"As we know, the impairment of intercellular communication in the follicles can lead to a decline in egg quality and even infertility," Agne Velthut-Meikas, associate professor at the Department of Chemistry and Biotechnology of the Tallinn University of Technology and the thesis supervisor, writes.

Why study microRNAs?

MicroRNAs are short, non-coding RNAs. This means that they have their own gene, but their sequences are not translated into a protein. Instead, microRNAs serve as regulators of the lifespan of other RNA molecules and of the corresponding protein synthesis processes.

Interest in microRNAs has increased significantly due to developments in sequencing technologies, which are enabling us to identify new RNA sequences from an increasing number of tissues. Thanks to this, microRNA sequencing has also progressed greatly in recent years, and we now know that the majority of gene expression in humans is regulated by microRNAs. In addition, microRNAs show potential in diagnostics, as these molecules have also been found in bodily fluids, packed in nanometre-sized (10-9 metres) extracellular vesicles.  The latter are secreted by the cells in our bodies and can be found in all bodily fluids, from blood plasma and urine to cerebrospinal fluid.

MicroRNAs as an intercellular language

Ilmatar Rooda, the author of the doctoral thesis that will be defended at the Department of Chemistry and Biotechnology, was interested in the role of microRNAs in human ovarian follicles – structures where the maturation of an egg cell is achieved by controlled molecular communication between different types of cells (see figure). Egg cell maturation occurs hand in hand with the increasing of the diameter of the follicle, which can expand by up to 2,500 times the diameter of individual cells before the follicle ruptures, i.e. before ovulation. It is clear that cells need to use different means to transmit signals to each other, and microRNAs can be one such means.

Studying granulosa cells, i.e. the body cells which surround an egg cell, in patients undergoing in vitro fertilisation revealed that microRNA genes are even present in the well-known fertility-regulating genes for the follicle-stimulating hormone receptor (FSHR) and for the aromatase protein (CYP19A1), which is involved in the production of the female sex hormone estradiol. These microRNAs regulate the production of proteins which play a key role in both the activation of follicle growth as well as altering contacts between cells, thereby enabling ovulation to occur. Thus, microRNAs add an extra layer of complexity to previous knowledge about how the ovaries function, which could prove important in both the treatment of infertility and in the development of fertility preservation procedures for young cancer patients.

Ilmatar’s work shows that microRNAs are also easily identifiable in follicular fluid collected from the intercellular cavity of ovarian follicles (follicular antrum). Notably, the microRNAs that are present there occur both inside extracellular vesicles as well as outside of them, and, in fact, their role depends on the very complex in which they are released from cells. In addition, Ilmatar’s doctoral thesis clearly shows that microRNAs that are packed into vesicles’ target genes which are involved in steroid hormone signalling, cell division, and immune system mechanisms. Meanwhile, microRNAs that occur in other protein–RNA complexes have no such clearly distinguishable relevance. This knowledge, in turn, leads to the hypothesis that the filling of excreted vesicles with microRNAs is a non-random intracellular process which is controlled in some way.

Key results of the thesis

When is an ovary called polycystic? It is when it contains a number of unovulated follicles in which egg cells are essentially trapped. For women, a lack of ovulation means an inability to conceive naturally, and polycystic ovary syndrome occurs in around one in ten women of childbearing age.

All in all, Ilmatar’s research is the first to simultaneously explore the differences between both intracellular and extracellular microRNAs in the follicles of egg donors and polycystic ovary patients. As it turns out, the microRNAs whose expression was impaired in the patients’ cells are involved in immune system regulation. MiRNAs packed into extracellular vesicles in polycystic ovary patients regulate the signalling pathway for insulin-like growth factor (IGF-1R). Immune system disorders and insulin-like growth factor pathway disorders have both been previously associated with the development of polycystic ovary syndrome. Ilmatar’s research suggests that intercellular communication via microRNAs also plays a major role in the normal functioning of the ovaries.

Ilmatar Rooda’s doctoral thesis was facilitated by collaboration between the Department of Chemistry and Biotechnology of TalTech and the Competence Centre on Health Technologies (CCHT). The thesis supervisors are Associate Professor Agne Velthut-Meikas (TalTech) and Professor Andres Salumets (CCHT). Assistance in the recruitment of patients for the studies was provided by Nova Vita Kliinik.    

The doctoral thesis can be accessed here.