The impact of age on single oocyte and their cumulus cells using single cell RNA sequencing
Keywords: single cell RNA sequening
immature oocyte
Prepubertal oocyte
Germinal versicle
oocyte transcriptome
oocyte quality
poor oocyte quality
poor oocyte comptence
women's health
oocyte RNA
cumulus cells
oocyte senescence
ageing
oocyte ageing

Oocyte quality is the major determinant of successful fertilisation and subsequent normal embryo development. Maternal age is the greatest influencing factor limiting the success of infertility treatment. Current assisted reproductive technologies (ARTs) are unable to recover the fertility of females in two categories: pre-pubertal females who have survived cancer and older women who have delayed childbearing several years beyond their peak reproductive capacity. In the first category, women are at high risk of premature ovarian failure because of gonadotoxic chemotherapy. Thus, ovarian tissue preservation becomes an integral component of the treatment plan. However, the progress of in vitro maturation (IVM) of the pre-pubertal oocytes remains poor. In terms of the second category, oocytes from older women are correlated with poor fertilisation, low implantation rates and spontaneous abortion. Both pre-pubertal and aged oocytes have a limited developmental competence for sustaining successful fertilisation and normal embryo development. Also, it is unclear whether follicular microenvironment, which support oocyte development, have contributed to diminish the quality of these oocyte. To improve the ART success rate, it is necessary to elucidate the mechanisms by which maternal age negatively affects oocyte quality, representing an extremely demanding task. Little is known about the transcriptional activity that underlies the impaired competence of oocytes at these ages.
Using single-cell RNA sequencing (sc-RNA-seq), this thesis investigated the impact of age on the transcriptome of single oocytes and their companion CCs at prepuberty, maturity and advanced age. This was achieved, first, by optimising a preparation protocol for generating high-quality cDNA libraries for Illumina sequencing. Sc-RNA-seq was then successfully performed in a single oocyte, providing an unprecedented depth of investigation, by generating 30 million reads from each sample. sc-RNA-seq revealed novel pathways, including retinoic acid receptor (RAR) metabolism and Rho signalling, suggesting that these pathways govern the transcriptional dynamics changes required to obtain competence. For aged oocytes, the dysregulation of RAR and mitochondrial L-carnitine was involved in the age-related deterioration. Interestingly, while CCs seemed to deteriorate as a function of advanced age, CCs associated with prepubertal oocytes did not reveal a distinct transcriptome in relation to those in young adults. Finally, the oocyte and CC sequencing data were validated using OpenArray real-time polymerase chain reaction (RT-PCR). In summary, single-cell transcriptomics provided insight into the intrinsic (oocyte) molecular changes and extrinsic microenvironment (CCs) associated with age. The aged follicular cells could contribute to the diminished developmental potential in oocyte senescence.