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Linking perturbed maternal environment during periconceptional development, due to diabetes, obesity or assisted reproductive technologies, and altered health during ageing

Final Report Summary - EPIHEALTH (Linking perturbed maternal environment during periconceptional development, due to diabetes, obesity or assisted reproductive technologies, and altered health during ageing.)

Executive Summary:
EpiHealth has fulfilled its main goal towards improving health of the human population by understanding the metabolic and epigenetic mechanisms in early development which create biological variation and have a long-term effect on the health of individuals. EpiHealth Identified important genetic pathways affecting the health of developing embryos in human and rabbit models of diabetic maternal environment. Furthermore, in human and mouse ART models main genetic and metabolic pathway alterations have been identified, adversely affecting the health of the progeny. The project linked for the first time using bioinformatics tools the longevity related pathways and those susceptible to early epigenetic perturbations to explain how early events influence the health and lifespan of individuals. Importantly, data sets available on patients born from ART in the last 20 yr have been linked and analyzed to help predict the importance of ART related variations in health across the entire lifespan.
The EpiHealth project was studying health areas with major societal and economic importance: (a) diabetes and obesity affecting a large proportion of society, for which long term and transgeneration consequences on babies born from affected mothers is still not fully understood; (b) ART, a much better defined system used in human and animal contexts and known to increase imprinting (e.g. gene methylation) related disorders and metabolic abnormalities in human babies and children. The results of the project need to be explored further, in order to define conditions of human ART which allows for improvements in the preimplantation environment to make ART even safer and improve ART-babies long term health. Basic mechanisms regulating epigenetic modification events have been studied intensively, resulting in novel discoveries enhancing our understanding of the key molecular players in early stages of development. Bioinformatic analyses of gene expression and large epigenetic datasets have been performed to identify key genetic pathways linking early developmental perturbations and reduced longevity, and several potential biomarkers have been identified for future studies and applications in medicine for disease prevention and health management in diabetic and ART pregnancies.

Project Context and Objectives:
The main goal of EpiHealth is to improve health of the human population by understanding the mechanisms and pathways in early development, with special emphasis on epigenetic changes and developmentally relevant metabolic signals, which create biological variation and have a long term effect on the health of individuals during their lifespan. Specific goals include: i) Identification of the main genetic pathways affecting the health of developing embryos in a diabetic or obese maternal environment (human and rabbit models); ii) Identification of the main genetic and metabolic pathways affected, and epigenetic and imprinting perturbations arising, in human and animal (mouse) ART models resulting in altered health of the progeny; iii) Discovery of the key genes and pathways affecting epigenetic and imprinting sensitivity in early stages of development in order to create intervention tools against epigenetic mis-programming; iv) Linking for the first time using bioinformatics tools the longevity related pathways and those susceptible to early epigenetic perturbations in order to explain how early events influence the health and lifespan of individuals. Data sets available on patients born from diabetic mothers or from ART in the last 20 yr will be analyzed to help predict the importance of these pathways and some of the resulting human biological variations in health across the entire lifespan; v) Studying the possibilities of early intervention by ameliorating the periconceptional maternal environment.
The EpiHealth project is studying two major health areas: (a) diabetes and obesity, a condition affecting a large proportion of society, for which long term and transgeneration consequences on babies born from affected mothers is still not fully understood; (b) ART, a much better defined system used in human and animal contexts and known to increase imprinting (e.g. gene methylation) related disorders and metabolic abnormalities in human babies and children. We propose that there will be some similarities between the diabetes induced developmental problems and those from human ART. The well-defined experimental conditions and accessibility of human ART allows for improvements in the preimplantation environment to make ART even safer. The project also aims to advise on the “optimal” maternal metabolic environment to alleviate adverse health effects and, furthermore, to test pathways and systems for active manipulation of the epigenetic programming of embryos. Basic mechanisms regulating epigenetic modification events is studied in relevant knock-out mouse models to identify and enhance our understanding of the key molecular players and allow direct intervention in early stages of development. Samples and data sets from periconception and perinatal stages of development are for the first time linked with those from healthy aged centenarians. Bioinformatic analyses of gene expression and large epigenetic datasets have been performed to identify key genetic pathways linking early developmental perturbations and reduced longevity.

