. The expression of decidualization markers, prolactin (PRL) and insulin like growth factor binding protein-1 (IGFBP1), was notably decreased following the knockdown of CD44v3, whereas the expression of both PRL and IGFBP1 increased after its overexpression in HESCs. Furthermore, the CD44v3-knockdown HESCs displayed significant deficiency in supporting trophoblast outgrowth in a coculture system of embryo implantation; however, overexpression of CD44v3 in HESCs promoted trophoblast outgrowth.ConclusionThe reduced expression of CD44v3 suppresses the proliferation and decidualization of HESCs, which might play a pivotal role in poor endometrial receptivity in women with RIF.

Project description:BackgroundDecidualization is a prerequisite for successful implantation and the establishment of pregnancy. A critical role of impaired decidualization in subfertility has been established. In human endometrial stromal cells (hESCs), Krüppel-like factor 12 (KLF12) and Nur77 are novel regulators of decidualization. We investigated whether KLF12 impaired the decidualization of hESCs in recurrent implantation failure (RIF) patients.MethodsEndometrial tissues and hESCs were collected from RIF patients (n = 34) and fertile controls (n = 30) for in vitro analysis. Primary hESCs isolated from RIF endometrial tissues were used to evaluate the biological functions of KLF12 and Nur77. In addition, their molecular mechanisms were investigated by adenovirus-mediated overexpression. Gene expression regulation was examined by real-time-quantitative PCR (qRT-PCR), immunostaining and luciferase reporter assay. Further, blastocyst-like spheroid (BLS) and blastocyst implantation models were performed to examine the roles of KLF12 and Nur77 during embryo expansion on hESCs.ResultshESCs from the RIF patients showed a poor decidual response, mainly characterized by decreased decidual prolactin (dPRL) secretion, impaired transformation and limited BLS expansion. In addition, KLF12 expression was increased in endometrial tissues from the RIF patients compared with those from the fertile controls, especially in stromal compartments. The opposite results were observed for Nur77 expression in these tissues. KLF12 repressed hESC decidualization by decreasing Nur77 expression. Mechanistically, KLF12 bound to a conserved site in the Nur77 promoter region. Nur77 overexpression significantly reversed the KLF12-mediated repression of dPRL expression, decidual transformation and BLS/blastocyst expansion.ConclusionsKLF12 impairs endometrial decidualization by transcriptionally repressing Nur77, and Nur77 overexpression reverses the poor decidual response of hESCs in RIF patients.

Project description:BackgroundSuccessful implantation is a complex process that is influenced by embryo quality, endometrial receptivity, immune factors, and the specific type of in vitro fertilization protocol used. DNA topoisomerase IIα (TOP2A) is a well-known protein involved in cell proliferation; however, its expression and effect on the endometrium in recurrent implantation failure (RIF) have not been fully elucidated.MethodsThe human endometrial tissues of healthy controls and patients with RIF were collected. A proteomic analysis was performed to evaluate the differentially expressed proteins between the RIF group and the fertile control group. The expression patterns of TOP2A in the human preimplantation endometrium of the patients with RIF were determined by immunohistochemical staining, Western blotting and qRT-PCR. TOP2A knockdown (sh-TOP2A) T-HESCs were generated using lentiviruses. The expression of TOP2A in T-HESCs was manipulated to investigate its role in decidualization. The TOP2A-related changes in decidualization were screened by mRNA sequencing in decidualized TOP2A knockdown and control T-HESCs and then confirmed by Western blotting and immunofluorescence staining. TOP2A-deficient mice were generated by injection of TOP2A-interfering adenovirus on GD2.5 and GD3.5.ResultsWe performed a proteomic analysis of endometrial tissues to investigate the potential pathogenesis of RIF by comparing the patients with RIF and the matched controls and found that TOP2A might be a key protein in RIF. TOP2A is ubiquitously expressed in both stromal and glandular epithelial cells of the endometrium. The data indicate that TOP2A expression is significantly lower in the mid-secretory endometrium of women with RIF. TOP2A expression was downregulated under stimulation by 8-bromo-cAMP and MPA. Ablation of TOP2A resulted in upregulated expression of decidual biomarkers and morphological changes in the cells. Mechanistic analysis revealed that TOP2A regulates the NF-κB signaling pathway in decidualized T-HESCs. The TOP2A-deficient mice exhibited lower fetal weights.ConclusionsOur findings revealed that abnormal expression of TOP2A affects decidualization and changes the "window of implantation", leading to RIF. TOP2A participates in the processes of decidualization and embryo implantation, functioning at least in part through the NF-κB pathway. Regulating the expression of TOP2A in the endometrium may become a new strategy for the prevention and treatment of RIF.

Project description:BackgroundSmall leucine-rich repeat proteins (SLRPs) are highly effective and selective modulators of cell proliferation and differentiation. Podocan is a newly discovered member of the SLRP family. Its potential roles in the differentiation of bovine muscle-derived satellite cells (MDSCs) and its underlying functional mechanism remain unclear. Our study aimed to characterize the function of the podocan gene in the differentiation of bovine MDSCs and to clarify the molecular mechanism by which podocan functions in order to contribute to a better understanding of the molecular mechanism by which extracellular matrix promotes bovine MDSC differentiation and provide a theoretical basis for the improvement of beef quality.MethodsBovine MDSCs were transfected with vectors to overexpress or inhibit podocan, and podocan protein was added to differentiation culture medium. qRT-PCR, western blotting, and immunofluorescence were performed to investigate the effects of podocan on MDSC differentiation. Confocal microscopy and western blotting were used to assess the nuclear translocation and expression of ?-catenin. An inhibitor and activator of ?-catenin were used to assess the effects of the Wnt/?-catenin signaling pathway on MDSC differentiation. We inhibited ?-catenin while overexpressing podocan in MDSCs. Then, we performed mass spectrometry to identify which proteins interact with podocan to regulate the Wnt/?-catenin signaling pathway. Finally, we confirmed the relationship between podocan and Wnt4 by co-immunoprecipitation and western blotting.ResultsPodocan protein expression increased significantly during bovine MDSC differentiation. Differentiation of bovine MDSC was promoted and suppressed by podocan overexpression or inhibition, respectively. Podocan was also shown to modulate the Wnt/?-catenin signaling pathway. Treatment of bovine MDSCs with ?-catenin inhibitor and activator showed that the Wnt/?-catenin pathway is involved in bovine MDSC differentiation. Furthermore, the effect of podocan on bovine MDSC differentiation was suppressed when this pathway was inhibited. We also found that podocan interacts with Wnt4. When Wnt4 was inhibited, podocan-induced promotion of bovine MDSC differentiation was attenuated through Wnt/?-catenin signaling.ConclusionPodocan regulates Wnt/?-catenin through Wnt4 to promote bovine MDSC differentiation.

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EHD1 impairs decidualization by regulating the Wnt4/?-catenin signaling pathway in recurrent implantation failure.

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EHD1 impairs decidualization by regulating the Wnt4/β-catenin signaling pathway in recurrent implantation failure.

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