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Integrated research on DEvelopmental determinants of Aging and Longevity

Final Report Summary - IDEAL (Integrated research on DEvelopmental determinants of Aging and Longevity)

Executive Summary:
In the IDEAL project we explored between early developmental conditions and late life events and the role the genome plays to mediate this link. The research in IDEAL was organized along seven workpackages (WP’s) conducted by 15 different groups working on humans, animal models and cell systems. Firstly the relation between early life adverse exposures and adult or late life physiological and molecular consequences was investigated for three systems: growth and metabolism, the immune and the reproductive systems (WP1-3) including a study on the plasticity of these systems throughout life and across generations (WP4). Secondly IDEAL focused on genetic control of the ageing process and the loss of epigenetic control during ageing taking normative, accelerated and delayed ageing into account (WP5 and 6). In WP 7 the hypotheses, experimental designs, and the acquired knowledge were put in the context of evolutionary theory by comparing systems and species and by theoretical modeling.
The epidemiological studies in human populations showed that diverse parental conditions (age, fertility and obesity) affect offspring health, and when those offspring become parents, can lead to a vicious transgenerational cycle of health problems with major gender effects. However, early life exposures in the population at large can be counteracted by the responses of our physiological systems. Birth weight discordant twins, for example, did not reveal major discordancies in adult health illustrating that natural variation in early life circumstances does not necessarily generate age-related morbidity. Plasticity of physiological system were observed across various species. Many of the physiological and genomic consequences of a high fat diet during development in mice, for example, could be reversed in middle age by lower fat diet. Also for human elderly, a lifestyle intervention changed metabolic health along with parameters of well-being. On the other hand our studies show how persistent the physiological and epigenetic changes that are introduced by harsh early environments can be. Especially the period around conception appears to be a sensitive window for establishment of epigenetic changes. Also the application of assisted reproductive technology (ART) can have persistent adverse health effects and epigenetic changes. IDEAL has created reference datasets of genes and methylated loci sensitive to such early exposures.
In the mouse studies we have shown that prenatal infection affects the development of the brain and behaviour and even metabolism. Later in life, modified metabolism has physiological consequences and can become transmitted across generations through altered gene expression and DNA methylation status of neurodevelopmental loci. Also physiological and epigenetic consequences of high fat diets during development can be transmitted to subsequent generations. Skeletal development of Xenopus was strongly affected by exposure to stress hormone in combination with thyroid hormone. In humans the late life effects of aberrant thyroid signalling during development was demonstrated by genetic and epigenetic variation affecting early endochondral ossification and the late risk of osteoarthritis. IDEAL research revealed that sex determination in worms and flies during development affects age-related pathology, some of which involve the gut microbiome and create sex specificity in the lifespan extension of dietary restriction. Mouse genes involved in steroidogenesis were shown to affect postnatal uterine maturation and could help to understand certain forms of female subfertility.
Finally IDEAL research revealed multiple interesting longevity loci. Some were associated with age-related physiological phenotypes and some even with birth weight, linking late and early life features of healthy ageing. Age-changes in potential biomarkers were observed for the nuclear genome (the epigenome, transcriptome), the mitochondrial genome, the gut microbiome and the immune system in stem cells, the glycome and the metabolome to associate with morbidity and mortality. This range of potential biomarkers that IDEAL delivers should be further tested in longitudinal studies as classification tools of absolute or individual morbidity and mortality risk and as monitoring tools in intervention studies. IDEAL results, especially those listing phenotypic consequences, methylation labile loci and genes in their expression responsive to early life conditions and ageing, can serve as reference for human and animal studies.

Project Context and Objectives:
Many lines of research including epidemiological studies in humans, observed a relation between the onset of age-related disease and (presumed) developmental conditions. In the human cohorts within IDEAL we have investigated which early life offspring features actually link to health problems later in the life course. We investigated also how paternal conditions (weight, age, reproductive capacity) modulate the health of the offspring and how reproductive history or offspring’s early life features affect the mother’s health. In the past 10 years, after a number of hall-mark papers published by IDEAL scientists before the project started, it became clear that the genome is involved in some of these early and late life links. All cells in an organism carry the same primary DNA sequence, the pattern by which this sequence is expressed (gene transcription), however, depends on the tissue, the life phase, and the environment including the intrinsic (inside the organism, such as ageing) and extrinsic (outside the organism, such as exposure to under-nutrition) environment. The expression of genes in response to such environmental conditions is regulated in the genome of cells and tissues by epigenetic control of the genome, responding to the environment throughout the life course. Epigenetic control is exerted by methylation of the primary DNA sequence, by chemical modification of the histon proteins around which the DNA strands are organized (coiled), and by expression of small and non-coding RNA sequences. The IDEAL project focused on the physiological and phenotypic consequences of a variety of developmental adverse conditions and of the ageing process itself on the regulation of the genome by measuring gene expression and characteristics of the epigenetic regulation (the epigenome).
So, in IDEAL we investigated consequences of early adverse exposures in different life phases, including development, adult life, and health effects late in life. We focused on consequences for growth and metabolism, for the immune system and the reproductive system. We also focused on the plasticity of these systems and the genes involved in the response of these systems. We wondered to what extent is the physiological consequence of an early exposure reversible and which pathways are crucial for such plasticity. Naturally we cannot answer such questions in humans alone since there is only the possibility of observational research and limited access of vital tissues in humans. By exploring different model systems and animal models in combination with human populations our objectives were to provide insight in the following issues:
1. To obtain insight into the role of epigenetic regulation in the response to developmental conditions in animals and its translation to human conditions;
2. To obtain insight into the role of epigenetic regulation in the response to developmental conditions in humans and for some conditions the animal model that resembles such responses;
3. To provide reference libraries of gene expression and epigenetic changes as a function of age and to provide insight into principles of epigenetic regulation across species;
4. To provide monitoring tools of epigenetic effects following developmental conditions;
5. To provide monitoring tools of epigenetic control during ageing and their meaning for physiological functions;
6. To provide pathways determining and promoting human longevity;
7. To provide pathways determining human health and longevity by influencing developmental processes;
8. To provide animal models for testing relevant compounds to modify epigenetic effects early or later in life.
We have created a first set of answers and databases on the nature of the early and late life relation and epigenetic control of the genome. We have also explored animal models and the human data to create a theoretical framework explaining in which evolutionary context plasticity, pleiotropy and developmental mechanisms may have evolved. IDEAL pioneered in the research field linking early and late life history by exploring a large diversity of animal models and systems, human health and disease. The field has since the start of IDEAL taken an enormous flight in the last 5 years. We have provided for this emerging field datasets on the molecular and genome wide consequences of early adverse exposure, the genes responding to such conditions and the phenotypic consequences. The data sets generated in the IDEAL project are put in public repositories and the accession numbers are listed in an NCBI umbrella (PRJNA309315) for BioProjects especially created for the IDEAL project (
IDEAL measured the physiological and phenotypic consequences in adulthood and later life of a diverse range of developmental conditions. These included for example prenatal infections, under- or over- nutrition and intrinsic parental factors (genetic background, age, reproductive capacity). We monitored the effect of these conditions and of the ageing process on the regulation of the genome by measuring gene expression and characteristics of the epigenetic regulation (the epigenome). Research performed in IDEAL was organized along seven workpackages and was performed by the following Principle Investigators and their research groups: Partner 1 (Slagboom/Beekman, Meulenbelt and Heijmans), Partner 2 (Cnattingius and Nyman), Partner 3 (Christiansen and Christensen), Partner 4 (Franceschi, Salvioli and Gargagnani), Partner 5 (Passarino), Partner 6 (Partridge, Gems and Beck), Partner 7 (Uller), Partner 8 (Levi and Sachs), P9 (Duboule), Partner 10 (Paweleck), Partner 11 (Hanson, Burdge and Lillicrop), Partner 12 (Steegenga, Zwaan), Partner 13 (Meyer), Partner 14 (Zwaan) P15 (Service XS) and P16 (Nordic Biosience). Here we will summarize the IDEAL work of these groups along the Work Packages WP1-7 and relate this work to the questions we asked at the beginning of the project (Figure 1).
Figure 1: The concept figure of the IDEAL project

