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. 2022 Oct;88(4):e13607.
doi: 10.1111/aji.13607. Epub 2022 Aug 15.

Identification of key genes and immune cell infiltration in recurrent implantation failure: A study based on integrated analysis of multiple microarray studies

Xue Feng  1 , Xiaolin Meng  1 , Shuaiqingying Guo  2 , Kezhen Li  2 , Lingjuan Wang  2 , Jihui Ai  1
Affiliations

Identification of key genes and immune cell infiltration in recurrent implantation failure: A study based on integrated analysis of multiple microarray studies

Xue Feng et al. Am J Reprod Immunol. 2022 Oct.

Abstract

Problem: Recurrent implantation failure (RIF) refers to a challenging topic in assisted reproductive technology (ART), the etiology of which may be attributed to impaired endometrial receptivity; however, the precise pathogenesis of RIF has not been thoroughly elucidated.

Method of study: Four RIF microarray datasets were obtained from the Gene Expression Omnibus database and integrated by the "sva" R package. The differentially expressed genes (DEGs) were analyzed using the "limma" package and then GO, KEGG, GSEA, and GSVA were applied to perform functional and pathway enrichment analysis. The immune cell infiltration in the RIF process was evaluated by the CIBERSORT algorithm. Finally, the hub genes were identified through the CytoHubba and subsequently verified using two items of external endometrial data.

Results: 236 genes were differentially expressed in the endometrium of the RIF group. Functional enrichment analysis demonstrated that the biological functions of DEGs were mainly correlated to the immune-related pathways, including immune response, TNF signaling pathway, complement and coagulation cascades. Among the immune cells, γδ T cells decreased significantly in the endometrium of RIF patients. In addition, the key DEGs such as PTGS2, FGB, MUC1, SST, VCAM1, MMP7, ERBB4, FOLR1, and C3 were screened and identified as the hub genes involved in the pathogenesis of RIF.

Conclusions: Abnormal immune response regulation of endometrium contributes to the occurrence of RIF, and γδ T cells may be the pivotal immune cells causing RIF. At the same time, the novel hub genes identified will provide effective targets for the prediction and therapy of RIF.

Keywords: hub genes; immune cell infiltration; recurrent implantation failure.

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Conflict of interest statement

The authors declare no conflicts of interest.

Figures

FIGURE 1
FIGURE 1
Principal component analysis cluster diagram of samples before and after batch normalization and volcano map of differentially expressed genes (DEGs). Principal component analysis of GSE103485, GSE111974, GSE25787, and GSE71331 datasets before (A) and after (B) normalization. (C) Volcano map of gene expression between RIF and the control group, and there were 151 up‐regulated and 85 down‐regulated genes in the RIF group. FC, fold change
FIGURE 2
FIGURE 2
Functional enrichment analysis of DEGs. (A) The top ten GO terms of DEGs, and GO analysis includes three functional groups: biological process (BP), cellular component (CC), and molecular function (MF). (B) KEGG pathway enrichment analysis of DEGs. Chord diagram visualizing the relationship between DEGs and important GO biological processes(C) and the relationship between DEGs and KEGG pathways (D). FC, fold change
FIGURE 3
FIGURE 3
GSEA and GSVA analysis. (A) GSEA of KEGG pathway. (B) GSVA and the differentially expressed analysis of pathways
FIGURE 4
FIGURE 4
Evaluation and visualization of immune cell infiltration. (A) Composition of 22 types of immune cells in each sample. (B) Correlation of immune cell types in the endometrium from all samples (RIF and normal pregnancy). The intensity of the color and the number in each square indicate the strength of the correlation; blue represents positive correlation, and red represents negative correlation. *p < .05, **p < .01, ***p < .001. (C) The proportion of various immune cell types in the RIF and control group. p‐values were obtained using the Mann‐Whitney U‐test
FIGURE 5
FIGURE 5
PPI network of DEGs and identification of hub genes. (A) PPI diagram of DEGs. Red nodes represent up‐regulated genes, while blue nodes represent down‐regulated genes, and the thickness of the connecting line represents the interaction score. (B) Ten hub genes. The color of the nodes represents the level of the score ranked by the Degree method using the CytoHubba plugin of Cytoscape, and the redder the color is, the higher the score is. The plot of the hub genes in the GO biological process pathway (C) and KEGG pathway (D), and the height of the column represents the relative mRNA expression of the genes
FIGURE 6
FIGURE 6
Validation of hub genes in endometrium through the menstrual cycle. Heatmap of hub genes in GSE4888 (A) and GSE98386 (B). Expression of hub genes in different menstrual phases of GSE4888 (C) and GSE98386 (D). Statistical analysis for group comparisons was performed using the Kruskal‐Wallis test (C), and Wilcoxon signed‐rank test (D). n.s. p ≥ .05, *p < .05, **p < .01, ***p < .001
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