MicroRNA signature changes in human ovarian follicle in relation to reproductive aging

Abstract

Small non-coding class of RNAs includes different types of molecules involved in different steps of RNA synthesis, processing, translation, as well as RNA modulation of transcription initiation, RNA degradation or protein synthesis block. Among these, microRNAs (miRNAs) represent the most studied and better characterized class of ncRNAs in terms of function and impact in human health and disease. miRNAs are short non-coding RNAs involved in the control of gene expression in different species. Specifically, miRNAs play a major role as master regulators of protein-coding genes. miRNA role in ovarian follicles has been characterized and the existence of miRNAs in human follicular fluid has been recently demonstrated. It was also reported that altered regulation of miRNA expression affects crucial pathways for follicle growth and oocyte maturation in several reproductive diseases. In addition, global miRNA analysis, on different tissues and organs and animal models has also shown that aging can influence mirna expression. Decreased female fertility with advanced maternal age has been widely documented. Although it is widely recognized that a key aspect that explains infertility in reproductive aging is the decline in oocyte quality, which also associates with higher risk of birth defects, genetic disorders and miscarriage, the molecular mechanisms underlying reproductive aging in female mammals are poorly understood. This thesis has aimed at evaluating impact of maternal age on miRNA expression profiles in human ovarian follicle and characterizing the pathways significantly affected by female ageing, identifying their regulator miRNAs. The manuscript includes three studies that for concision, they will be referred in the text, by Roman numerals, as Study I, II and III respectively. The Study I has been focused on the characterization of microRNAs in human follicular fluid (FF), the Study II on the miRNome changes in Follicular fluid exosomes from women of two different age groups; while miRNA identification in human MII oocyte and their expression profile changes in relation to reproductive aging have been shown in the Study III. Firstly, it was ascertained whether miRNAs are cargo of FF exosomes and whether they are involved in the regulation of follicle maturation. At a later stage, TaqMan Human microRNAs cards were performed to verify differently expressed miRNAs in FF respect with plasma samples, collected from 15 healthy women who underwent to intracytoplasmic sperm injections (ICSI). 37 miRNAs had significantly higher expression levels in human FF. 32 are carried by exosomes and involved in critically important pathways for follicle growth and oocyte maturation. Specifically, nine of them target and negatively regulate mRNAs expressed in the follicular microenvironment encoding inhibitors of follicle maturation and meiosis resumption. In order to reveal the contribution of miRNAs to female reproduction aging, we examined their expression changes during aging in human follicular fluid, using TLDA technology. Different miRNA distribution was found in FF exosomes from old and younger women. We detected about 50 miRNAs in FF exosomes which showed highly significant differences related to aging and were predicted to regulate ECM-receptor interaction, PI3K-Akt, p53, TGF-beta, HIF-1 and mTOR signaling pathways. Finally, we identified 57 miRNAs constantly expressed in 12 MII oocytes and 12 miRNAs displaying altered regulation in women of advanced reproductive age. By computational approach we explored the possible functions of differentially expressed miRNAs inside human germ cells, and confirmed experimentally miRNA differential expression in murine MII oocyte. Finally, a significant negative correlation miRNA-targets was found for miR-29a, miR-203 and specific mRNAs, that have been ascribed responsible for mechanisms modulating epigenetic changes underlying compromised oocyte quality caused by advanced maternal age.