Basically IDEAL research focused on two major phases in life. Firstly research as performed on the relation between early life exposure and adult or late life consequences (WP1-4) and secondly on genetic control of and epigenetic erosion by the ageing process taking normative, accelerated and delayed ageing into account (WP5 and 6). In WP7 the hypotheses, the experimental designs, and the acquired knowledge were put in the context of evolutionary theory.
The main observations will be discussed per workpackage and topic for which IDEAL made relevant observations by studying humans and animal models.
In WP1 we explored the effect of parental and birth factors on offsprings health early and late in life. We also studied exceptional adverse conditions such as malnutrition early in life and the effect on parameters of growth and metabolism and epigenetic modifications. In animal models we mimicked early life stress conditions and investigated effects on setpoints of skeletal development in relation with epigenetic control (gene expression and DNA methylation) especially of thyroid signaling pathways. In human studies we investigated how genes involved in thyroid metabolism play their role in setpoints of skeletal development on one hand and the development of a late life disease such as osteoarthritis on the other. By investigating the HOX gene cluster in mice, vital for skeletal development of all mammals, we deepened our understanding of how epigenetic modification affects the control of gene expression.
In WP2 we investigated the relation between the immune system, development and ageing. We investigated in mice the consequences of prenatal immune challenges for behavior, metabolism and immune-senescence of the brain. We also investigated in mice the role of epigenetic regulation in clonal expansion and contraction of T cells. In humans we investigated the relation between cellular immune markers and ageing, comparing normative, decelerated and accelerated ageing. Special emphasis was directed to the change in gut microbiome (GM) composition in relation to age (in humans) and development (in worms and flies). The latter study included the response of GM on administration of metformin, a type 2 diabetes drug that is being tested in clinical trials for potential beneficial influences on metabolism also in non-diabetics.
In WP3 we focused on sex specific effects of development on lifespan, we investigated what the contribution of different tissues is to sex differences in invertebrate lifespan. We also investigated the control of steroidogenesis during development and ageing.
In WP4 we investigated the plasticity of systems, by changes in expression of genes and physiology, to variable nutritional conditions during development or early adulthood. We studied the effects on regulation of growth, metabolism, reproduction and response to stressors. We also investigated plasticity response genes and the transmission to next generations of the alterations in gene expression, DNA methylation and physiology induced by environmental conditions.
In WP5, having learned from WP1 how control of gene regulation is created during development and whether this regulation is sensitive to developmental conditions, in WP5 we investigated whether or not such control is maintained with ageing under normal, pathologic and postponed ageing conditions.
In WP6 we focused on identifying genetic loci associated with longevity, the relation of such loci with early, mid and late life phenotypes. In addition we investigated potential biomarkers of biological age.
In WP7 we investigated the evolutionary context of IDEAL findings and this workpackage integrates the data through the effective use of bioinformatics and through theoretical modelling.
In WP8 we organized a variety of dissemination activities related to various IDEAL specific and general results and knowledge. The activities were targeted at the scientific communities of basic scientists, of medical scientists and health professionals, to policy makers and/or society as a whole. For more extensive description of these activities see the separate section on dissemination.
In WP9 the daily management was in the hands of a managerial assistant, a scientific director , the coordinators (Slagboom and Zwaan) and a fully functional secured SharePoint for data and document exchange. We had weekly meetings with the management board and regular WP leaders meetings to monitor progress within each of the WPs. The IDEAL website: explains the aims of the IDEAL project, lists the publications, and reports about the IDEAL annual meetings. Contributions of IDEAL participants in popular media can be viewed at the Media page .

Project Results:
A summary of the knowledge created during 5-year IDEAL research.
1. Early development and Late consequences in relation to growth and metabolism (WP1)
1.1 The parental factors, birth weight, ART and offspring’s health.
The Cnattingius group investigated the role of genetic background and environment for pregnancy complications and early life events in human populations. The group performed studies in nation-wide register data in Sweden, which enabled the study of maternal and paternal genetic factors for reproductive outcomes and offspring health. Secondly a database was built of twins with known zygosity born 1926-85, including information twin’s birth characteristics, offspring birth characteristics, health and diseases among twins.
This extensive research revealed the following interesting results. Maternal and paternal genetic factors are of importance for the development of preeclampsia, fetal growth, and placental degenerative disorders (Wikström et al., 2012). The Nyman group in Sweden observed that leukemia in children associates to paternal age (Sergentanis et al., 2015). The intergenerational influence on birth weight the Swedish groups appears to be to a large extent due to genetic factors. High birth weight predisposes to obesity and gestational diabetes: a vicious circle across generations (Cnattingius et al., 2012) . Very preterm born infants are at increased risk of cerebrovascular and ischemic heart diseases in young adulthood (Altman et al., 2012; Ueda P et al. 2014). The group further found that early term birth and moderately fetal growth restriction are independent risk factors for infant mortality. Further studies were performed into the relation of birth size and mothers health characteristics (Hajiebrahimi M et al., 2014). Placental abruption increases mothers subsequent risk of cardiovascular mortality (DeRoo L et al., 2015). The association between reproductive factors and risk of premenopausal breast cancer was studied in depth and appears to be influenced by familial (genetic and early environmental) factors (Hajiebrahimi et al, 2015) and cancer risk was found to be similar in twins and singletons (Chen et al., 2016). Offspring birth weight does not influence mother’s risk of premenopausal breast cancer. Finally the group concluded that in females from opposite-sexed twin pairs, the influence of birth weight on breast cancer risk is minor, and male co-twins birth weight does not influence this risk (Hajiebrahimi et al., 2013).
Another aspect of the offspring’s consequences of parental health parameters is at stake when assisted reproductive technology (ART) has to be applied when parental reproductive capacities are limited. ART is an early life condition potentially affecting early and late life health of offspring and mother. The Nyman group investigated risk factors affecting the outcome of an ART treatment, the growth of the children and epigenetic modifications in ART versus spontaneously conceived children. In the offspring, the Nyman group observed an increased cancer risk among children born after assisted conception. (Iliadou et al., 2014) and that autism and mental retardation are increased among offspring born after in vitro fertilization (Sandin et al., 2013). In addition, the group studied in registers the short and long term effects of infertility, fertility treatments on children’s and couples health. A major and complicated undertaking supported by the IDEAL project was to set up a cohort study of couples undergoing assisted reproduction technology (ART) in Stockholm and Uppsala (the Uppstart study, By studying this cohort and a control cohort, the research group found that for the mother, IVF treatments are associated with higher mammographic breast density, and that a diagnosis of depression/anxiety are associated with lower rates of pregnancy and live births (Fert & Ster , 2016). In addition the group found a significant risk for depression, allergies and constipation among twins at 9 and 12 years of age conceived through ART in comparison to normally conceived twins. However, after multiple testing correction, only the association between IVF and constipation remained. An association was also found with infant mortality (manuscript under preparation). The group found evidence that urinary incontinence in adulthood can have a fetal origin in results collected in a nationwide twin study. Finally the group aimed to link these observations to epigenetic changes that may be observed in ART offspring as compared to controls.
In preparation of the epigenetics studies in Uppstart a review on epigenetics and ART was published (Iliadou AN et al., 2011). The Nyman group in collaboration with the Heijmans group now investigates the epigenetic effects of IVF and its relation with foetal growth and postnatal development in the largest IVF-epigenetic study today. Cord blood was investigated from 81 children conceived by IVF and 75 control children from the “Born-into-life” cohort (collaboration with Dr. Almqvist). Genome-scale DNA methylation data has been generated using the Illumina 450k array. A first genome-wide analysis was perfomed revealing DNA methylation differences associated with IVF treatment. At 49 CpG dinucleotides a mean difference in methylation between IVF children and controls was found (Figure 2) and these sites map to genes involved in among others cellular differentiation, mitosis and neurogenesis. Additional bioinformatics approaches and analyses are currently on-going to establish the link between these differences and health parameters.

