Comprehensive Analysis of circRNA-Mediated ceRNA Regulatory Networks in relation to Recurrent Implantation Failure.

Recurrent implantation failure (RIF), as a common condition of embryo implantation failure, has always been a major challenge in assisted reproductive technology. Although RIF is not clearly defined, it is widely accepted that RIF is a failure experience that cannot be successfully implanted in the presence of three or more transfers with high-quality embryos or ten or more transfers of embryo [ 1 , 2 ]. Embryo factors affecting the pregnancy rate have always been emphasized to be closely related to the cause of RIF [ 3 ]. When other parameters such as the number and quality of oocytes and embryos have been used as success parameters, RIF is particularly frustrating for couples. However, the interaction between the embryo and the endometrium, concerning the cell growth and differentiation of the embryo and endometrium, is an important process affecting implantation [ 4 ]. Recent evidences indicated that endometrial receptivity dysfunction primarily contributed to the occurrence of RIF [ 2 ], and related-biomolecules levels, such as MUC1 [ 5 ], HOXA-10, and E-cadherin [ 6 ], were found to be altered in women with RIF. However, the underlying mechanism of RIF has not been fully revealed in previous studies, and hence further research is urgently needed to find biomarkers for the diagnosis and prognosis of RIF. Extensive studies have demonstrated that messenger RNA (mRNA) encoding protein and noncoding RNA (ncRNA) regulating cell physiology and shape cellular functions such as microRNA(miRNA), long noncoding RNA (lncRNA), and circular RNA (circRNA) were involved in biological processes [ 7 , 8 ]. CircRNA, as a new class of ncRNA, exhibits high stability, abundance, and tissue specificity. It participates in the process of gene translation into protein and interacts with RNA binding protein through sponging miRNA [ 9 ]. Differentially expressed circRNAs were revealed in patients with RIF, indicating the potential of circRNA as biomarkers for clinical diagnosis and treatment of RIF [ 10 ]. Construction of competing endogenous RNA (ceRNA) regulatory networks mediated by circRNA to explore candidate genes and circRNA mechanism in various diseases has been established. The hsa_circ_0011385 was confirmed as a ceRNA associated with pathogenesis of bladder cancer via construction of circRNA-miRNA-mRNA regulatory network [ 11 ]. As for patients with RIF, hsa_circ_0038383-mediated ceRNA network based on prediction of circRNA–miRNA and miRNA–mRNA pairs was constructed [ 12 ]. Nevertheless, the regulatory mechanism of ceRNA network in RIF is still lack of numerous studies. The present study obtained differently expressed mRNAs, miRNAs, and circRNAs from three databases including GSE111974 , GSE121219 , and GSE147442 and discussed the prominent circRNAs and their molecular mechanisms in RIF.

The GEO database was searched to obtain eligible microarray datasets which must be sourced from human endometrial tissue samples, profiled by same technology, and supplemented with clear series matrix files and gene symbols. Three eligible microarray datasets, GSE111974 , GSE121219 , and GSE147442 , were employed to screen DE-mRNAs, DE-miRNAs, and DE-circRNAs. The GSE111974 dataset, generated on the GPL17077 platform, contains endometrial tissue samples obtained from 24 patients with RIF and 24 fertile control patients. The GSE121219 , generated on the GPL18058 platform, includes endometrial tissue samples derived from 8 RIF patients and 10 matched controls. GSE147442 , generated on the GPL21825 platform, involves endometrial tissue samples sourced from 8 RIF patients and 8 fertile control patients. The value of log2-fold change |log2FC| ≥ 1 and the adjusted p value < 0.05 both were used to evaluate differential expression. The visualization of DE-mRNAs, DE-miRNAs, and DE-circRNAs was presented on the volcano plots and heatmaps. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were carried out for DE-mRNAs in endometrial tissue samples between RIF and fertile control. GO terms are typically classified into the biological process (BP), cellular component (CC), and molecular function (MF), and the KEGG pathway database encompasses biochemical pathways obtaining metabolic and regulatory pathways where the enrichment of DE-mRNAs was determined by p value < 0.05 using the “clusterProfiler” software package in the R/Bioconductor. A PPI network was built on the basis of DE-mRNAs in endometrial tissue samples between RIF and fertile control using the STRING tool, whose visualization was accomplished using Cytoscape software (v3.9.0). The high confidence for the interaction score was set as 0.4. The Cytoscape plugin “MCODE” was used to identify significant modules and hub genes, with cut-off criteria of degree ≥10. A computer-based miRNA-mRNA target prediction was performed by mapping hub genes with degree ≥10 in the PPI network into the starBase database. Venn intersection was carried out to screen overlapping miRNAs between targeted miRNAs and DE-miRNAs ( GSE121219 ) in endometrial tissue samples between RIF and fertile control. The intersection follows the principle of miRNA-mRNA regulation, namely, miRNAs posttranscriptional gene silencing by guiding mRNA degradation or translational repression. A computer-based circRNA-miRNA target prediction was also performed by mapping overlapping miRNAs above into the starBase database. Venn intersection was carried out to screen overlapping circRNAs between targeted circRNAs and DE-circRNAs ( GSE147442 ) in endometrial tissue samples between RIF and fertile control. The intersection follows the principle of ceRNA hypothesis that circRNAs share miRNA binding sites and compete for posttranscriptional control of mRNAs.

