The researchers of a Nature Aging paper used multi-omics approaches to investigate cellular, molecular, and genetic drivers of human ovarian aging [1].
An early decline
A decline in ovarian function occurs before a similar decline in other organs. Already in their mid-to-late thirties, women can experience symptoms such as fertility problems or increased frequency of offspring disabilities [2]. Fertility-related issues are not the only symptoms: this decline in ovarian function also impacts the proper functioning of other bodily systems, leading to various health problems and impacting lifespan [3, 4].
Sequencing ovarian cells
In this new study, the researchers used human ovarian tissues from four young (23-29 years) and four reproductively aged (49-54 years) healthy donors and performed two types of sequencing: one that provides a gene expression profile and another that provides information about chromatin accessibility across the genome.
Sequencing data analysis identified eight clusters representing all major somatic cell types in the ovary. The proportion of each cell type differed between young and aged ovaries, suggesting an impact of aging on ovarian architecture.
In aged ovaries compared to young ovaries, the researchers observed significantly decreased numbers of components of ovarian follicles, granulosa, and theca cells, as well as blood vessel and lymphatic endothelial cells. Since the number of follicles decreases with age, it is unsurprising that the number of cellular components that build them also would decrease. Only epithelial cell numbers were higher in aged ovaries, and the authors suggest that it is linked to “lifetime ovulation-induced rupture and repair.”
Comparing the changes in gene expression in ovarian tissues and eight age-matched human tissues indicated “high coordination in aging-related transcriptomic changes” among ovarian cells but not in cells from other tissues. Therefore, the researchers believe that the nature of ovarian aging is distinct from that of other tissues.
Ovary-specific mTOR
Identifying differentially expressed genes (DEGs) indicated that most changes among aging-associated DEGs were similar and shared among cell types; however, some were cell type-specific.
Analysis of pathways and ovarian aging-specific genes suggested mTOR signaling as an ovary-specific pathway with a central role in ovarian aging. This is particularly interesting given that previous research has shown that mTOR can delay mouse ovarian aging [5] and that mTOR has a sex-specific effect on lifespan extension, being more potent in females [6].
Aging-related changes
Aging results in many changes on the molecular level. The researchers in this article looked into cellular senescence and cellular communication. Aged ovarian tissues showed an increase in one of the senescence markers and presented with a subset of upregulated senescence-associated secretory phenotype (SASP) genes.
On the other hand, the researchers generally observed reduced intracellular communication with age. However, there were some differences between different cell types, with epithelial cells experiencing more and more robust interactions with all cell types.
Researchers described pathways, such as ones essential for follicle development and growth, that were higher in young granulosa cells and oocytes but decreased in aged cells. However, there were also pathways related to cell adhesion and fibroinflammation that showed higher communication probability in most cell types in aged ovaries.
Losing identity with age
The researchers investigated the master transcription factor networks that define cell identity since cell identity loss is an age-related change that cells undergo. First, they identified transcription factor motifs related to each ovarian cell type and compared their activity in young and aged cells. They noted significantly decreased motif activity of identity-associated transcription factors in most cells during ovarian aging. The exception was epithelial cells that had increased identity-associated transcription factor activity.
Additional proof of age-related cell identity loss came from analysis of the expression of the top 100 cell-type-specific genes. For young ovarian cells, the expression of those genes was high except for smooth muscle and epithelial cells. In aged granulosa, immune, and theca cells, the level of cell-type-specific genes was low, suggesting cell identity loss.
Building regulatory networks
The researchers used the data they obtained to build cell-type-specific transcription factor regulatory networks for human ovarian aging in different ovarian cell types. This network highlighted the important role of the CEBPD protein in human ovarian aging. “CEBPD target genes were enriched in key aging processes, including mTOR signaling, MAPK signaling and cellular senescence, across multiple cell types.”
