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Using human genomics to decipher biological mechanisms underlying reproductive ageing and fertility in women


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Authors

Stankovic, Stasa 

Abstract

Women are born with a non-renewable ovarian reserve, which is depleted throughout reproductive life. When this reserve is exhausted, they experience menopause and cease ovulating. Importantly, menopause timing is highly variable and can impact health outcomes in later life. One in 100 women experience menopause before the age of 40. As natural fertility begins to decline 10 years prior to menopause, the age of menopause impacts reproductive options for many women, leading to increased demand for fertility treatments, which have low success rate. This is especially important as more women delay childbearing. Endocrine and imaging tests used in the clinical setting only record changes in ovarian function that have already taken place, thus disabling early prediction and timely identification of women with reduced reproductive lifespan. Human genetic studies have attempted to overcome this problem by identifying genetic markers associated with menopause timing and thus providing substantial insight into the biological mechanisms governing ovarian ageing. However, previous approaches have been largely restricted to assessing common genetic variation, leaving many aspects of the trait biology unexplored. This dissertation describes five distinct projects that advance our understanding of the genetic determinants of female reproductive ageing by employing state-of-art genomic and proteomic technologies with robust functional models. Chapter 3 uses whole exome sequence data to identify rare protein-coding variants associated with menopause timing in ~120K women in the UK Biobank (UKBB), and implicates five novel ANM genes with effect sizes up to ~5 times larger than previously discovered for common variants. Notably, heterozygous loss of ZNF518A shortens reproductive lifespan by delaying puberty timing in girls and reducing ANM by nearly 6 years in carriers, an effect larger than any variation currently tested in clinical genetics for premature ovarian ageing. Furthermore, I provide evidence that ZNF518A is a master transcriptional regulator of ovarian development and establishment of the ovarian reserve in foetal life, thus highlighting novel mechanisms involved in ANM aetiology. I also identify a new cancer predisposition gene, SAMHD1, which has a comparable effect size in women and men to well-established genes such as CHEK2, further reinforcing the link between cancer and reproductive ageing. Finally, I show that mothers with genetic susceptibility to earlier ovarian ageing have a higher rate of de novo mutations in their offspring. This provides direct evidence that female germline mutation rate is heritable and highlights a mechanism for maternal effects on offspring health. Chapter 4 extends the exome sequence analysis to an extreme form of early menopause, i.e. POI, which is often considered a monogenic disorder, with pathogenic mutations reported in ~100 genes. However, such reports are based
5 on small numbers of individuals without independent replication, or/and no functional validation. I systematically evaluate the penetrance of these reported genes in ~120K UKBB women, 2,231 of whom reported ANM before age 40. In this largest study of POI to date, I find limited evidence to support any previously reported autosomal dominant gene. For nearly all these genes I could rule out even modest penetrance, with 97.8% of all identified protein truncating variants found in reproductively healthy women with ANM over 40. In addition, I demonstrate novel haploinsufficiency effects in studied POI genes, including TWNK and SOHLH2. Collectively my results suggest that most POI cases are likely oligogenic or polygenic in nature, which has major implications for future clinical genetic testing and counselling. Chapter 5 presents the first proteogenomic study for the ANM targeting 4,775 distinct proteins measured from plasma samples of 10,713 European ancestry individuals in the Fenland study. Although this analysis did not identify robust protein candidates associated with ANM, it demonstrates the potential of such approaches to discover new biomarkers. Chapter 6 presents the largest genomic meta-analysis for age at menarche on ~566,000 women of European ancestry and 696 genomic loci that contribute to regulation of menarche timing. I use this data to explore biological mechanisms and overlap between genetic architectures of reproductive health outcomes. I provide the first evidence on the enrichment of DDR mechanisms for menarche timing, indicating the involvement of DDR in regulation of both extremes of reproductive lifespan, i.e. menarche and menopause. In addition, I report first gene candidates that I speculate may act via oocyte-specific mechanisms to modify reproductive longevity. I also highlight DDR and other novel mechanisms, including ribosome biogenesis, which impact multiple reproductive health outcomes, such as polycystic ovarian syndrome (PCOS), twinning and number of children (NEB). Finally, I demonstrate the first population genomic evidence on the role of DDR related mechanisms in various anthropometric, metabolic and reproductive health outcomes, indicating that DDR could act as a marker of health outcomes beyond cancer. Combining human genomic evidence with cutting edge CRISPR technology and the In vitro gametogenesis system, in Chapter 7 I investigate the role of PARP-1 in proliferation of primordial germ cells during the establishment of the ovarian reserve. I demonstrate suggestive evidence on the role of PARP-1 in decreasing ANM in women and, paradoxically, that deletion of PARP-1 increases the efficiency of primordial germ cell production in vitro. I speculate that, despite the initial increase in primordial germ cells in the PARP-1 knockout, the quality of these cells could be compromised, thus ultimately limiting the functional ovarian pool. Collectively, these findings provide significant insights into the biological processes of reproductive ageing in women and have the potential to guide future experimental work aimed towards identification of new therapies for enhancing reproductive function and preserving fertility in women, as well as designing intervention strategies to prevent or diminish menopause-related health outcomes.

Description

Date

2022-09-11

Advisors

Perry, John
Ong, Ken

Keywords

menopause, fertility, genomics

Qualification

Doctor of Philosophy (PhD)

Awarding Institution

University of Cambridge
Sponsorship
MRC (MC_UU_00006/2)