The raw data of GSE111974 were differentially analyzed, and mRNAs with a value of log2-fold change |log2FC| ≥ 1 and the adjusted p value < 0.05 were considered differentially expressed between RIF and fertile control. We then identified 350 DE-mRNAs consisting of 200 upregulated mRNAs and 150 downregulated mRNAs in endometrial tissue samples of RIF compared with those of fertile control, and all were displayed by the volcano plot ( Figure 1(a) ) and heatmap ( Figure 1(b) ). To evaluate the main functional pathways in RIF, we conducted GO annotation and KEGG pathway analyses of 350 DE-mRNAs between RIF and fertile control. After GO analysis, we found that 350 DE-mRNAs were significantly enriched in 69 GO terms including 60 terms belonging to BP, 3 terms belonging to CC, and 6 terms belonging to MF ( Table 1 , p < 0.05). After KEGG pathway analysis, we found that 350 DE-mRNAs were significantly enriched in 19 KEGG pathways ( Table 2 , p < 0.05). We subsequently construct a PPI network by mapping 350 DE-mRNAs between RIF and fertile control into the STRING database ( Figure 2 ), in which 6 hub genes were identified with degree ≥10, namely, KDR, AGT, POSTN, PTGS2, TOP2A, and RRM2. KDR, AGT, POSTN, TOP2A, and RRM2, were downregulated in endometrial tissue samples of RIF compared with those of fertile control, while PTGS2 was upregulated in endometrial tissue samples of RIF compared with those of fertile control. We searched the starBase database for putative miRNAs based on KDR, AGT, POSTN, PTGS2, TOP2A, and RRM2. Then, we differentially analyzed the raw data of GSE121219 and identified 43 DE-miRNAs in which there were 22 upregulated miRNAs and 21 downregulated miRNAs in endometrial tissue samples of RIF compared with those of fertile control, and all were displayed by the volcano plot ( Figure 3(a) ) and heatmap ( Figure 3(b) ). Three miRNAs (hsa-miR-424-5p, hsa-miR-195-5p, and hsa-miR-29b-3p) targeted KDR and are differentially expressed in RIF. Two miRNAs, hsa-miR-29b-3p and hsa-miR-30c-5p, target POSTN and differentially expressed in RIF. Two miRNAs (hsa-miR-3142 and hsa-miR-30c-5p) targeted RRM2 and are differentially expressed in RIF. Subsequently, we differentially analyzed the raw data of GSE147442 and identified 1048 downregulated circRNAs along with 920 upregulated circRNAs in endometrial tissue samples of RIF compared with those of fertile control, all displayed by the volcano plot ( Figure 4(a) ) and heatmap ( Figure 4(b) ). Concurrently, we independently mapped hsa-miR-424-5p, hsa-miR-195-5p, hsa-miR-29b-3p, hsa-miR-30c-5p, and hsa-miR-3142 into the starBase database to obtain putative circRNAs. We selected overlapping ones between DE-circRNAs and putative circRNAs that must show the same expression patterns as KDR, POSTN, and RRM2 in RIF by Venn functional intersection. Accordingly, a total of 10 circRNAs, hsa_circ_001572, hsa_circ_001884, hsa_circ_001375, hsa_circ_001449, hsa_circ_000029, hsa_circ_001168, hsa_circ_000210, hsa_circ_001484, hsa_circ_001698, and hsa_circ_000089, stood out. Based on the above DE-miRNA and hub gene interactions, DE-circRNA and DE-miRNA interactions, we constructed 15 groups of the ceRNA network which were involved in RIF ( Figure 5 ).

