Dr Jane Cleal: Functional gene mapping of the endometrial phenotype involved in successful pregnancy: combining 3D imaging and single cell transcriptomics

The endometrium, is the lining of the womb in which the embryo implants at the start of pregnancy. The endometrium must be in the right state to accept the embryo at the correct time during a woman's monthly cycle; this is referred to as 'receptive endometrium'. If the endometrium is not in the right state to allow the embryo to attach then pregnancy is less likely to occur. If the endometrium continues to have these problems, this can lead to subfertility: our work will help the 1 in 3 couples in the UK with subfertility. We think that the endometrium can also be 'over-receptive', where it allows attachment more easily and therefore allows attachment of poor quality embryos. These are unlikely to survive and will later result in pregnancy loss: miscarriage. Improving pregnancy success in women with subfertility and repeated miscarriage requires increased understanding of the endometrium at the molecular level: i.e. what does the endometrium look like in the optimal 'receptive state', which allows healthy embryos to attach and grow successfully into a baby. Genes contain the instructions for the cell, and which genes are turned on or off determine what type of cell it is and how well it can do its role. This project will identify the genes in endometrium from women who cannot become pregnant (non-receptive endometrium) and from women with repeated occurrence of miscarriage (over-receptive endometrium). We aim to identify genes that are incorrectly switched on or off in the endometrium of women who have problems becoming pregnant or those with multiple early pregnancy losses compared to women that do not. Previous studies have tried to answer these questions but they did not produce results that agreed. We think this is because it is difficult to collect identical samples of endometrium for comparison, as not all samples will come from exactly the same place or go to exactly the same depth. Because the endometrium is made up of many different cell types, and each cell type has different genes, any differences in the sample cell population may affect the results and make differences important to fertility difficult to spot. One aim is therefore to find a way of accounting for sampling differences in the endometrial samples. The first step in understanding the basis of these conditions is to identify how and why the genes change and what the changes mean biologically. To address this we aim to use 3D imaging to understand how the endometrial cells types are arranged in space and where the genes whose expression change are physically located. How the research will be carried out: We have samples from the lining of the womb from women who suffer from inability to become pregnant, repeated pregnancy loss and normal. To study fertility related problems (subfertility/recurrent implantation failure, repeated pregnancy loss and normal) we will use modern sequencing techniques to measure the levels of all the genes switched on in our endometrium samples. These methods accurately capture all of the genes that are switched on in a tissue rather than the less precise methods from earlier studies. In order to understand what the levels of genes in our endometrial samples mean, it is important to have more information on the cell types in the endometrial samples. Using our newest state-of-the-art methods (Drop-Seq) we can get a gene signature for each individual cell in an endometrial sample. Once we know the cell-specific genes within normal endometrium, we can then apply this to our clinical samples. We can then use the knowledge on how genes change in each cell type, to adjust for differences seen in the whole sample of tissue, making the overall result much more accurate. We also need to understand what the changes in gene expression mean to the human body. To address this we aim to use our established novel, 3D imaging techniques to understand how the endometrial cells types are arranged in space and where the genes whose expression change are physically located. By looking at samples from healthy woman or those with a history of subfertility or miscarriage, we can identify the important cells or structures needed for a receptive endometrium. Together these experiments will provide a more accurate set of gene markers that relate to how receptive a woman's womb lining is to the implanting embryo, and potentially help predict whether a successful pregnancy will occur. This work will provide much-needed targets for interventions to improve pregnancy outcomes. What happens next: By adjusting for variation in cell types, we hope to achieve the first robust gene signature associated with endometrial receptivity and likely pregnancy success. In addition, using our 3D imaging of cell types in the endometrium we will be able to see where the relevant changes in gene expression are happening. We will share these results in the research community, by attending scientific conferences, and publishing our work in peer-reviewed high impact publications. Our research will also bring doctors and researchers one-step closer to being able to provide a more effective treatment to screen, diagnose and treat women with difficulties in conceiving and