Figure 2. Manhattan-plot of IVF epigenome-wide analysis; each dot shows a DNA methylation site that was tested for mean differential methylation between 81 IVF children and 75 controls.
1.2 Epigenetic control during development and early adverse conditions
The relation between early adverse exposure and late effects was investigated under extreme conditions such as the Dutch Hungerwinter (malnutrition, stress) and in twins discordant for birth weight. The Heijmans group investigated the Dutch Famine study to map on a genome-wide scale how the prenatal environment induces persistent epigenetic differences at the level of DNA methylation that may be the molecular explanation for the established effect of prenatal famine exposure to long-term metabolic health. For instance, persons prenatally exposed to the Hunger Winter conditions develop high blood pressure and obesity later in life. By using two independent technology platforms the Heijmans group provided consistent evidence that early gestation is the critically sensitive period in development for epigenetic responses . The analysis highlighted that DNA methylation changes occur near genes involved in development, growth and metabolism, in line with the later-life phenotypic consequences of famine exposure (Tobi et al., 2014, 2015).
The group also systematically catalogued dynamic DNA methylation in fetal development and cell differentiation to identify genomic regions whose regulatory activity is captured by DNA methylation (Slieker et al., 2013, 2015). Comparison of the methylomes of adult and fetal human tissues resulted in a catalogue of DNA methylation marks associated with regulatory activity that drive normal development and highlighted a role of DNA methylation in the modification of enhancer activity and alternative splicing, relevant steps in the control of gene expression.
The Christiansen group investigated birth-weight discordant twins. In contrast to the Barker hypothesis, the extremely birth weight discordant monozygotic twins show no evidence of later life adverse effects of low birth weight on parameters such as glucose metabolism, the pituitary-thyroid axis, bone metabolism, serum lipid levels, or body fat mass (Frost 2013). Genome-wide methylation profiling in adult blood from birth weight discordant twins revealed no differences in DNA methylation of single CpG sites, but an association to quantitative birth weight discordance was found for a region covering two genes reported to be involved in metabolism (Tan et al., 2013; Tan et al., 2014; Chen et al., 2916). Thus, birth-weight, as a proxy for pre-natal conditions, may result in small but persistent epigenetic modifications and based on the lack of effects on health phenotypes these changes are likely to reflect the “ghost of development past”.
Effects often speculated upon from the comparison of same-sex with opposite-sex twins have not been observed; the group found no evidence of a masculinizing effect on female twins with male co-twins (Ahrenfeldt et al., 2015) e.g. there is no impact on academic performance or hormone sensitive cancer risk later in life.

1.3 Early developmental conditions and skeletal development
The Sachs group mimicked abnormal early life conditions in an animal model to define how early life epigenetic set-points are modified by stress-like conditions and what the consequences are for gene expression. There are close and interesting parallels between the Amphibian metamorphosis process and the period shortly before and after birth (perinatal period) in mammals. These two phases are marked by the action of thyroid hormones and glucocorticoids. Induction of these two critical endocrine pathways during the perinatal period was proposed to have many consequences from early life to the elderly. The group investigated the response of Xenopus tropicalis tadpoles treated by thyroid hormones and stressed by glucocorticoid treatment to identify for which genes the expression was affected by these abnormal early life conditions (Grimaldi et al., 2013, Sachs et al., 2015 and Buisine et al., 2015).

Figure 3: Signalling pathway network. KEGG pathways with at least one differentially expressed gene were extracted and connected. On the left, the loose component and on the right, the dense component (network of pathways)
Thyroid hormones and glucocorticoids have tissue-specific effects (epidermis, liver and hind-limb buds were compared) on the regulation of gene expression. Many identified genes have already been shown to be involved in aging and histone or DNA modifications.The group established a bioinformatics tool that constructs a signalling pathway network from the transcriptome data and using this tool they explored at what hubs and bottlenecks the operation of the network could become disrupted (Figure 3). In this way the group linked the effect of stress to the presence of transcripts coding for components of DNA repair machinery in the limb. Thus, due to the high rate of cell proliferation in this organ, the variations of transcript could lead to an increase in the number of errors during replication of DNA, introducing mutations.
Transcriptome analysis also showed transcript variations that could lead to a decrease of DNA methylation and to a decrease of gene transcription through the increase of specific histone modifications (H3K27 tri-methylation). Thus, the group investigated the epigenetic effect of these two treatments by investigating DNA methylation, using the whole genome methylome mapping method, MethylCap-Seq. DNA regions were identified that showed altered DNA methylation, these are called differentially methylation regions (DMR).
The Meulenbelt group investigated in human cells and tissues what the relation is between the thyroid hormone pathway and a frequent age-related disease that affects the skeleton: osteoarthritis (OA). This is a common joint disease among the elderly leading to pain and stiffness of the joints because the aging process affects cartilage and bone in the joint. The group aims to unravel how enhanced thyroid hormone signalling that was observed in mature joint (articular) cartilage with age affects the cells in cartilage. Such cells (chondrocytes) in old joints have a tendency of changing their characteristics into a state that the cells usually exhibit during the development of the skeleton. These so called maturational arrested articular chondrocytes change to the state (morphology) they have in the growth plate and by doing so confer risk to the onset of OA. During development the skeleton is first generated as cartilage and after a process of endochondral ossification is changed into bone. After maturation of the skeleton at the end of its development, genes become silenced in the mature chondrocytes by epigenetic mechanisms. The Meulenbelt group investigated if such epigenetic control changed during ageing. To this end they performed analysis of diseased and healthy cartilage tissues from patients and in addition cell-model based (Figure 4) and mouse-model based research. The result supported the hypothesis that loss of epigenetic silencing of thyroid signaling with age in combination with environmental challenges such as mechanical stresses contribute essentially to the onset of OA. Furthermore, they provided additional support that interfering with intracellular thyroid hormone levels could be a powerful way to oppose the pathological events that are occurring in OA as a result of biomechanical burden. (Bomer et al., 2015; 2016).

