Age-Related Transcriptomic Profiling of Human Granulosa Cells Reveals mRNA-microRNA Regulatory Network Associated with Key Ovulation Dynamics [miRNA-Seq]

Advanced maternal age (AMA) patients experience decreased success from assisted reproductive technologies (ART), attributed to quantity and quality of oocytes, which is significantly influenced by the health of surrounding granulosa cells (GCs). In this study, we compared mRNA and microRNA (miRNA) transcriptomes between young (<32 y.o.) and AMA (>38 y.o.) patients' GCs to identify ovarian aging molecular signatures. Granulosa cells were isolated from 18 patients' follicular fluid, and RNA was isolated for sequencing and subsequent bioinformatics analysis. We identified 293 and 21 differentially expressed genes (DEGs) and miRNAs (DE miRNA), respectively, between young and AMA GCs (P value < 0.05, FDR < 0.25, Fold Change > 1.5). Highly expressed mitochondrial-encoded genes, MT-ND3, MT-ND6, and MT-CYB are upregulated in young GCs. Pathway analysis indicates DEGs play a role in inflammation, cytokine signaling, extracellular matrix (ECM) remodeling, and angiogenesis. Some key DEGs include CXCL8, IL1B, NLRP3, SIGIRR, ANGPT2, ADAM8, and ADAMTS14. Additionally, target gene prediction and pathway analysis of DE miRNA indicates both up- and downregulated miRNA in young GCs are likely targeting genes associated with cell signaling, mitochondrial function, oxidative stress, apoptosis, and senescence pathways in addition to cytokine signaling, angiogenesis, and ECM remodeling. To further investigate regulatory mechanisms, we looked at the convergence of DEGs with predicted target genes of DE miRNA and identified miR-483-3p, miR-1268a, miR-4497, miR-7704, miR-135a-5p, miR-1261, and miR-4791 as key regulators of pathways involved with inflammation, ECM, and angiogenesis. The gene expression data suggests aged GCs have an impaired ability to elicit the same pro-inflammatory response combined with a dysregulation of angiogenesis and ECM remodeling when compared to young GCs, and miRNA may be playing a key role in the regulation of these key ovulatory events. Overall design: Human granulosa cells were isolated from patients' follicular fluid collected during hormone-stimulated oocyte retrieval cycles. Patients were split into two groups: young and aged. The young group consisted of patients under 32 years old, and the aged group consisted of patients over 38 years old. Patients with a BMI greater than 25, endometriosis, or polycystic ovarian syndrome were excluded from the study. RNA was isolated from a minimum of 1 million cells from each patient. Patient RNA samples were pooled into groups of three to limit individual variation. Each group contained nine patients pooled into three biological replicates.