In recent years, an increasing number of women choose to delay pregnancy, which reduces the success rate of natural pregnancy, leading to an increase in the demand for assisted reproductive technology. Thanks to the advancement of ovarian stimulation, in vitro fertilization embryo transfer has been extensively used as an effective method to treat infertility from the previous evidence [ 13 , 14 ]. However, RIF still remains a challenging issue in assisted reproductive technology, which makes medical staff and patients frustrated. Therefore, new approaches for RIF are urgently needed. As reported in previous studies, RIF is closely related to endometrial receptivity and related proteins in the endometrium expressed abnormally in patients with RIF [ 5 , 15 ]. miRNA is a small endogenous RNA and a key regulator of posttranscriptional gene expression and candidate genes acting as biomarkers in diseases [ 16 ]. Wang et al. revealed that several miRNAs such as miRNA489, miRNA199 A, and miRNA369-3P were considered as the key regulatory factors during RIF through targeting mRNAs [ 17 ]. Furthermore, a study presented by Zhou et al. showed that the abnormal expression level of circRNAs in the endometrium of RIF patients was detected [ 18 ]. More and more studies have focused on the function and mechanism of circRNAs by constructing the ceRNA network since ceRNA hypothesis was first proposed [ 19 , 20 ]. In this study, we constructed a circRNA–miRNA–mRNA regulatory network based on three eligible microarray datasets ( GSE111974 , GSE121219 , and GSE147442 ). It was found that 350 DE-mRNAs were identified in endometrial tissue of RIF patients and fertile control and were performed by GO and KEGG analyses to evaluate the main functional pathways in RIF. We observed that these DE-mRNAs were significantly enriched in 69 GO terms and 19 KEGG pathways. As for GO terms, DE-mRNAs were mainly the enrichment in “axon development,” “apical part of cell,” “endopeptidase inhibitor activity,” and “endopeptidase regulator activity.” KEGG pathway analysis indicated that the phospholipase D signaling pathway and calcium signaling pathway were the top two pathways for these DE-mRNAs. Although there were few reports on phospholipase D directly related to RIF, Kashir et al. manifested that defects of phospholipase C exhibited negative impacts on RIF patients undergoing fertility treatments [ 20 ]. Calcium plays a key role in the reproductive process from germ cell maturation to placental development, and the calcium signaling pathway participates in establishment of embryo implantation and pregnancy [ 21 , 22 ]. Subsequently, we identified 6 hub genes from the PPI network and interaction between DE-miRNAs and hub genes in RIF. The analysis showed that levels of five genes (KDR, AGT, POSTN, TOP2A, and RRM2) were decreased, while PTGS2 was increased in endometrial tissue of RIF compared with those of fertile control. In addition, three miRNAs (hsa-miR-424-5p, hsa-miR-195-5p, and hsa-miR-29b-3p) targeting mRNA KDR were differentially expressed in RIF. Two miRNAs (hsa-miR-29b-3p and hsa-miR-30c-5p) targeted mRNA POSTN and were differentially expressed in RIF. Two miRNAs (hsa-miR-3142 and hsa-miR-30c-5p) targeted RRM2 and are differentially expressed in RIF. Andraweera et al. [ 23 ] revealed that KDR expression was reduced in placentae of females with adverse pregnancy outcomes such as preeclampsia, gestational hypertension, and small for gestational age infants. A study on bovine preimplantation embryonic development pointed out that mRNA POSTN was identified in in vitro culture media as an indicator of embryo quality [ 24 ]. RRM2 has been widely explored in cancer diseases, and its expression was found to be increased in women with endometriosis-associated ovarian cancer while reduced in women with endometriosis [ 25 ]. Based on the DE-circRNA and DE-miRNA interactions in RIF, there were several circRNAs showing same expression patterns as KDR, POSTN, and RRM2 in RIF were identified, including hsa_circ_001572, hsa_circ_001884, hsa_circ_001375, hsa_circ_001449, hsa_circ_000029, hsa_circ_001168, hsa_circ_000210, hsa_circ_001484, hsa_circ_001698, and hsa_circ_000089. Actually, the circRNAs we found were different from the previous evidence revealing circRNAs (hsa_circ_0058161, hsa_circ_0033392, hsa_circ_0030162, hsa_circ_0004121, hsa_circ_0034642, and hsa_circ_0034762) participate in pathogenesis of RIF [ 26 ]. It suggested that our circRNAs might be novel biomarkers of RIF. In this study, a novel circRNA-miRNA-mRNA regulator network related to RIF was identified, and 10 circRNAs, including hsa_circ_000029, hsa_circ_001168, hsa_circ_000210, hsa_circ_001484, hsa_circ_001698, and hsa_circ_000089, hub-genes, consisting of POSTN, KDR, and RRM2, were involved in the development of RIF. However, the results were only relied on public dataset analysis with small samples, and further clinical experiments are essential to verify our prediction. Moreover, the regulation of circRNAs on miRNAs and miRNAs on hub genes remains unknown. In summary, this work might contribute to explore the initiation and progression of RIF and develop potential treatments for RIF.