Figure 4: Graphical representation of the experimental set-up for the determination of the effect of the epigenetic landscape of cartilage from hBMSCs and hPACs, retrieved from operation material obtained during hip or knee joint replacement surgery and grown in three dimensional cell culturing systems.
The group subsequently investigated whether, in general, epigenetic mechanisms underlie the generally observed loss of maturational arrested state of chondrocytes in articular cartilage with age and OA disease. As such a stepwise integration of transcriptomic and epigenetic data in relation to OA disease states of the mature articular cartilage was applied. The results implied that changes in epigenetic control lead to expression differences of genes but in fact only at a limited number of genes. This occurred specifically at genes involved either in maintaining the chondrocyte phenotype or in adversely pursuing the endochondral ossification lineage. The results indicate that chondrocytes in end-stage disease have lost their ability to epigenetically control expression of genes essential to skeletal development, consequently regaining their growth plate morphology and starting to proliferate, with resulting debilitation of cartilage, a well-described hallmark of OA (denHollander et al., 2015).
In addition to the focus on the role of thyroid hormone on skeletal development and deterioration, the Duboule group investigated in detail how the epigenetic regulation of the Hox genes determined the development of the mouse skeleton. During the IDEAL project they finalized their studies concerning the mechanism of recruitment and erasure of various epigenetic marks during the embryonic development at the HoxD gene locus. This work is currently prepared for for publication (Darbellay et al., 2016).
2. Early development and Late consequences in relation to the immune system (WP2)
2.1 Consequences of immune challenges during early development
The antenatal period is highly sensitive to the damaging effects induced by environmental insults such as infections. Considerable efforts have been made by human epidemiological studies to delineate the role of prenatal infection in neuropsychiatric and neurological disorders with developmental components. Maternal infection during pregnancy has been repeatedly implicated in neurodevelopmental brain disorders such as schizophrenia, autism, and bipolar disorder. Human epidemiology cannot establish causality in this clinically relevant context, however. The overall objective of the Meyer group in IDEAL was to explore the neuronal and behavioural consequences of prenatal immune challenge using mouse models of viral-like immune activation.
Intense prenatal immune activation indeed interfered with normal brain development and causes long-term neuronal and behavioral deficits, some of which show a progressive worsening across aging (Giavanoli et al., 2013 and 2016;Pacheco Lopez et al., 2013; Richetto 2014 and Meyer 2015; Labouesse et al. 2015). Prenatal exposure to immune challenges at low intensities do not induce these pathologies per se, but can still increase the offspring’s more susceptibility to additional environmental stressors such as psychological trauma in puberty leading to an earlier onset of cognitive and behavioural abnormalities. These experiments strongly support a causal role of immune-mediated neurodevelopmental abnormalities in major psychiatric and neurological disorders. Research during the IDEAL time was key for the identification of mechanisms underlying the induction of brain pathology following prenatal exposure to intense immune challenges and further enabled the identification of the synergistic neuropathological effects of low-intensity prenatal immune challenges and exposure to traumatizing experiences during peripubertal development. The latter achievements led to the formulation of a novel hypothesis proposing that the adverse effects induced by prenatal infection reflect an early entry into developmental brain disorders, but the specificity of subsequent disease or symptoms is likely to be influenced by the genetic and environmental context in which the prenatal infectious process occurs.
At the moment the world has great attention for the consequences of Zika infections for pregnant women. The IDEAL coordinator visited Brazil and indicated that in the IDEAL project the Meyer group in collaboration with the Slagboom lab investigated the epigenetic consequence of prenatal infection. The prenatal immune activation caused epigenetic changes in specific genes and locations. The Meyer group firstly investigated DNA methylation changes associated with the prenatal infection. They found increased prefrontal levels of 5-methylated cytosines (5mC) and 5-hydroxymethylated cytosines (5hmC) in the promoter regions of the GAD1 and 2 genes. These genes encode the 67-kDa isoform of the GABA-synthesising enzyme glutamic acid decarboxylase (GAD67) and the 65-kDa GAD isoform (GAD65), respectively. These effects were accompanied by elevated GAD1 and GAD2 promoter 25 binding of methyl CpG-binding protein 2 (MeCP2) and by reduced GAD67 and GAD65 mRNA expression. Moreover, the epigenetic modifications at the GAD1 promoter correlated with prenatal infection-induced impairments in working memory and social interaction. These findings highlight that hypermethylation of GAD1 and GAD2 promoters may be an important molecular mechanism linking prenatal infection to presynaptic GABAergic impairments and associated behavioural and cognitive abnormalities in the offspring.
2.2 The gut microbiome
The Gems group investigated the impact of the gut microbiome on the development and ageing of the nematode Caenorhabditis elegans. It was shown that applying the drug metformin in the food increased the lifespan and ageing rate of the worms by altering the gene expression and physiology of the gut bacterium Escherichia coli with which the nematode was co-cultured. (Cabreiro et al. 2013). Metformin is a drug used in the treatment of type-2-diabetes and has recently been admitted to be tested as a clinical trial of healthy individuals to investigate a more general beneficial metabolic health effect. In fact, the research of the Gems group lead to revealing the first known death cause for C. elegans. The group found that ~40% of C. elegans die relatively early in the wave of ageing-related death from pharyngeal infection which can be prevented by blocking bacterial proliferation, and by reducing pharyngeal pumping rate (e.g. in eat-2 mutants). Mechanical senescence of the pharyngeal cuticle may lead to loss of barrier function, and bacterial infection, gangrenous degeneration of the pharynx, organismal infection and death.
The effect of the gut mircobiota on immmunosenescece was further investigated in human centenarians (representing decelerated ageing), their offspring and Down syndrome patients (representing accelerated ageing). The composition of the gut microbiome was indeed associated with immune-senescence and inflammaging (ageing of the immune system) (Biagi et al.,, 2014).

3. Early development and Late consequences in relation to reproduction (WP3)
3.1 Biological determinants of fertility
The Levi group has focused on elucidating regulatory pathways involved in human reproduction focusing on a specific genes ,Dlx5/6 and Foxl2. These genes are directly implicated in the control of steroidogenesis and are crucial for normal reproduction. In both males and females the aging process is associated with reduced steroidogenesis. Elucidating the molecular mechanisms that control and maintain levels of steroid hormone levels is, therefore, a central issue to understand not only reproduction, but also the aging process. To study the function of these genes in reproductive physiology and on tissue homeostasis we have generated conditionally mutant mice, which have permitted to inactivate Dlx5/6 or Foxl2 in different tissues and in different moments of life. In humans the group examined a large Premature Ovarian Insufficiency (POF)-affected family to study a possible association to the genomic region which includes Dlx5/6 (CabureEt et al. 2012). The genetic (linkage and homozygosity) analysis of a large consanguineous Middle-Eastern POF-affected family presenting an autosomal recessive pattern of inheritance revealed two regions significantly explaining the POF on chromosome 7p21.1-15.3 and 7q21.3-22.2 of which the latter includes the Dlx5 and Dlx6 genes.
In the mouse studies (Bellesort et al., 2015, 2016) the group observed that conditional deletion of Foxl2 in the postnatal uterus resulted in infertility. During postnatal uterine maturation in the absence of Foxl2 they observed a severely reduced thickness of the stroma layer and a hypertrophic, disorganized inner myometrial layer (IML). The clinical relevance of these findings derives from the fact that in humans, thickening of the IML (also called “junctional zone”) is associated with reduced fertility, endometriosis, and adenomyosis. Our data, therefore, reveal a new role for Foxl2 (which is a gene associated to a form of POF known as BPES) in postnatal uterine maturation and could help to understand certain forms of female subfertility. Using a combination of mutant mouse models the group further found the origin of the facial defect associated with Foxl2 mutations and BPES. Postnatal inactivation of Dlx5/6 in the uterus results in sterility without any obvious ovarian involvement. The uteri of Dlx5/6-deleted mice present very few uterine glands and numerous abnormally large and branched invaginations of the uterine lumen. Furthermore, we have shown that DLX5 is highly expressed in human endometrial glandular epithelium and that its expression is affected in endometriosis. The group concluded that Dlx5 and Dlx6 expression determines uterine architecture and adenogenesis and is needed for implantation. We further observed that in endometriosis, changes in epithelial morphology are accompanied by a strong reduction of the expression of DLX5. Remarkably, in human endometriosis the epithelia lose their columnar organisation and present an altered morphology, which resembles closely that observed in conditionally inactivated mice. DLX5 and DLX6 must be regarded as interesting targets for future clinical research on endometriosis. To investigate the link of these genes with late life disease we have generated adult mice in which Dlx5 and Dlx6 were conditionally ablated in bone using osteocalcin-cre. The phenotyping of these mice shows severe osteopenia that increases with age. The RNAseq transcriptome has been generated and analysis is on-going. Combination of these results with lineage experiments should result in unveiling new possible pathways involved in osteoporosis.

3.2 Biological determinants of sex differences in ageing invertebrates (insects and worms)
In developed countries, men die of age-related pathologies on average some 5-6 years earlier than women although women spend on average more years in disability. Moreover, the incidence of many individual age-related diseases is different between men and women. Although for humans these sex differences have not always been present, in animal models sex differences have been consistently observed.
Many differences between male and female originate from differences in developmental processes occurring, particularly in utero and during puberty. Thus, the origins of sex differences in ageing pathology likely originate in early sex determinative developmental processes. One objective of research in WP 3 of IDEAL has been to better understand how sex determination specifies sex differences in ageing and late-life health. This research has focused on tractable model organisms (the nematode C. elegans and the fruit fly Drosophila). Interestingly, some treatments that extend the healthy lifespan of laboratory animals are more effective in females than in males. These include dietary restrictions such as putting animals on a low-calorie diet, or reducing how much protein or sugar they eat. One possibility is that plasticity in ageing is larger in females than in males, which hypothesis may also be tested in humans.