Age at menopause
Age at natural menopause (ANM) impacts female fertility and other health aspects. Multiple genome-wide association studies (GWASs) underscore the importance of genetics in determining ANM, with almost all of the locations in the genome (genomic loci) associated with ANM located in the non-coding regions and related to cell type-specific gene regulation.
The researchers aimed to better understand the relationship between ANM-associated gene variants and ovarian aging. They used data from previous studies to identify ANM-associated variants and compared them to the putative enhancers and promoters they identified in this study. Ths search for regulatory elements again pointed to the involvement of mTOR signaling and DNA damage response.
The researchers focused more deeply on one of the genetic variants related to the latter, located in the HELB gene’s putative promoter, which was associated with delayed ANM. HELB encodes DNA helicase B, a negative regulator of homologous recombination. The researchers identified one genetic variant that could decrease the activity of transcription factors to bind to the HELB promoter and potentially affect its expression. This could potentially affect genome maintenance and delay ANM.
In-depth functional analysis into HELB expression regulation, using human stem cells differentiated into ovarian cell types, confirmed that a late ANM-associated HELB regulatory genetic variant reduced HELB gene expression.
Further, the post-GWAS analysis allowed the authors to identify candidate gene regulatory variants and their putative target genes associated with ANM and narrow the list to ovarian tissue-specific hits. This list can inform future studies of non-coding variants that impact human ovarian aging.
From genetic insights to therapies
The authors of this study created resources that can be used to further the understanding of female ovarian aging. Their analysis already pointed out a few critical observations, such as “upstream factors (for example, CEBPD) and downstream effectors (for example, mTOR signaling) contributing to ovarian aging.”
The importance of mTOR and its role in ovarian aging can be a potential target for developing future therapies to slow it down. However, to achieve that, a deeper mechanistic understanding of molecular processes is necessary.
Additionally, while this study brought valuable new tools, future studies should include larger sample sizes with women of different ages and reproductive life stages. The stages of the menstrual cycle should also be considered as a variable.
Literature
[1] Jin, C., Wang, X., Yang, J., Kim, S., Hudgins, A. D., Gamliel, A., Pei, M., Contreras, D., Devos, M., Guo, Q., Vijg, J., Conti, M., Hoeijmakers, J., Campisi, J., Lobo, R., Williams, Z., Rosenfeld, M. G., & Suh, Y. (2024). Molecular and genetic insights into human ovarian aging from single-nuclei multi-omics analyses. Nature aging, 10.1038/s43587-024-00762-5. Advance online publication.
[2] Nagaoka, S. I., Hassold, T. J., & Hunt, P. A. (2012). Human aneuploidy: mechanisms and new insights into an age-old problem. Nature reviews. Genetics, 13(7), 493–504.
[3] Muka, T., Oliver-Williams, C., Kunutsor, S., Laven, J. S., Fauser, B. C., Chowdhury, R., Kavousi, M., & Franco, O. H. (2016). Association of Age at Onset of Menopause and Time Since Onset of Menopause With Cardiovascular Outcomes, Intermediate Vascular Traits, and All-Cause Mortality: A Systematic Review and Meta-analysis. JAMA cardiology, 1(7), 767–776.
[4] Cargill, S. L., Carey, J. R., Müller, H. G., & Anderson, G. (2003). Age of ovary determines remaining life expectancy in old ovariectomized mice. Aging cell, 2(3), 185–190.
[5] Heng, D., Sheng, X., Tian, C., Li, J., Liu, L., Gou, M., & Liu, L. (2021). Mtor inhibition by INK128 extends functions of the ovary reconstituted from germline stem cells in aging and premature aging mice. Aging cell, 20(2), e13304.
[6] Harrison, D. E., Strong, R., Sharp, Z. D., Nelson, J. F., Astle, C. M., Flurkey, K., Nadon, N. L., Wilkinson, J. E., Frenkel, K., Carter, C. S., Pahor, M., Javors, M. A., Fernandez, E., & Miller, R. A. (2009). Rapamycin fed late in life extends lifespan in genetically heterogeneous mice. Nature, 460(7253), 392–395.
