Molecular Signatures Correlated With Poor IVF Outcomes: Insights From the mRNA and lncRNA Expression of Endometriotic Granulosa Cells

Abstract

The outcomes of in vitro fertilization (IVF) for endometriotic women are significantly worse than for patients without ovarian endometriosis (OEM), as shown by fewer retrieved oocytes. However, the exact pathophysiological mechanism is still unknown. Thus, we conducted a prospective study that analyzed mRNA and lncRNA transcriptome between granulosa cells (GCs) from patients with fewer retrieved oocytes due to OEM and GCs from controls with male factor (MF) infertility using an RNA sequencing approach. We found a group of significantly differentially expressed genes (DEGs), including NR5A2, MAP3K5, PGRMC2, PRKAR2A, DEPTOR, ITGAV, KPNB1, GPC6, EIF3A, and SMC5, which were validated to be upregulated and negatively correlated with retrieved oocyte numbers in GCs of patients with OEM, while DUSP1 demonstrated the opposite. The molecular functions of these DEGs were mainly enriched in pathways involving mitogen-activated protein kinase (MAPK) signaling, Wnt signaling, steroid hormone response, apoptosis, and cell junction. Furthermore, we performed lncRNA analysis and identified a group of differentially expressed known/novel lncRNAs that were co-expressed with the validated DEGs and correlated with retrieved oocyte numbers. Co-expression networks were constructed between the DEGs and known/novel lncRNAs. These distinctive molecular signatures uncovered in this study are involved in the pathological regulation of ovarian reserve dysfunction in OEM patients.

Keywords: RNA sequencing; female infertility; granulosa cells; lncRNA; mRNA; ovarian endometriosis; retrieved oocyte numbers.

Figure 1

mRNA profiling of granulosa cells (GCs) from patients with infertility associated with ovarian endometriosis (OEM). (A) The x-axis of the volcano map represents log₂(fold change (FC)) value of total detected genes, while the y-axis represents their −log₁₀(p value) value. The significant FC_up differentially expressed genes (DEGs) are presented in the upper right quadrant (red), and the significant FC_down DEGs are shown in upper left quadrant (blue). (B) Two-dimensional principal component analysis (PCA) of DEGs from the transcriptomes of RNA-seq data. The PC1 separates the two groups (MF versus OEM). The PC2 separates the samples according to the gene expression pattern. Green color indicates samples from the male factor (MF) group, and red color indicates samples from the OEM group. (C) Correlation heatmap between DEGs and the clinical characteristics of RNA-seq GC samples. The ordinate represents DEGs, and the abscissa is clinical characteristics (*p < 0.05, **p < 0.01, ***p < 0.001) (N_MF = 6, N_OEM = 5).

Figure 2

Functional enrichment analysis. (A) Gene Ontology (GO) bubble chart for DEG enrichment. (B) Kyoto Encyclopedia of Genes and Genomes (KEGG) bubble chart indicating the enrichment of DEGs. Rich factor is defined as S gene number divided by B gene number. “S” was annotated as the number of genes with significant difference in specific GO/KEGG categories; “B” was the number of genes for a specific GO/KEGG (N_MF = 6, N_OEM = 5).

Figure 3

DEGs selection for validation. (A) Purple circle represents DEGs with |log₂(FC)| ≥ 1, p < 0.08 and FPKM > 0.8 in all samples from this study, yellow circle represents reported genes from the DEGs that are related to GCs/oocyte/follicular development, green circle represents genes from the DEGs are intersected with reported original transcriptomic sequencing data of GCs and OEM-affected oocytes. (B) The log₂(FC) value of selected DEGs for validation (N_MF = 6, N_OEM = 5).

Figure 4

OEM-associated DEGs validation and correlation analysis with retrieved oocyte numbers. (A–K) Validation of selected DEGs by expanding clinical samples through real-time quantitative polymerase chain reaction (qPCR) amplification demonstrate significant differences between the OEM and MF groups. (N, sample size). (L–U) Pearson correlation analyses between relative expression levels of the validated DEGs and the retrieved oocyte numbers (r, Pearson correlation coefficient). (*p < 0.05, **p < 0.01, ***p < 0.001).

Figure 5

LncRNA profiling of GCs from patients with infertility associated with OEM. (A) Volcano map analysis of differentially expressed known lncRNA indicates significantly upregulated lncRNAs (red), downregulated significantly lncRNAs (blue), and non-significant lncRNAs (gray). (B) Correlation heatmap between differentially expressed known lncRNA and the clinical characteristics of RNA-seq samples. The ordinate represents known lncRNA, and the abscissa is clinical characteristics. (C) Co-expression network of clinical characteristics and known lncRNAs through Weighted Gene Co-expression Network Analysis (WGCNA); Cytoscape was used to visualize the network diagram (|correlation coefficient| > 0.5, p < 0.05). (D) Volcano map analysis of differentially expressed novel lncRNA. (E) Correlation heatmap between differentially expressed novel lncRNA and the clinical characteristics of RNA-seq samples. (F) Co-expression network of clinical characteristics and novel lncRNAs through WGCNA; Cytoscape was used to visualize the network diagram (|correlation coefficient| > 0.5, p < 0.05) (*p < 0.05, **p < 0.01, ***p < 0.001) (N_MF = 6, N_OEM = 5).

Figure 6

Co-expression analysis of mRNA and lncRNA. (A) Co-expression network from validated DEGs and known lncRNAs through WGCNA; Cytoscape was used to visualize the network diagram (|correlation coefficient| > 0.9, p < 0.05). (B) Co-expression network from validated DEGs and novel lncRNAs through WGCNA; Cytoscape was used to visualize the network diagram (|correlation coefficient| > 0.9, p < 0.05). (C) Venn diagram of co-expressed known lncRNAs with clinical characteristics and validated DEGs. Purple circle represents co-expressed known lncRNA with clinical characteristics, while yellow circle means co-expressed known lncRNA with all validated DEGs (|correlation coefficient| > 0.5, p < 0.05). (D) Venn diagram of co-expressed novel lncRNAs with clinical characteristics and validated DEGs. Purple circle represents co-expressed novel lncRNA with clinical characteristics, while yellow circle means co-expressed novel lncRNA with all validated DEGs (|correlation coefficient| > 0.5, p < 0.05) (N_MF = 6, N_OEM = 5).