Unravelling the causes of declining uterine function with age

Context
The earliest sign of advancing age experienced by women is a decline in their fertility. Maternal age not only affects the risk of having a baby with chromosomal abnormalities (such as Down's syndrome), but also increases the frequency of suffering from various pregnancy complications such as miscarriage, pre-eclampsia or preterm birth. The reason why these pregnancy complications are more prevalent in older women are not well understood, as they are most commonly not associated with chromosomal defects of the baby. Advanced maternal age is an immense societal challenge as many women delay child bearing until later in their lives, with over 1 in 5 babies born to women over 35 in the UK. It is extremely important to better understand why these complications increase with age, and explore potential avenues for future treatments.

Using the mouse as a model of how age affects womb function and pregnancy outcome, we have found that the cells lining the womb (called endometrium) that support implantation of the embryo, undergo a significant decline in their ability to respond to pregnancy hormones with increasing age. This reduced response correlates with widespread changes to the way DNA is packaged in these cells. Packaging of DNA is regulated by a layer of information superimposed onto the DNA sequence itself, known as the 'epigenome', that controls which genes can be switched on and off. We find that in aged endometrial cells many important genes have an altered "epigenetic signature", which prevents them from being switched on at the right time. In this proposal, we are aiming to acquire crucial new knowledge into the precise nature and progression of these epigenetic alterations and how they may affect endometrial cell and thus womb function.


Aims
The overarching aim of this project is to study how age affects the behaviour and function of the cells lining the womb, i.e. the endometrium. We will address three specific objectives:

1) Use precisely characterised samples throughout the second half of the reproductive life course in the mouse (with optimised experimental technology to keep animal use to an absolute minimum) to identify robust changes to the epigenome of endometrial cells with age, and correlate these with the functional decline in hormone responsiveness.

2) Building on these insights, use carefully characterised human endometrium collected from women who have gynaecological complaints (e.g., uterine fibroids, that are commoner as women age), to assess the most promising candidate genes and pathways in the endometrium of women of older reproductive age. This objective will take advantage of innovative cell culture methods that allow us to maximise the informational output we can gain from each sample.

3) Validate the critical role of identified candidate genes in cell culture assays for inducing, or preventing, ageing-associated molecular changes. Assess whether well-established drugs that interfere with the identified genes/pathways may halt or even reverse the age-associated decline in endometrial cell function, as first lead into potential future therapeutic applications.


Potential applications and benefits
A detailed understanding of the molecular changes that occur during the ageing process is of fundamental importance for basic bioscience and medical research. Female reproductive decline is a particularly good example to study these effects.

The immense benefit of this study is that it will help elucidate why the womb is less capable of adapting to pregnancy in older women. This knowledge forms the stepping stone for therapeutic interventions in the longer term. We will indeed make first steps into this direction, by testing whether drugs that target specific pathways we identify can help improve endometrial function. These are exciting prospects for future research to significantly reduce the need for health care provision and alleviating economic burden.

The dynamic capacity of the uterine endometrium to decidualise in response to pregnancy hormones declines dramatically with age. We have shown this to be true in the mouse where decidualisation failure is a major cause of developmental arrests in pregnancies of older females. Similarly, evidence from uterine pathologies in humans, such as fibroids and adenomyosis that are more prevalent in women of advanced age and also increase the risk of pregnancy complications, points to shared age-associated changes in both species insofar as endometrial cells acquire a progesterone-resistant phenotype.

Based on our extensive preliminary data, we hypothesise that significant changes to the epigenome are a key factor underpinning the decline in endometrial function. In particular, we find a marked de-regulation of a gene family involved in protein (including histone) citrullination, an epigenetic modification that antagonises multiple other histone marks and as such may trigger a cascade of epigenetic rearrangements.

In this proposal, we aim to use primary mouse endometrial cells for an in-depth characterisation of the progression of epigenomic changes during ageing (24-52 wks), integrated with transcriptomic changes and functional assessment of these cells. These data will inform the analyses of carefully characterised human endometrial tissue, enabled by the Co-I. For this, we will take advantage of novel organoid culture methods that we helped establish and that will be most powerful to maximise the depth of information gained from each biopsy. We will assess distinct endometrial compartments separately (stroma and glands, isolated and upon co-culture) in innovative 3R approaches. Finally, we will prove direct roles of identified candidate genes and pathways in CRISPR activation and deletion assays, and assess whether chemical inhibition of specific epigenetic modifiers can help restore the decidualisation response as a lead into future therapeutic strategies.

The main groups of people who will benefit from this research are
1- Academic scientists in basic bioscience and biomedical research,
2- Clinical scientists and clinicians in Women's Health and Obstetrics & Gynaecolgy,
3- Healthcare providers,
4- Higher Education,
5- Scientific communication professionals,
6- Pharmaceutical industry, and
7- The general public as a whole.

They will benefit directly through the wealth of new knowledge arising from this research.

Academic scientists will benefit from the novel insights on the impact of ageing on cellular function and epigenetic constitution. The mechanistic aspects that we focus on in our proposal have not been studied before in this context, and may serve as a paradigm for ageing research, as we are addressing some of the earliest cellular changes that have severe impact on health and wellbeing, specifically to women. Clinical. Clinical scientists will also benefit from these findings. This is in particular the case if our targeted approaches of interfering with the accumulation of age-associated aberrant epigenetic marks are successful.

We therefore expect that in the longer term, our research will have significant translational potential that will be of benefit to healthcare providers and the general public as a whole. Any commercial potential will be exploited and managed by Babraham Institute Enterprise (the wholly owned trading arm of BI). The collaboration agreements will preclude unreasonable restrictions that restrict or delay data sharing. A potential key industrial partner in this context is Bayer AG with whom both PI (MH) and Co-I (HC) currently have, or have had, research collaboration agreements and/or grant funding in place.

Researchers and scientific communication professionals will benefit through the timely dissemination of our results, as ongoing advances of the project will be presented at national and international conferences. The results of this work will be published in peer-reviewed open access journals or deposited in open access databases. The integrated genome-wide transcriptional and epigenomic network data will be made available on open-access servers, which will enable us and other researchers to expand the dataset in an interactive manner. Opportunities for intellectual property development and commercial exploitation are provided by novel insights into mechanisms of epigenome regulation, methodological advances and in the longer term potentially cell and embryo culture protocols. The lay audience will benefit from presentations, press interviews and/or public lectures, and through the social impact of the results of the study.

The project involves in-depth training of the staff, which will make them highly desirable for subsequent employment in the academic, health care and commercial research sector, and on scientific advisory boards. They in turn help in teaching and outreach activities, and engagement with students and high school pupils on regular occasions such as Schools Day, which ensures that the impact of our work to the (higher) education sector. We actively engage in training the next generation and fostering scientific enthusiasm through visits at schools, participating in the Researchers in Residence Scheme, and School's Day Projects.

More generally the work will enhance the knowledge economy and promote the health of a variety of scientific disciplines. The work is innovative, cross-disciplinary and at the forefront of biomedical science, which will enhance the UK's scientific reputation.