Integrative Analyses of Uterine Transcriptome and MicroRNAome Reveal Compromised LIF-STAT3 Signaling and Progesterone Response in the Endometrium of Patients with Recurrent/Repeated Implantation Failure (RIF)

Intimate two-way interactions between the implantation-competent blastocyst and receptive uterus are prerequisite for successful embryo implantation. In humans, recurrent/repeated implantation failure (RIF) may occur due to altered uterine receptivity with aberrant gene expression in the endometrium as well as genetic defects in embryos. Several studies have been performed to understand dynamic changes of uterine transcriptome during menstrual cycles in humans. However, uterine transcriptome of the patients with RIF has not been clearly investigated yet. Here we show that several signaling pathways as well as many genes and microRNAs are dysregulated in the endometrium of patients with RIF (RIFE). Whereas unsupervised hierarchical clustering showed that overall mRNA and microRNA profiles of RIFE were similar to those of endometria of healthy women, many genes were significantly dysregulated in RIFE (cut off at 1.5 fold change). The majority (~75%) of differentially expressed genes in RIFE including S100 calcium binding protein P (S100P), Chemokine (C-X-C motif) ligand 13 (CXCL13) and SIX homeobox 1 (SIX1) were down-regulated, suggesting that reduced uterine expression of these genes is associated with RIF. Gene Set Enrichment analyses (GSEA) for mRNA microarrays revealed that various signaling pathways including Leukemia inhibitory factor (LIF) signaling and a P4 response were dysregulated in RIFE although expression levels of Estrogen receptor α (ERα) and Progesterone receptor (PR) were not significantly altered in RIFE. Furthermore, expression and phosphorylation of Signal transducer and activator of transcription 3 (STAT3) are reduced and a gene set associated with Janus kinase (JAK)-STAT signaling pathway is systemically down-regulated in these patients. Pairwise analyses of microRNA arrays with prediction of dysregulated microRNAs based on mRNA expression datasets demonstrated that 6 microRNAs are aberrantly regulated in RIFE. Collectively, we here suggest that dysregulation of several major signaling pathways and genes critical for uterine biology and embryo implantation may lead to uterine abnormalities in patients with RIF.具有植入能力的囊胚与容受性子宫之间的亲密双向互作，是成功实现胚胎植入的先决条件。在人类中，复发性/重复性植入失败（recurrent/repeated implantation failure, RIF）的发生可能源于子宫内膜容受性改变、子宫内膜基因表达异常，以及胚胎存在遗传缺陷。既往已有多项研究探索人类月经周期中子宫转录组的动态变化，但目前针对RIF患者的子宫转录组仍未得到清晰阐释。本研究发现，RIF患者的子宫内膜（RIFE）中存在多条信号通路以及大量基因和微小RNA（microRNAs）的表达失调。尽管无监督层次聚类显示，RIFE的整体mRNA和微小RNA表达谱与健康女性子宫内膜相似，但RIFE中有大量基因出现显著表达失调（筛选阈值为1.5倍变化）。其中，约75%的差异表达基因包括S100钙结合蛋白P（S100 calcium binding protein P, S100P）、趋化因子（C-X-C基序）配体13（Chemokine (C-X-C motif) ligand 13, CXCL13）以及SIX同源框1（SIX homeobox 1, SIX1）均呈现下调趋势，提示这些基因的子宫表达降低与RIF相关。对mRNA芯片进行的基因集富集分析（Gene Set Enrichment Analyses, GSEA）显示，尽管雌激素受体α（Estrogen receptor α, ERα）和孕激素受体（Progesterone receptor, PR）的表达水平在RIFE中未出现显著改变，但包括白血病抑制因子（Leukemia inhibitory factor, LIF）信号通路和孕酮应答在内的多条关键信号通路在RIFE中出现失调。此外，信号转导与转录激活因子3（Signal transducer and activator of transcription 3, STAT3）的表达与磷酸化水平均有所降低，且与贾纳斯激酶（Janus kinase, JAK）-STAT信号通路相关的基因集在这类患者中呈现系统性下调。通过对微小RNA芯片进行两两分析，并结合mRNA表达数据集预测失调的微小RNA，本研究发现RIFE中有6种微小RNA出现异常调控。综上，本研究提示，对子宫生物学和胚胎植入至关重要的多条主要信号通路及基因的表达失调，可能导致RIF患者出现子宫异常。