P-710 Single-cell Proteomic Analysis of Human Oocytes Reveals a Decreased Abundance of Meiosis and Proteostasis Regulators with Advanced Maternal Age

Abstract Study question How does maternal age affect the proteomic content of her oocytes during their final meiotic progression? Summary answer Compared to young oocytes, oocytes obtained from women of advanced maternal age (AMA) show a decreased abundance of meiosis and proteostasis regulators. What is known already It is well established that AMA leads to a decline in oocyte number and quality, key limiting factors of female reproductive success. Poor oocyte quality is tied to aneuploidies and alterations in cytoplasmic fitness, however the precise mechanisms that underlie oocyte aging remain unknown. Currently knowledge has been largely restricted to transcriptomic studies with no information on the proteome of oocytes obtained from women of AMA. Single-cell proteomic studies may shed light on these mechanisms, as proteins and (post)-translational modifications regulate events unfolding at the oocyte-to-embryo transition in transcriptionally silent oocytes. Study design, size, duration A total of 67 oocytes, obtained from 52 women undergoing controlled ovarian stimulation cycles between May 2021 and May 2022, were included in the study. We obtained 18 fresh germinal vesicle oocytes (GVs) and 17 vitrified in vivo matured metaphase II oocytes (MIIs) from young women (oocyte donors), and 18 GVs and 14 MIIs from AMA. Participants/materials, setting, methods The oocytes were obtained from oocyte donors (n = 27) and AMA patients (n = 25) with a mean age of 24 ± 3.9 years and 39 ± 1.8 years respectively. Single denuded oocytes were snap frozen and their proteomic profiles were generated using the mass spectometry based method plexDIA . Mann-Whitney U test (fold change >|1.5|, adjusted p-value <0.05), Spearman correlation (rs ≥|0.4|, p-value <0.05) and ggplot were used in R to compare the different groups. Main results and the role of chance A total of 2105 proteins were quantified across oocytes stages and age groups. Most of proteins were present in oocytes independently of their maturation state and related to vital processes, such as cellular respiration and energy metabolism. Fifty-four proteins varied in abundance between immature and mature oocytes, including RS14, YBOX2 and RL10A (translational regulation), CLUS and AHSA1 (post-translational modifications) and proteins involved in meiosis progression (WEE2, AURKA, BUB1B, SPDLY). Notably, we observed a negative correlation between protein levels and maternal age for meiosis regulators (e.g.1433E, CDK1; rs: -0.7 and -0.5), meiotic spindle proteins (e.g. DYL2, CAPZB, ARP3, GCP3; rs: -0.6, -0.6, -0.5, -0.5 respectively) and for 5 proteasome subunits (PRS8, PRS6A, PRS10, PSA6, PSMF1; rs: -0.4 to -0.7). The proteasome is a degradation machinery with multiple roles, including meiosis regulation and proteostasis maintenance. Additional proteins of the proteostasis network were found to decline with age (e.g., TCPH, TCPA; rs: -0.5, -0.5). Loss of proteostasis is a common feature of aging and diseases. We also observed a positive correlation between mitochondrial and stress response proteins and maternal age (e.g. ATP5L, TMX1; rs: 0.6, 0.5). Limitations, reasons for caution Unlike GVs that were collected fresh, MII oocytes underwent vitrification and warming before being included in the study due to clinical protocols. These procedures may have unknown effects on the proteome. Wider implications of the findings We show that a woman’s age correlates with changes in her oocyte proteome, which may contribute to the deterioration of oocyte quality through dysregulation of meiosis and disturbance of proteostasis. Proteins showing differential expression in oocytes from women of AMA, such as proteasome, may become targets for improving oocyte quality. Trial registration number Not applicable