The Gems lab conducted a detailed survey of sex differences in pathology in C. elegans, taking advantage of the optical transparency of this organism to view pathology in situ. It was found that in the female sex, major pathologies developed soon after reproduction, including distal gonad disintegration, uterine tumour formation, intestinal atrophy and steatotic yolk accumulation. Remarkably, none of these pathologies were seen in males. This led to identification of female-specific drivers of pathology, as follows: high levels of apoptosis occurs in the female (but not male) germ line, contributing to atrophy; unfertilized oocytes initiate futile embryogenetic programmes generating tumours (males do not possess oocytes); and conversion of intestinal biomass into yolk causes atrophy and yolk steatosis (males do not synthesized yolk). A genetic trick was then used to make male worms with female guts. This sexual transformation was sufficient to cause gut atrophy and yolk steatosis.
Parallel studies were conducted by the Partridge group in the fruit fly D. melanogaster. Stem cells can help to repair old and damaged tissue because when they divide, they can form a cell that can specialize into one of several mature cell types. Previous studies have shown that stem cell activity in the gut affects how long female flies live for. The Partridge lab looked in detail at the guts of male and female fruit flies as they aged. This revealed that female guts deteriorate as the flies grow old because stem cells in the gut divide too often and form small tumours. These stem cells help young females grow and repair their guts, but start to turn against them as they age. However, as in worms, male guts stay well maintained and do not show the same signs of ageing, they seem to have a better barrier function, even at older ages.
Females fed less food had guts that aged more slowly, suggesting that females live longer on a restricted diet because this improves their gut health. Then, as in worms, male flies were made with female guts. These feminized males had more gut tumours than normal males, but they also experienced a greater increase in lifespan when placed on a restricted diet. This is likely to be because the poorer condition of their gut meant there was more scope for the diet to improve their health. Given the importance of the gut as an immunogenic barrier tissue, this led the group to examine sex differences in the response to immune challenge over ageing, and found that despite their better maintenance of gut integrity, males have high, age-related systemic inflammation and a poor response to infection at older ages, leading to a sepsis-like response, and death.

These results reveal new commonalities in sex differences in ageing, particularly greater pathology in the female intestine, which may echo human sex differences in ageing in current societies. For example, just as worms convert intestinal tissue into yolk, mammals (including women) consume their own bone to provision milk with calcium, a mechanism linked to osteoporosis later in life; moreover, during ageing women, like flies, are more prone to cancer of the intestinal tract. These studies begin to reveal how sex-specific mechanisms of development and reproduction give rise to sex-specific senescent pathologies.

3.3 Evolution of sexually dimorphic longevity in humans
Discussions of sex differences in ageing generated a new theory addressing the following questions: Why do humans live longer than other higher primates? Why do women live longer than men? What is the significance of the menopause? Answers to these questions may be sought by reference to the mechanisms by which human aging might have evolved. Here, an evolutionary hypothesis is presented that could answer all three questions, based on the following suppositions. First, that the evolution of increased human longevity was driven by increased late-life reproduction by men in polygynous primordial societies. Second, that the lack of a corresponding increase in female reproductive lifespan reflects evolutionary constraint on late-life oocyte production. Third, that antagonistic pleiotropy acting on androgen-generated secondary sexual characteristics in men increased reproductive success earlier in life, but shortened lifespan. That the gender gap in aging is attributable to androgens appears more likely given a recent report of exceptional longevity in eunuchs. Yet androgen depletion therapy, now used to treat prostatic hyperplasia, appears to accelerate other aspects of aging (e.g. cardiovascular disease). This suggests that reducing androgen levels during puberty and early adulthood reduces aging rate, but late-life androgen depletion does not.
This identifies a potential therapeutic window for interventions to improve late-life health in men. A major overall aim of this work in the long-run is to inform development of interventions (e.g. dietary, pharmacological) optimized for men or women, to reduce development of senescent pathologies in later life thereby improving late-life health. These interventions could, in principle, include early life interventions (e.g. in diet) and, more broadly, lead to understanding of the origins of senescent pathologies.

4. The phenotypic and molecular plasticity of organisms in response to environmental conditions and its connection to evolution (WP4).
4.1 Induced plasticity in response to Dietary interventions in mice
The Steegenga group explored the phenotypic plasticity of aging mice. For this purpose they created an aging mouse cohort in which the mice were exposed to different dietary interventions ranging from healthy (calorie restriction, CR) to unhealthy (medium-fat, MF) diet. In addition, they applied an intermittent (INT) diet alternating weekly between calorie restriction and the MF diet ad libitum, to explore whether this constant challenge increased healthy aging. To test phenotypic plasticity diet switch groups were created whereby the mice at middle (12 months) or old (24 months) age were transferred from a healthy to an unhealthy diet or vice versa. During the study a panel of physiological parameters were monitored. After sacrifice biochemical, morphological and molecular (transcriptomic and epigenetic) analyses have been carried out. Apart from comprehensive phenotyping of the mice, the group focussed on the diet-induced effects on the liver and colon during aging.
The group made the following observations. At middle age (12M) the beneficial effects of the intermittent diet were found to exceed the effects of a CR diet, but at old age (24M), a CR diet was found to have stronger beneficial effects on survival and to improve liver and colon health better than the INT diet. Weekly intermittent calorie restriction strongly improved the adverse health effects caused by a MF diet. In addition it was observed that transferring mice from an unhealthy to a healthy diet at the age of 12M, the expression of most genes in the liver adopted to the healthy diet. However, a small subset of genes was irreversibly affected by the MF diet including genes playing a role in liver cancer. So it can be concluded that also at old age the phenotypic plasticity still appeared to be high. A CR-MF diet switch in 24M old mice showed that adaptation of the majority of the differentially expressed genes. Changes in DNA methylation in differentially expressed genes were detected that might be responsible for the irreversible changes in gene expression.