Project Results:
Two models have been studied in WP1: the rabbit (WP1A) and the cellular (WP1B) models. The overall goal of the rabbit model is to study the effects of diabetes on blastocyst physiology and development. Blastocysts from diabetic rabbits suffer from diabetes-like metabolic changes which result, among others, in a delayed development. The insulin receptor signaling is altered. Compensatory mechanisms come in action, such as an increased synthesis of IGF1 and IGF2 and an increased expression of adiponectin and adiponectin receptor1. Maternal diabetes leads to non-enzymatic protein modifications not only in the mother but also in their blastocysts. As early as at day 6 p.c. specific AGE modifications are found in trophoblast and embryoblast cells in blastocysts from diabetic pregnant rabbits.

In the cellular model human and mouse stem cells have been analysed. In first sets of experiments it was shown that irrespective of their origin mouse cell lines (mESC, NT-ESC) were highly similar in respect to their pluripotency signatures. Human embryonic stem cells were investigated and revealed to be more sensitive to oxygen stress (induced by H2O2 treatment in vitro) than control somatic cell lines. ROS detection and lipid peroxidation assays were optimized. Furthermore, gene expression studies of hESC lines have shown several genes upregulated following H2O2 exposure. Targeting vectors have been constructed for generating fluorescent reporter cell lines.

WP2 is dedicated to (1) human studies using existing databases, datasets and stored samples (WP2A) and (2) mouse experimental studies (WP2B) with parallel Tasks to investigate effects of maternal oocyte age, embryo cryopreservation and prolonged embryo culture on short- and long-term developmental potential.

In WP2A, existing microarray databases derived from human oocyte, 4-8 cell and blastocyst stages have been evaluated using a range of bioinformatics approaches for the differential expression of genes both important in development to these stages and also signaling pathway critical in environmental sensing and developmental programming.

In WP2B, experimental protocols for assessing the effects of ART factors and treatments have been optimised in our mouse model. Results indicates that older oocytes tend to generate slightly more advanced embryos but with fewer blastomeres. Prior embryo cryopreservation tended to reduce the rate of embryo development coinciding with fewer blastomeres within blastocysts. Offspring from ageing oocytes (older mothers) tend to grow faster but no effect of cryopreservation on postnatal growth has been observed.
In WP3 KRAB zinc finger proteins are studied, as findings implicate this extensive group of transcriptional repressors in regulating epigenetic stability early in development, impacting on health, disease and longevity. Using mouse embryonic stem cells we utilised a lentiviral system to overexpress tagged zinc finger proteins for use in ChIP-seq experiments. Cell lines have been constructed for 5 KRAB zinc finger proteins and libraries for next generation sequencing were generated and analysed. Novel insights into the role of several KRAB ZF proteins have been obtainted, including identification of novel characteristics in some of them.

WP4 The main aim of this WP was to identify epigenetically regulated genes that are involved in human ageing. To this end, methylation profiles from two human samples, namely a discovery (n=125; age-range: 20-104 yrs) and a validation cohort (n=142; age-range: 26-102 yrs) were investigated to determine age-related changes in the methylation patterns. We found 538 age-related differentially methylated CpG sites (DMSs) that were consistent in both cohorts. Of the 538 DMSs, 80% exhibited hypomethylation with age, a finding that is consistent with the published literature. Subsequent integration of these DMSs with the transcriptome data led to the identification of 144 genes that exhibited significant correlation (p-value < 0.001) between gene expression and DNA methylation. Furthermore, the cis-mQTL analysis showed that the DNA methylation of 73 DMSs is influenced by genetic variants.

Comparing the age-related methylation profiles with those from placenta and cord samples (data from WP2; the comparative analysis was performed by WP5) revealed that at least one gene, which showed differential methylation with age, is also involved in early human embryonic development. The two DMSs found in that gene were hypomethylated in long-lived individuals and one of them showed a significant correlation with lower mRNA expression.

WP5 has implemented a computational infrastructure capable of providing bioinformatics analysis as required for EpiHealth WPs. The system comprises a server-based computing and data storage environment within which analysis pipelines for the principal sources of EpiHealth high-throughput profiling data are implemented. The system also includes facilities for secure data transfer and dissemination via web-based portals, and makes full use of academic-derived annotation resources and analysis software.
Combined analyses of data from several WPs revealed correlations between diabetic stage embryo alterations, human ART embryos and KRAB ZF proteins. Furthermore, some correlations in longevity study genes and early ART-related epigenetic alterations have been found. The project formulated some recommendations for future ART practice and data recordings, and the novel results are under publication.

