O-033 Molecular insights into the decline in oocyte quality with advanced maternal age: findings from comprehensive single cell -omics analysis of 112 human oocytes

How does maternal age impact the molecular composition of oocytes during their final meiotic progression?

Maternal age significantly correlates with proteomic changes during oocyte maturation, particularly in proteostasis and meiosis related proteins, with minimal impact on transcriptome and DNA-methylation patterns.

Oocyte quality declines with maternal age, resulting in diminished developmental competence and higher aneuploidy rates. However, the molecular mechanisms behind this decline in oocyte quality remain elusive. Transcriptomic studies have revealed few differentially expressed genes in advanced maternal age (AMA) oocytes, suggesting that age-related changes in oocyte quality result from a complex interplay of molecular factors, rather than a single cause. However, comprehensive methylation and proteomic data are still lacking. We employed advanced -omics to assess the effect of maternal age on the molecular signature of human oocytes, focusing on DNA-methylation, transcriptome and proteome changes during the final meiotic progression.

This study included a total of 112 oocytes obtained from young (<35 years, n = 35) and AMA women (>37 years, n = 55), who were recruited in the study from October 2021 to October 2023. Both germinal vesicle (GV, n = 68) and metaphase II (MII, n = 44) oocytes were analysed. Additionally, 19 immature oocytes (GV and metaphase I) from 9 young women were used for validation.

Parallel single-cell bisulfite and RNA sequencing was applied to 44 oocytes (26 GV-Young, 6 MII-Young, 8 GV-AMA, 4 MII-AMA) and single-cell proteomics to 68 oocytes (18 GV-Young, 18 MII-Young, 18 GV-AMA, 14 MII-AMA). Additionally, 10 GV and 9 MI oocytes were treated with the proteasome inhibitor MG-312 (0 µM, 10 µM) for 6 hours, followed by rescue in vitro maturation (rIVM) for 36 hours. Chromosomal distribution was assessed by immunocytochemistry.

Our analysis revealed no significant changes in DNA methylation patterns in either GV nor MII oocytes associated with AMA. Also, very limited changes were detected in the transcriptome, with transcript levels of only 5 genes in GV oocytes and 7 genes in MII oocytes detected as being significantly changed. In contrast, proteomic analysis, particularly in GV oocytes, revealed significant age-related changes, notably in proteins participating in the proteostasis network (signalosome complex, UCHL1) including chaperones (TRiC-complex, HSP7C, STIP1), and in the cell cycle, including signal transduction factors (1433E, integrins) and cytoskeleton regulators (DYL2, CAPZB, ARHGG) (Rs ≤ |0.5|, p ≤ 0.05). The proteasome complex, which plays a crucial role both in meiosis and the proteostasis network, was found to decline with age; changes were evident in several subunits of the complex (e.g., PRS8, PRS6A, and PRS10; Rs ≤ -0.56, p ≤ 0.05). Compared to controls, treatment of immature oocytes with the proteasome inhibitor MG-132 resulted in either maturation failure or chromosome mislocalization in metaphase plate, further validating the essential role of the proteasome during oocyte maturation. In MII oocytes, 7 proteins showed alterations with age, including the oocyte-specific marker DDX4, which significantly declined in abundance (Rs =-0.6, p ≤ 0.05).

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 transcriptome and proteome.

Our findings suggest that age primarily affects oocyte quality at the post-transcriptional level, potentially through meiosis dysregulation and proteostasis disruption. We also demonstrate the proteasome's vital role in oocyte maturation, suggesting that targeting the proteasome complex may improve oocyte quality in AMA women.

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