RNA-Seq reveals women’s fertility may decline with age due to impaired RNA levels

Age may adversely affect women’s fertility by impairing levels of RNA molecules which in turn alter the function of genes involved in key biological pathways during the final maturation stage of a human egg cell, according to the findings of a new study published today in the journal Aging Cell.

Researchers from the Centre for Genomic Regulation (CRG), the Centro Nacional de Análisis Genómico (CNAG-CRG) and Clínica Eugin sequenced the RNA molecules, also known as the transcriptome, within oocytes to understand which genes are affected in their activity by age. They used single-cell sequencing to analyse the transcriptome of 72 individual oocytes from 37 donors between 18 and 43 years of age.

Single‐cell transcriptome profiling of human oocytes

(a) Schematic representation of the experimental design. Briefly, 37 women were recruited. The mean woman age was 28.8 years (SD = 7.7, range 18–43), and from each woman we included between 1–4 GV oocytes. GV oocytes were analysed directly as GV (n = 40) right after denudation or, after 30 h in G2^TM medium, as in vitro matured metaphase II (IVM‐MII, n = 32) oocytes. Their transcriptome was compared by single‐cell RNA‐sequencing analysis (Smart‐seq2). (b) Exemplary pictures of a germinal vesicle (i) and a IVM‐MII (ii) oocyte included in the study. Scale bar = 100 µm. (c) Oocytes cluster according to their maturation stage. (d) Differentially expressed genes between the GV and the IVM‐MII groups are represented in red. Labels correspond to the top10 differentially expressed genes in each category after filtering for fold change 2 (avg_log2FC > 1) and sorting markers according to their p‐value (cutoff = 0.01). Total number of variables: 12,431. (e) Example of two GV markers (RPL39L: Ribosomal Protein L39‐Like; PAIP2: Poly(A) binding protein Interacting Protein 2) and two IVM‐MII markers (MED30: MEDiator complex subunit 30; PAXIP1: PAX Interacting Protein 1). (f) Gene Ontology enrichment analysis of each maturation stage. The top 5 activated GO terms are shown. p‐values were adjusted using the FDR method. GeneRatio: number of genes related to the GO term/total number of significant genes. GV: Germinal Vesicle; IVM‐MII: in vitro matured metaphase II, PB: Polar Body

They found that the number of transcripts for genes involved in chromosome segregation and RNA processing increased progressively with age, while the number of transcripts related to mitochondrial metabolism decreased.

However, these age-related changes to the transcriptome only occurred when egg cells reached their final stage of development during in vitro maturation. The transcriptome was less affected by age in immature egg cells. According to the researchers, the findings suggest that age may influence an oocyte’s ability to process gene products critical for the last steps of their development.

Further analyses revealed a number of potential master regulator genes, which are genes that occupy the very top of a regulatory hierarchy, that are affected by age. Future work will test whether these genes play a critical role in oocyte aging.

“Here we show that the final step of oocyte maturation itself might be negatively affected by age, which is critical for reproduction because it provides the material early embryos need to develop normally and survive,” says Bernhard Payer, AXA Professor of Risk prediction in age-related diseases and Group Leader at the Centre for Genomic Regulation (CRG) and co-author of the study. “What we don’t know yet, is which of these changes are merely a consequence of the aging process and which may directly contribute to the quality drop in oocytes with age.”

According to Holger Heyn, Group Leader at the Single Cell Genomics team at the Centro Nacional de Análisis Genómico (CNAG-CRG), part of the CRG, “our sequencing technologies to analyze individual cells are extremely powerful to describe even subtle differences between cells. This was particularly important here when dissecting the impact of aging on oocytes. This work is a great example of the power of interdisciplinary science. Joining expertise from the clinics with high-resolution cellular profiling and advanced computational biology was key for the success of the study.”

The researchers also used donor height and weight information to assess the impact of body mass index (BMI) on the transcriptome. Unlike age, abnormal BMI mostly affected the transcriptome of immature oocytes. According to the authors, the finding suggests that the fertility decline caused by age might have different root mechanisms than the one caused by abnormal BMI.

Women’s fertility generally declines with age. One of the main reasons for this is due to depleted ovarian reserves, as baby girls are born with all the oocytes, from which mature eggs will develop, during their lifetime. Another reason is that the quality of eggs lowers with age, which is thought to be one of the main reasons for higher rates of infertility after the age of 35. Being overweight or underweight has also been associated with poor oocyte quality and reproductive outcome.

The authors conclude that though more studies are required, their findings may result in the future development of new diagnostic tools to better assess oocyte quality in reproductive medicine, as well as potential drug treatments that modulate the affected pathways to rejuvenate aged oocytes.

Source – Center for Genomic Regulation