4.2 Transmission of induced phenotypic and molecular plasticity to subsequent generations
There is evidence that induced phenotypes such as the ones discussed above, can be passed on across generations. The underlying mechanism however remains unknown. Such trans-generational effects are of relevance to understanding patterns of health and disease and may have implications for optimal health during ageing. Dietary fat is known to influence patterns of cardio-metabolic disease. The purpose of the experiments of the Burdge/Hanson/Lillicrop groups was to determine whether feeding diets with distinct and increased amounts of fat over four generations of female mice modified markers of metabolic function and to investigate the role of epigenetic changes in any metabolic dysregulation. A feeding trial in obesity-prone mice was conducted in which females were fed one of a range of diets with different fat contents (5% to 21% by weight) throughout the life course over four generations (indicated as F1-F4). Subsequently in young adults and ageing mice the (early and late) phenotypic and molecular consequences were measured by markers related to cardio-metabolic health, the expression of the transcriptome and the DNA methylome of tissues relevant to any metabolic changes, and to determine whether any induced epigenetic changes present in tissues of interest were also present in embryos.
The effect of diet and generation on cardio-metabolic phenotype: there was little effect of diet or generation on phenotypic markers in young adults. However, there was a significant increase in whole body, liver and heart weight in ageing mice in the F3 highest fat group compared to the lowest fat group in the F1 and F3 generations. Fasting plasma glucose concentration was significantly increased with ageing in the lowest fat group in the F3 generation and in all generations in the highest fat group. Ex-vivo vasoconstriction was increased in ageing females in the highest fat group in the F3 generation. Conclusions: increasing dietary fat may induce metabolic compensation in young adults that persist over three generations. However, such compensatory effects declined during ageing in a manner contingent on dietary fat and generation.
The effect of diet and generation on hepatic gene expression: to investigate how dietary fat modified phenotypic outcomes in ageing mice, the liver transcriptome was measured in young adult and ageing female mice in F1 and F3 generations. Seven per cent of the measured genes differed between F1 ageing versus young adult mice fed the lower fat (LF) diet, while 0.5% genes differed with ageing in the higher fat (HF) group. About 4% of measured genes differed between diets in F1 young adult mice. Ageing also altered 83% of genes that were also altered by diet in F1 HF young adults. About 3% of measured genes differed between generations in young adult LF mice, while 0.09% differed between generations in the HE group. The pathways altered by diet in F1 HF mice and by generation in the LE group were associated with ageing; in particular up-regulation of cholesterol biosynthesis. Conclusions: these findings show that higher fat consumption altered the regulation of the liver transcriptome towards a pattern in young adults similar to that in ageing mice, but the time course extended over generations contingent on the level of dietary fat.
The effect of diet and generation on hepatic DNA methylation: next the group investigated how dietary fat modified the methylome and how these changes related to changes in transcription and phenotype with respect to diet (high or low fat), age (animals sacrificed at day 90 and 456) or generation (focusing on F1 and F3). DNA methylation was assessed using MeDIP-seq to ensure a wide coverage of the hepatic methylome to identify differentially methylated regions (DMR). Comparison of mice fed the low fat (LF) versus high fat (HF) diet in the F1 generation revealed 35 differentially methylated regions, while in the F3 generation there were 14 DMRs identified between the two dietary groups. The comparison of methylation in F1 compared to F3 found 6 DMRs in the LF dietary group, while in the HF dietary group there were 108 DMRs. Ageing interestingly induced the greatest change in the HF group where there were 288 DMRs compared to 48 DMRs in the LF group. Similar pathways were altered by ageing in both groups. These were related to cholesterol biosynthesis, mitochrondrial dysfunction, and mTOR regulation, but the extent of dysregulation was greater in the HF then the LF group. Conclusions: These findings show that feeding a HF diet significantly alters the pattern of DNA methylation during ageing and although similar pathways were altered in both dietary groups this was exacerbated by the HF diet.
In summary the findings showed that although young mice were able to tolerate a range of different amounts of fat in their diet, this tolerance was lost in adult mice, which showed reduced ability to control the concentration of glucose in blood and to regulate their blood pressure that was exacerbated by consuming a high fat diet. Investigation of the function of genes in their liver, which is central to controlling the amount of glucose in blood, showed that mice fed a high fat diet aged more rapidly than those fed a low fat diet and that mice fed the low fat diet aged progressively over several generations. Similarly, a key process in gene regulation, DNA methylation, was altered across generations and that this effect was greatest in mice fed a high fat diet. However, the methylation changes observed across generations did not directly link to changes in gene expression. An analysis of the blastocysts further indicated that methylation changes in the liver were not directly passed on to subsequent generations but rather during differentiation and maturation of hepatocytes.
Transmission of effects to subsequent generations was also studied in the mouse model of prenatal viral-like immune challenge by the Meyer group. Following the parallel of the vicious cycle with respect to overweight mothers and children studied in mice and men in IDEAL, the parallel question fro the immune challenge in mice would be whether any consequences of prenatal infection in mothers is passed on to next generations. Hence the first generation (F1), second (F2), and third (F3) generation offspring was generated to study possible trans-generational effects of in-utero immune challenges. F2 and F3 offspring developed abnormalities in depression-like behaviour not present in the F1. These effects were transmitted via the paternal lineage. For a number of candidate genes, DNA methylation was measured in the midbrain of F1, sperm of F1 and midbrain of F2 of poly(I:C) exposed animals (mimicking infection) and controls. Indeed methylation changes were found in different generations, affecting different genes in different ways.
4.3 Plasticity in invertebrates, a fast model for evolution.
All organisms with a separation between germ line and the somatic part of the body undergo the process of ageing. Organisms that age faster, not only die faster, but generally also develop faster and reproduce earlier. This mode of life is to some extent genetically determined; flies live shorter than mice and mice live shorter than humans. However, the environment also plays a crucial part. The environment does not only consist of the adult environment, but development and even the maternal and paternal environment can affect the health of offspring. The extent to which lifespan is affected by developmental factors is determined by the adult environment. For instance, low birth weights only relate to much higher chances for late life diseases when during adulthood individuals are obese. The predictive adaptive response (PAR) was proposed to provide a ultimate, evolutionary, explanation for this. It was hypothesised that human foetuses evolved to use information from the womb as a kind of “weather forecast” for adult life. When this forecast is wrong, i.e. when developmental resource availability did not match the adult environment, this will lead to diseases. Previous evolutionary theoretical models suggested that environments in which such a response would evolve should change in a highly predictable manner.
Indeed, some insects live in such predictable environments. For instance, seasonal butterflies live in dry and wet season conditions that always alternatingly follow each other in time. Therefore, the Zwaan group hypothesised that butterflies would be a good model for testing the PAR hypothesis. The group first determined experimentally that larval and adult conditions led to improved lifespan and reproduction if matched, which was corroborated with a model that showed that this could evolve under the field conditions for this species (Van den Heuvel et al., 2013). However, adult resource availability of, for instance, fruit-flies can be poorly predicted by larvae as resources have a high turnover rate in natural condition. Indeed as predicted, larval and adult nutrition did not lead to improved traits (as in the butterflies) when conditions were matched (May et al., 2016 in prep).
The group further investigated the effect of dietary restriction (DR) using a similar theoretical model (Van den Heuvel et al., 2016, in prep) which predicted that flies would have to be able to adjust their reproductive output quickly upon nutrition variation which was verified experimentally (Van den Heuvel et al., 2014). Interestingly, similar to the PAR experiment with fruitflies, an effect was found of early life conditions on late life traits. The group concludes that early life events can alter life histories of organisms in a long-lasting way. Furthermore, gene expression studies on the flies under different larval and adult conditions indicated that variation in body proportion (allometry) is likely to be involved in the life-long signature of larval nutritional conditions of adult gene expression. Moreover, despite the fact that the expression of many genes was affected by larval and adult conditions, variation for only a few genes could be related to the observed adult phenotypes for lifespan and reproduction (May et al., 2016, in prep.). The ability to conclude this is a consequence of including three diets in our design. Had we included only two in a classical case-control analysis we would have reached contrasting conclusions on the relationship between gene expression and lifespan for these ten clusters. For example if we used poor and control only, it would have been logical to conclude infer that the genes up and down-regulated in poor-raised flies relative to control contribute linearly to the phenotypic differences observed between the two, however, had we compared control to rich, we would have concluded that the relationship between expression levels and phenotypic values was in the opposite direction. The risk of such misguided interpretation in a two-factor design strongly warrants the inclusion of three or more environments, especially when establishing the causality between the life history (lifespan, ageing) phenotype and some measure of molecular and/or genetic variation is difficult, strenuous or even impossible. In any case, the work is one of the first, if not the first, to partition the variation in gene expression to developmental and adult diets, and their interactive effects with age. Moreover, the effects of “developmental exposures” on epigenetic and/or gene expression marks are often measured in late-life and lack (for understandable reasons) a longitudinal component. We showed that long-lasting effects on gene-expression can be found, but that linking them “as is” to observed phenotypes is likely to lead to spurious conclusions. The potential that many of the observed differences in molecular (epi) genetic and even physiological measures may merely reflect the “ghost of development past” should serve much more as null-hypothesis than it does in the current literature.
Gene expression levels of different DR studies were compared and found strikingly different results (Zandveld et al., 2016, in prep.). The group hypothesised that this is related to differences in age specific declines in reproduction between flies in different labs, experiment or genotypes. Both these results indicate that observed variation in gene expression and/or molecular epigenetic markers (such as DNA methylation) late in life do not necessary causally relate to observed health, disease and ageing phenotypes such as in the human Dutch Hunger Winter cohort.
Because development and lifespan are connected, it was investigated whether developmental conditions could restrict the evolution of lifespan in an experimental evolution (EE) setup. Therefore late reproducing flies (as these also live longer) were selected under different nutritional larval conditions. Longer lifespan evolved quite similar under different larval conditions, however, the relationship with development time and the effect of larval food was altered (May et al. 2016, in prep.). It was concluded that therefore development does not restrict but does influence how the lifespan phenotypes is shaped and optimised. Additionally flies were evolved on different adult nutritional levels, which optimized their reproductive output on the food they evolved on. Furthermore, development time was altered strengthening our conclusion that development and lifespan are intertwined in fruit-flies, but not necessarily in a restrictive way (Zandveld et al., 2016, in prep.). The group determined genetic variation that was generated in the genome of the flies during 70 and 140 subsequent generations of evolution for the late and early reproducing lines respectively. Highly reproducible and consistent changes in the frequencies of gene variants were that associated with adaptations to developmental nutrition, to selection on age at reproduction, and/or to their interaction. Those variants corresponded to various genes, among which those of the nutrient sensing TOR/INS pathways. This set of highly relevant and well replicated lines is a set of resources that could be developed through the IDEAL project that opens many opportunities for the future to study the role of natural genetic variation (subtle effect variants) and its genetic and environmental interactions in determining lifespan and ageing in the fruit fly. Such knowledge on how evolutionary forces shape the genetic architecture of a complex phenotype such as ageing can help guide and interpret data from for instance human genome wide association studies.
Insulin is a hormone that regulates the processing of nutrition in all eukaryotic organisms. In adult flies there are four active insulin hormones that have similar functions. It was investigated whether lifespan under DR would be altered in flies with mutated inactive insulin hormones. The mutant flies were smaller, lived much longer and reproduced less, while the response to food of these traits remained similar (Zandveld et al., 2016, in prep.). Although insulin levels affect lifespan and reproduction, it is nutrition that dictates the effect of insulin by its levels. In nature genetic effects are much smaller. It was tested whether fish that are genetically different in life history traits also were different in insulin (related) genes. Indeed fish populations that reproduced more generally differed in insulin genes while other genes in the genome did not (Van den Heuvel et al., 2-16, in prep.). Furthermore, we showed that individual fungi of the species Podospora anserina differed in their genetically determined response to glucose. The level of glucose at which the fungus reproduces at a cost of lifespan differ genetically. This genetic variation was associated with the type of substrate they were isolated from (Zandveld et al., 2016, in prep.).
Hence, some theories that were initially based on human populations could be tested in model organisms because of their ease of manipulation and/or because of their relevant ecology and biology. The crucial conditions under which such, often only verbally developed, theories are realistic. In these model organisms the relationships between development, lifespan, genetics and environment can be tested. For instance, using multiple organisms, one can most powerfully test how a PAR depends on the predictability of the environment, by having a positive (the butterflies) as well as a negative control (fruit flies). The latter has a more similar predictability of the environment when compared to humans. While humans are organisms with arguable one of the “slowest” life histories, and flies one of the “fastest”, the relevant ecological parameters determining the predictability of their life histories are very comparable, resulting in similar outcomes with respect to the connection of development and lifespan.
The gender aspects were associated with the sex-specific effects of the environmental and genetic manipulations and measurements. Males and females play different in a species’ life history, such as the much larger reproductive investment of females as compared to males, and the stronger role of sexual selection (male-male competition; female choice) in males. This is expected to affect sex-specific mortality, lifespan and ageing. Indeed ageing research should focus on both sexes.
5. Age-related changes in epigenetic control from cell systems to the whole organism (WP5).
5. 1 Gene expression and DNA methylation in mouse haematopoietic stem cell ageing.
The Beck group observed that ageing of primary haematopoetic stem cells especially included changes in migration and adhesion genes. For performing epigenetics analysis, the group developed sequencing- and array-based platforms for genome-wide DNA methylation (methylome) analysis in 2013 and ‘nanoMeDIP-seq’, a method for methylome analysis using low DNA concentrations (Taiwo et al., 2012) to understand the functional consequences of DNA methylation on phenotypic plasticity of haematopoietic cells during ageing. By using this innovative methodology the DNA methylation profile of the whole genome (methylome) from rare bone marrow cells using very small amounts of DNA became feasible.
To study the role of DNA methylation in somatic stem cell aging, murine haematopoietic stem cells isolated from young, medium-aged and old mice were analysed using the nanoMeDIP-seq (Taiwo et al., 2013). With age, a global loss of DNA methylation of approximately 5%, but an increase in methylation at some CpG islands was observed. Just over 100 significant (FDR <0.2) aging-specific differentially methylated regions were identified in the cells, including polycomb repressive complex-2 target genes Kiss1r, Nav2 and Hsf4 . This means that with ageing the repression of gene expression in specific regions of the genome of stem cells may be altered. Especially the observation that migration and adhesion genes were epigenetically altered may have relevant consequences since an important role of immune cells in blood produced by the stem cells is their fast migration.
For genome-wide DNA methylation analysis of larger samples sizes e.g. as part of epigenome-wide association studies (EWAS), the group also developed an analysis pipeline for analysis of large datasets. This 450k Chip Analysis Methylation Pipeline (ChAMP) (Morris et al., 2013 and 2014) is an integrated analysis pipeline implemented as Bioconductor package in R, offering a choice of the most popular normalisation methods, a novel caller of differentially methylated regions, detecting copy number aberrations and detection of 5-hydroxymethylcystosine (Feber et al. 2014 ; Stewart et al., 2014). In collaboration with other IDEAL partners, ChAMP was used for EWAS on ageing (Steegenga et al. 2014) and genotype-epigenotype interactions(van Dongen et al., 2016). To inspire other research groups to work with the methodology the Beck group organized two 450k DNA methylation workshops in 2012 and 2013 .
5.2 Changes in molecular pathways during ageing. Systems, clocks and variability