maintaining their pregnancy. Track Record of applicants Dr Jane Cleal: Lecturer in Epigenetics Faculty of Medicine (FOM), University of Southampton (UoS). She co-leads the Southampton Placental Group and is focussed on understanding changes in gene expression in placenta and maternal tissues during pregnancy. With the aim of translating this into interventions to improve pregnancy outcome and fetal growth. Her extensive work on amino acid transport in the human placenta led to significant advances in understanding how amino acids are transported to the fetus. Her placental gene expression studies highlight a role for vitamin D in regulating placental gene expression and she was awarded PhD studentships in 2011 and 2015 to study this. This provided preliminary transcriptomic data using Next Generation Sequencing methodology and the establishment of human placental cell culture (following cell separation) in Southampton, which forms the basis of her current BBSRC funded project. Dr Cleal currently co-supervises a PhD student with Professors Cheong and Lewis. This project combines the techniques they have established in human placenta with Professor Cheong's clinical background to investigate the 3D structure of the human endometrium. This novel work, along with their collaboration with Dr Patrick Stumpf who runs the Drop-Seq laboratory in the FOM, provides the preliminary data for this current application. Professor Ying Cheong: Is Professor of Reproductive Medicine in the UoS. She is also Medical Director of Complete Fertility Southampton. She has a track record in research in endometrial receptivity, implantation, recurrent miscarriage and endometriosis. She is the lead for the Hampshire and Isle of Wight CRN. She holds multiple NIHR, MRC and commercial grants and is an experienced researcher. She will lead on the research design, conduct and recruitment of women. Professor Susan Laird: Professor of Reproductive Biology, Sheffield Hallam University with over 25 years research experience studying endometrial structure and function, and particularly its role in recurrent miscarriage and infertility. Her work has linked the use of endometrial primary cell culture and whole tissue analysis to gain a greater understanding of the cellular control of endometrial function. Recent work has included a collaboration with a group in Hong Kong, which used RNA-Sequencing to show differential endometrial gene expression in women with recurrent implantation failure and recurrent miscarriage. She is also a co-applicant of the multi-centred endometrial scratch trial for women undergoing first time IVF funded by the NIHR. Professor Rohan Lewis: Professor in Placental and Integrated Physiology in the FOM, UoS. His research focuses on integrated approaches to placental transport, which take into account interactions of nutrient and drug transporters placental structure and blood flow. Working with the Faculty of Engineering he has developed computer models of these processes and tested them experimentally, leading to advances in our understanding of amino acid, lipid and cortisol transfer across the placenta. To make these models more realistic Prof Lewis has developed 3D imaging approaches to model a tissue's structure. This work has been funded by 3 BBSRC grants and the European Union. Mr Mostafa Metwally: Consultant Gynaecologist in Reproductive Medicine and Surgery at the Jessop Wing, Sheffield Teaching Hospitals and Honorary Senior Clinical Lecturer at the University of Sheffield. He has a record of accomplishment in clinical research in women with recurrent miscarriage and has published and presented his work at many national and international meetings. Currently he is the principal applicant of the multi-centred endometrial scratch trail for women undergoing first time IVF funded by the NIHR. Collaborator: Dr Patrick Stumpf, an established postdoctoral researcher, runs the Drop-Seq laboratory within the FOM, UoS and has provided a letter of support. He has significant expertise in Drop-Seq, having developed the protocol in Southampton with MRC funding. He works closely with The Bioinformatics Core Facility in the FOM and together they have put in place the data analysis services for this project with appropriate resources including bioinformatics protocols and cutting-edge software. Research environment and facilities: Laboratory work will take place in the Institute of Developmental Sciences (IDS), one of the UK's leading research centres for the study of fetal, placental and maternal biology, which is funded by the UoS and British Heart Foundation. This building is purpose-built fully equipped and provides a state-of-the-art research environment with excellent core resources and in close vicinity to the Princess Anne Maternal Hospital where clinical samples will be collected. The Biomedical Imaging Unit is a central imaging facility of the FOM available for research and diagnostic purposes. It is an open access multiuser facility with a range of light and electron microscopes supported by seven fulltime imaging staff. The MRC funded innovative Drop-Seq set-up for the cell-sequencing experiments is in close proximity to the IDS labs and is run by word-leaders in this technique. The UoS WISH Facility is a service provider for the sequencing analysis that we require for the cell experiments.