145 Altered microRNA profiles in aged human follicular fluid extracellular vesicles reflect response to oxidative stress

Advanced maternal age (AMA) infertility patients have difficulty conceiving, which is often a result of poor oocyte quality. The oocyte quality is significantly influenced by the surrounding microenvironment of the follicle. This microenvironment, which constitutes the follicular fluid (FF), contains extracellular vesicles (EVs) that act as important mediators of cell-to-cell communication. Here, we investigated differences in microRNA (miRNA) profiles of FF-EVs between young (<32 years old) and AMA (>38 years old) patients to understand how aging affects the follicular environment at a molecular level. By analyzing the miRNA transcriptomes of FF-EVs, we aimed to identify specific miRNAs affected by age to uncover their potential roles in regulating pathways critical for oocyte development. FF was collected from 18 patients (nine in each group) during oocyte retrievals under IRB approval (no. 20142468). Patient selection criteria were based on age (<32 or >38 years), BMI < 25, and no diagnosis of endometriosis or polycystic ovarian syndrome. EVs were isolated from FF by differential ultracentrifugation and size exclusion chromatography. Samples were pooled in groups of three (n = 3 each). RNA was isolated from EVs using the Exosomal RNA Isolation Kit (Norgen Biotek Corp.), according to the manufacturer’s instructions. RNA was sent to NovoGene for library preparation and sequencing. The Illumina NovaSeq platform was used for single-end 50 bp sequencing of small RNA. Bioinformatics analysis and differential miRNA expression were performed on QIAGEN CLC Genomics Workbench software. Target gene prediction and pathway analysis were performed using Ingenuity Pathway Analysis software (QIAGEN) and DAVID Bioinformatics software (National Institutes of Health), respectively. We identified 23 differentially expressed miRNAs between young and aged FF-EVs (P value <0.05, FDR < 0.10, fold-change > 2). Pathway analysis of target genes indicated that miRNAs upregulated in young FF-EVs, including miR-1-3p, miR-503-5p, miR-508-3p, and miR-139-3p, are likely involved in targeting genes associated with cellular senescence, as well as key signaling pathways such as PI3K-Akt and MAPK, which are responsible for regulating cell proliferation, metabolism, and cell survival. In contrast, miRNAs upregulated in aged FF-EVs, including miR-194-5p, miR-192-5p, miR-19a-3p, and miR-205-5p, not only target the PI3K-Akt signaling pathway but also the advanced glycation end products (AGE)-receptor in the AGE (RAGE) signaling pathway. Targeting AGE-RAGE signaling by miRNAs in aged FF-EVs is interesting due to it being implicated in reactive oxygen species generation and its dual role in apoptotic cell death and cell survival. Dysregulation of the AGE-RAGE pathway can lead to pathological conditions. Our analysis indicates that aged FF-EV miRNA cargo is potentially altered in response to the increased ROS environment. Such changes may indicate an adaptive response to mitigate oxidative damage and a subsequent impact on oocyte quality. This research will potentially identify miRNAs and their target genes or pathways that may be used as targets for therapeutic treatments to mitigate the impacts of aging on oocyte quality for future AMA fertility patients.