The immune system during ageing.
Immunity is compromised in older people, not only making them more susceptible to new infections, but also reducing their ability to defend themselves against previously-encountered pathogens, persistent pathogens and possibly also cancer. To a certain extent, genetic background controls the immune ageing process but there is also a major impact of epigenetics resulting from immune modulation as a result of a lifetime of exposure to pathogens and environmental influences such as pollution and nutrition. The goal of the Pawelec group in IDEAL was to establish immune signatures representing good immunological health in different cohorts of elderly people and to determine the influence of genetic variation and exposure to Cytomegalovirus (CMV) on these. The group found that CMV but not other common persistent herpes viruses is one major driver of the evolution of immune signatures associated with health and mortality in old age (Janssen et al., 2015; Derhovanessian et al., 2015; 2014). Monozygotic and dizygotic twin comparisons indicate a high level of heritability of the impact of CMV, consistent with data also derived from investigations of the Leiden Longevity Study and the offspring of centenarians (Mortensen et al., 2012; Goldeck et al., 2016). Epigenetic manipulation to restore the function of an immune system exhausted by a lifetime´s exposure to pathogens and other influences could be more rapidly deployed. Our results suggest that a major factor contributing to immune ageing is infection with a common herpes virus called Cytomegalovirus (CMV). This virus usually causes no symptoms unless the host is immunosuppressed, but requires constant immune control that occupies a large fraction of the infected person´s immune resources. Treating CMV infections might contribute to rejuvenating the immune system. Moreover, identifying the genes which from our studies on familial longevity, centenarian offspring and identical twins appear to protect against the effects of CMV on the immune system may enable us to replicate the effects of these genes in that majority of the population which does not possess them. The Pawelec and Slagboom groups found long-lived families to have lower gene expression levels of IL7R in their blood as compared to controls (Passtoors et al. 2015) which may be one of the genes involved in healthy ageing.

