Circular RNA expression profiling of human granulosa cells during maternal aging

Advanced maternal age was closely associated with decreased pregnancy rates and increased spontaneous miscarriages. Even seeking assisted reproductive technology (ART), only 12.2% of women aged 41-42, and 4.2% of women older than 42 years achieved live births with their own oocytes, which were remarkably lower than that of women younger than 35 years (40.1%). Elucidating underlying molecular signatures that link maternal age and fecundity decline is particularly important for developing predictive markers and therapeutic targets for improving reproductive outcomes in aging women. Circular RNAs are a unique class of non-coding RNAs with a stable structure formed by special loop splicing, and they contribute to post-transcriptional regulation of gene expression by binding specifically to miRNAs and sequestering miRNAs to terminate suppression of their targets. Increasing evidences indicate that circular RNAs play important roles in many age-related diseases, such as cardiovascular disease, nervous system disorders, and cancer. However, the role of circular RNAs in reproduction and their contribution to ovarian aging remains to be discovered. The aim of this study was to characterize circRNA expression profiles of granulosa cells according to maternal age, identify age-related changes that could potentially reflect oocyte competence, and constitute non-invasive tools to explore follicular micro-environment quality and to predict ART outcomes, thus improving the personalized patient’s care. Circular RNAs profiles in granulosa cells obtained from IVF patients (n=6) was analyzed by a human circRNA microarray. Samples from women with advanced age (AA,≥ 38 years) were compared to those from women with young age (YA,≤ 30 years). Significantly differentially expressed circular RNAs discovered in the microarray were validated in additional 20 AA and 20 YA samples by real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Granulosa cells from another independent cohort (80 women range from 22 to 48 years old) were analyzed by qRT-PCR to further verify the association of diminished ovarian function with candidate circular RNAs. Our results indicated that circRNA_103827 and circRNA_104816 expressions in granulosa cells were negatively correlated with the number of top quality embryos and pregnancy outcomes. Bioinformatics analysis revealed that both circular RNAs were potentially involved in glucose metabolism, mitotic cell cycle, and ovarian steroidogenesis. Therefore, age-related up-regulation of circRNA_103827 and circRNA_104816 might be potential indicators of compromised follicular micro-environment which could be used to predict IVF prognosis, and improve female infertility management. Six samples were analysed. Three samples of granulosa cells from women with advanced age (AA, ≥ 38 years) were compared to three samples from women with young age (YA, ≤ 30 years).