The baseline characteristics of the six patients are listed in Supplemental Table 2. Age, duration of fertility, body mass index, type of infertility and antral follicle count were comparable between the two groups. No statistically significant difference was reported in the basal hormone levels. Combined with the higher serum E2 concentration on the day of HCG (4126.67±1293.18 pg/mL vs 103.33±53.63 pg/mL, p = 0.0058) and clinical symptoms after eggs removal, patients in COH group were hyper ovarian responders and they later developed OHSS. The lower serum LH concentrations on the day of HCG (1.55±0.32 vs 21.12±11.41 mIU/mL, p =0.0411) resulted from the pituitary desensitization in GnRH agonist cycles.
RNA-Seq data analysis
Transcriptome analysis based on RNA-seq and PCA showed the clustering of the samples in each group (Figure 1A). There were 2796 DEGs (p-value with FDR correction < 0.05), including 1870 upregulated and 926 downregulated genes in COH (Supplementary Data 1). Figure 1B shows volcano plots providing an overview of the differential status of gene expression.
GO and KEGG pathway analysis was performed on the DEGs. Enriched Go terms are showed in Figure 2A B and arranged and displayed according to biological processes, cellular components, and molecular functions. For the cellular component annotation classification, both up- and down-regulated genes were mainly localized in the membranes, endomembrane system, vesicle, nucleus, and extracellular region. Concerning the molecular function, both up- and down-regulated genes were mainly associated with binding, catalytic activity, and signal transduction. The enriched biological processes for the up- and down-regulated genes were mainly involved in biological regulation, metabolic processes, response to stimulus and multicellular organismal process. KEGG pathway analysis showed that the top 20 KEGG pathways were significantly enriched and are shown in Figure 2C. Among these pathways, the immune system (complement and coagulation cascades) and signal transduction (PI3K-Akt signaling pathway) were found to be the most enriched. Besides, the Ras signaling pathway, MAPK signaling pathway, and WNT signaling pathway were also enriched.
To further investigate the DEGs relationships, a co-expression network of 640 DEGs (p-value < 0.01) based on protein-protein interaction (PPI) is shown in Figure 3A. Four key genes, including C3, CXCL8, CXCL12, and PENK were considered to have important regulation and control ability. A total of 221 DEGs related to EMT, 83 DEGs involved in endometrial receptivity and 57 DEGs associated with both functions were screened (Figure 3B, Supplementary Data 2). The six up-regulated transcripts with the highest FC in EMT were MMP1, SOHLH2, ELOVL3, LBP, IL1B, and IGFBP1. The six down-regulated transcripts identified as receptivity-associated genes were NLRP5, SLC7A4, SEPRINB9, CD36, CREB3L1, and ATP6V0A4.
DNAm profiling analysis
Genome-wide DNAm profiles were studied by reduced-representation bisulfite sequencing (RRBS-Seq). The mean genome-wide methylation levels of the six samples in C, CG, CHG, and CHH are shown in Supplementary Table 3. CG sites (mCG) modified by methylation were found to be stable and fully methylated. The methylated CHH sites (mCHH) were unstable and were in a hemi-methylated or hypo-methylated state.
The DMRs statistics between NOR and COH groups are shown in Figure 4A. The violin plots showing the distribution of methylation in each locus on 10 genes revealed significant differences in methylation (Figure 4B). The region-level analysis of all CG revealed 38 significant DMRs, of which 20 exhibited increased (associated with 10 genes) and 18 decreased (associated with 10 genes) methylation (Supplementary Table 4). The most significant DMRs included CG in the upstream region of RAP1B and the gene-body of NPAS3. RAP1B was also one of the genes highlighted in the site-level analysis (Supplementary Data 3).
GO and KEGG pathway analyses were performed to characterize the genes annotated to DMRs. In site-level analyses, no GO terms were significantly represented (Supplementary Data 4). This is typically mirrored by region-level analyses (Supplementary Data 5). Pathway enrichment analyses for the same gene lists showed enrichment in 12 pathways in site-level analysis, including insulin signaling, Rap1 signaling, renal cell carcinoma, neurotrophic signaling, N-Glycan biosynthesis, measles, Jak-STAT signaling, transcriptional misregulation in cancers, endocytosis, cytokine-cytokine receptor interaction, HTLV-I infection, PI3K-Akt signaling pathway. No enrichment was seen in region-level analysis; however, genes showing a correlation between methylation and gene expression were enriched for the Wnt signaling pathway.
Screening for gene function and expression and selected TSPAN4, NPAS3, RAP1B, and TGFB3 for qPCR was performed to evaluate the correlation between DNAm and transcriptome (Supplementary Figure 2). In this study, there was a limited correlation between methylation and transcriptome during COH.