Potential Impact:
The expected impact, a very strong contribution to the understanding of human biological variation across the lifespan in health and disease was the central element of the EpiHealth project. EpiHealth has united research teams focused on early events in development, periconceptional and periimplantational stages with that of longevity gene research from samples of very old individuals. The approach was unique, as the chosen focus, human ART and diabetes/obesity conditions are not only common in the European population, but well defined, and in the case of ART they allow an active intervention, too. There are existing databases, which allow data mining for population and public health information, combined with a well recorded clinical intervention. EpiHealth is focused on nutritional changes and micromanipulation, ROS exposure and genetic reprogramming. The project revealed correlations between diabetic stage embryo alterations, human ART embryos and KRAB ZF proteins. Correlations in longevity study genes and early ART-related epigenetic alterations have been found. The project generated recommendations for future ART practice and data recordings, and the novel results are under publication.
Overall, the EpiHealth project is expected to have a major strategic impact on the field by the following aspects:
Social problem solving: Overweight affects 30-80% of adults in the WHO European Region and up to one third of children. The recent explosion of the worldwide pandemic of metabolic syndrome (MetS) (24 % of US adults), with subsequent development of obesity, type 2 diabetes (T2D) and CVD, compromises progress made in reducing the morbidity and mortality of CVD in recent years. Future applications of the new preventive or correcting measures to reduce adverse effects of early life events on adult health is expected to be the method of choice for modern medicine in order to progress towards a more effective and socially sensitive form of medicine and to prolong the lifespan and the quality of life for elderly people.
Reinforcing competitiveness: The EpiHealth project is integrating multidisciplinary research and is expected to have a positive long-term impact on a broad spectrum of medical research landscape in Europe by generating various animal and cellular model systems for further basic and applied modeling research and applications in a complex medical field of Developmental Origins of Health and Disease. Basic knowledge on epigenetic mechanism and programming, cell pluripotency and differentiation will advance research on understanding gene function and epigenetic effects and help medical teams to develop new therapies. Increasing the output of research and expanding the comparative knowledge of gene regulatory systems will make results more amenable to therapeutic and diagnostic applications for health, and hence to clinical trials and commercial exploitability. Clinical applications of the results in human ART and for diabetic patients would have a direct effect on the health of future generations. The validation of the novel cellular models allows implementation of 3Rs in future research, by replacing animal models with cellular ones. Furthermore, some of the discoveries in EpiHealth revealed novel potential biomarkers of early development and embryo health, which will merit further validation and development towards commercially viable assays. These assays would have profound implications in improving the safety of ART and the health of babies born from diabetic pregnancies. Commercialization of these results would reduce health care cost, create new jobs and would increase competitiveness of the European economy.
Dissemination to the scientific community included almost 100 events, most of them in form of full papes and conference presentations. The project used web and social media based approaches as well, and public dissemination and training activities helped to educate the public and allowed feedback of the society towards the scientific community.

List of Websites:
www.epihealth.biotalentum.eu
Prof Andras Dinnyes. Andras.dinnyes@biotalentum.eu

Attached documents : Furthermore, project logo, diagrams or photographs illustrating and promoting the work of the project (including videos, etc...) as well as the list of all beneficiaries with the corresponding contact names can be submitted, provided the consortium ensures that all necessary authorisations have been obtained and that the publication of the information by the Commission does not infringe any rights of third parties (e.g. commercial interests, including intellectual property, or privacy and the integrity of the individuals, in particular in accordance with Community legislation regarding the protection of personal data).
List of PIs:
Andras Dinnyes/BIOT/: andras.dinnyes@biotalentum.hu
Tom Flemming /USH/: T.P.Fleming@soton.ac.uk
Bernd Fischer /MLU/: bernd.fischer@medizin.uni-halle.de
A.Fergusson- Smith /UCAM/: afsmith@mole.bio.cam.ac.uk
Daniel Brison /UNIMAN/: Daniel.Brison@manchester.ac.uk
Almut Nebel /CAU/: a.nebel@mucosa.de
Giovanna Lazzari /AVANT/: giovannalazzari@avantea.it