Decidualization 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. Endometrial 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. hESCs 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. KLF12 impairs endometrial decidualization by transcriptionally repressing Nur77, and Nur77 overexpression reverses the poor decidual response of hESCs in RIF patients.

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. 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. 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. 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. Construction and management of the Nanjing multi-center biobank. No. 2013-081-01 . Registered 10 Dec. 2013.

Phosphorylation is a classic post-translational modification that regulates protein function. Proteins typically contain multiple potential phosphorylation sites, which can be modified by various kinases at different locations. Studying phosphorylation changes of target proteins in disease often requires phospho-specific antibodies. However, commercial options may be limited to a single site or entirely unavailable. Here, a method is described to detect changes in phosphorylation modifications of target proteins in clinical samples using the Phos-tag gel electrophoresis and to identify detailed phosphorylation sites through in vitro kinase assays. Phosphorylated proteins bound to Phos-tag exhibit slower migration rates in SDS-PAGE gel electrophoresis, enabling semi-quantitative analysis of phosphorylation changes in disease tissues based on the mean pixel intensity of the slowly migrating bands. By combining Phos-tag SDS-PAGE with immunoblotting using pan-specific antibodies against multiple candidate proteins, researchers can efficiently identify target proteins with phosphorylation changes. Following the screening of candidate kinases, in vitro kinase assays are performed with the target protein, and the resulting phosphorylated products are subjected to mass spectrometry for precise site identification. This method does not require specific phosphorylated protein antibodies, allowing large-scale screening of tissue samples to identify altered phosphorylation states in disease. Furthermore, the identified phosphorylation sites can be used to develop specific antibodies for quantitative and localization analysis in disease tissues, providing deeper insights into their functional roles.

Phosphorylation is a classic post-translational modification that regulates protein function. Proteins typically contain multiple potential phosphorylation sites, which can be modified by various kinases at different locations. Studying phosphorylation changes of target proteins in disease often requires phospho-specific antibodies. However, commercial options may be limited to a single site or entirely unavailable. Here, a method is described to detect changes in phosphorylation modifications of target proteins in clinical samples using the Phos-tag gel electrophoresis and to identify detailed phosphorylation sites through in vitro kinase assays. Phosphorylated proteins bound to Phos-tag exhibit slower migration rates in SDS-PAGE gel electrophoresis, enabling semi-quantitative analysis of phosphorylation changes in disease tissues based on the mean pixel intensity of the slowly migrating bands. By combining Phos-tag SDS-PAGE with immunoblotting using pan-specific antibodies against multiple candidate proteins, researchers can efficiently identify target proteins with phosphorylation changes. Following the screening of candidate kinases, in vitro kinase assays are performed with the target protein, and the resulting phosphorylated products are subjected to mass spectrometry for precise site identification. This method does not require specific phosphorylated protein antibodies, allowing large-scale screening of tissue samples to identify altered phosphorylation states in disease. Furthermore, the identified phosphorylation sites can be used to develop specific antibodies for quantitative and localization analysis in disease tissues, providing deeper insights into their functional roles.