The gut microbiome and immunosenescence.
The Franceschi group was especially involved in the study of gut microbiota modifications during human aging and its possible relationships with immune-senescence. In this regard, the group was the first to report the composition of gut microbiota (GM) as well as the expression of microbial genes in extreme old age (centenarian persons), highlighting the relative abundance of pathobionts in GM of centenarians and the correlations of these microbial genera with specific blood and urine metabolites particularly abundant in these subjects (Biagi et al., 2010 and 2011; Rampelli et al., 2013). This metabolomic and lipidomic analysis allowed the identification of specific metabolomic signatures of aging and longevity (Collino et al., 2013; Montoliu et al., 2014). A further development of this research line was the analysis of GM in persons with accelerated aging rate, i.e. Down’s Syndrome (DS) patients, where it was hypothesized that an altered GM composition could be among the possible causes of the elevation of inflammatory responses. Surprisingly enough, the GM of these persons does not differ very much from that of non-trisomic controls, except for some subdominant genera (Parasporobacterium, Sutterella). However, the abundance of this last microorganism significantly correlated with the Aberrant Behaviour Checklist (ABC) total score, allowing us to hypothesize a possible role for this microbial genus in behavioural features in DS (Biagi et al., 2014).

Genome wide DNA methylation changes with age : clocks and variability.
The Heijmans group performed large-scale analysed of methylome data to chart the impact of ageing on epigenetic control. An integrated analysis of the methylome and transcriptome of more than 2000 individuals demonstrated widespread age-related increases in epigenetic variability in line with a loss of epigenetic control with age. Crucially, this accrual of epigenetic variability was linked to the expression of genes of well-established relation to the ageing processes such as DNA damage response, senescence and apoptosis. A complementary analysis of public methylation data on seven human tissues, revealed that many of the discrete DNA methylation changes accumulating with age (i.e. which do not increase in variability) are tissue specific but that the type of genomic regions that are affected are shared between tissues (namely polycomb-repressed CpG islands and enhancers). The group found many trans effects of the methylation changes, meaning that the expression of genes was affected at locations other than those where the epigenetic alterations took place. Trans effect are highly relevant in the gene expression regulation of entire pathways.
The Christiansen group analysed epigenome-wide changes in DNA methylation in longitudinal samples from eth same subjects namely elderly twin pairs followed for 10 years. Their analysis shows changes in a number of sites mainly related to cellular signalling events and synaptic transmission. Twin modelling indicates that the majority of changes were explained solely by individuals unique environmental factors indicating that the blood methylome is responsive especially to the unique environmental exposure of individuals.
The Franceschi group was among the first to propose a model to analyse the high-dimensionality data stemming from the Infinium 450k Illumina microarray for the detection of methylation of about half million of CpGs along the human genome (Bacalini et al., 2015), and was able to identify the methylation level of specific CpGs of the ELOVL2 gene as the most powerful and reproducible marker of chronological age (Garagnani et al., 2012). Persuing this research, the group participated to other studies with the group of Steve Horvath on the validation of the model of the “epigenetic clock”. In particular, it resulted that 82 centenarians and their offspring have a methylation age younger (8.6 years) than expected based on their actual chronological age (Horvath et al., 2015). In addition the Franceschi group investigated the Horvath clock in whole blood from 29 individuals with Down Syndrome (DS), their mothers and their unaffected siblings. The data were analysed together with others obtained in three additional datasets from whole blood, brain tissue and buccal epithelium. DS subjects were found to exhibit a highly significant age acceleration effect in three independent datasets of blood and brain tissue but not in buccal epithelium.
In middle age, the Slagboom group, however, did not observe a difference in DNA methylation age as calculated by the Horvath clock, between long living families and controls in a study of 2300 individuals. So the prominent physiological age differences with respect to healthy ageing between these groups on which Slagboom et al., published extensively were not detected by the Horvath clock. Gargagnani and Deelen investigated the relation between these physiological parameters and DNA methylation at the ELOVL2 gene and found no association to any of the parameters relevant to healthy ageing such as lipid, thyroid, glucose metabolism, cognitive decline, and mortality in middle age, familial longevity. The algorithm of multiple loci by which the epigenetic clock was proposed to assess DNA methylation (DNAm) age was found to be associated with increased mortality in old twins as shown by the Christiansen group (Christiansen et al., 2016). In a 10-year follow up of middle-aged twins, however, DNAm age could predict neither cognitive function nor decline. It is known that cognitive and physical decline established by standard functional tests in middle age are informative since the do predict mortality. From the above we conclude that the DNAm age biomarker is either not informative in middle age but only at higher ages or diseases populations, or not informative on biological age aspects other than mortality.
The relation between epigenetic changes with age and specific functional decline was studied in depth by the Meulenbelt and Christiansen group. As described above for WP1, the Meulenbelt group found changes in DNA methylation with age in cartilage that indicated dysdifferentiation to occur at a well-known osteoarthritis locus which associated to the disease indeed when affected and non-affected cartilage was compared. In twin studies, the Christiansen group is currently investigating the relation between micro RNAs and DNA methylation at longevity loci on one hand and phenotypic measures such as cognition and bone parameters on the other.
ServiceXS/GenomeScan has performed Illumina 450K assays for the epigenetic studies of IDEAL partners especially for WP 1 and 5. The SME focused further on innovating its methylation and hydroxymethylation technique. Even lower input amounts and strongly degraded FFPE samples (Peak at ~225 bp) can be reliably measured as a commercial service. They provided data and customer information to IDEAL partners to further develop DNA methylation data Quality Control workflows (MethylAid). These services were used for two IDEAL partners for whom the studies were performed under ISO17025.
6. The genetic basis for longevity and biomarkers of biological age and healthy ageing (WP6).
6. 1 Genetic basis for healthy ageing and longevity
The molecular and genetic basis for ageing and longevity was studied basically in two different ways. First of all genetic association studies were performed including the study of candidate genes and genome wide scans. The basis for such studies is that the frequency of genetic variants either in candidate genes or at loci regularly distributed across the whole genome is compared between long living cases and a younger aged control group. Loci at which the variant frequency differs significantly between the cases and controls are considered associated to longevity. Such loci can be further investigated functionally to identify the relevant gene(s) at that locus that contribute to longevity, or the reverse, to mortality. A second genome wide approach had been performed in the FP7 GEHA (Genetics of Healthy Ageing) study including linkage analysis which is an approach based on family members, since the GEHA study included nonagenarian sibling pairs. This linkage analysis was finalized during the IDEAL project. Several candidate gene association studies were performed and in addition a genome wide analysis of copynumber variation in the genome was studied.
The second approach was to monitor epidemiological data of the normative or healthy aging population in diverse EU countries to identify biomarkers indicative of the biological ageing rate and predictive of morbidity and mortality. The type of biomarkers that were investigated included MT DNA copynumber, transcriptome profiles, glycomics and metabolomics data.
All groups collaborated in the longevity genetics studies. In a genome wide genetic association study (GWAS) the frequency differences of genetic variants in human cases and controls is investigated at over a million of loci across all chromosomes. Such a GWAS was performed previously for the identification of disease loci with great success and in IDEAL applied to identify loci in the genome that may contribute to longevity (the capacity to attain a long lifespan). To this end more than 20,000 elderly cases of ages above 85 years and 60,000 controls younger than 65 years were investigated in a collaboration, led by the Slagboom group, of existing epidemiological studies of IDEAL groups and other EU research groups. This study also included the majority of the nonagenarians collected during the FP7 EU-GEHA (Genetics of Healthy Ageing) project. This joint IDEAL effort revealed significant association with longevity for two loci in the genome: the TOMM40/APOE/APOC1 and the 5q33.3 locus (Deelen et al., 2014). Further analysis revealed that association at the first locus could be explained by genetic variation at the APOE gene, known to contribute to increased mortality risk by one allele APOE4 and decreased risk by another APOE2. We subsequently collaborated with longevity research groups in China and the US and found in that study again that the locus at chromosome-5 associated to the longevity phenotype in addition to the APOE and IL-6 locus (Zeng et al., 2016) which locus had been identified as longevity locus previously by the Franceschi group in Italian centenarians.

The novel locus on chromosome 5 identified by the IDEAL consortium was associated in a meta-analysis of multiple studies to survival beyond 90 years, the rare variant and surrounding sequence at this locus contributing to protection from stroke, cardiovascular events and in middle age high blood pressure. In individuals older than 75 years, however, the association with all-cause mortality is not influenced by blood pressure. This means that at higher ages, the 5q33.3 locus appears to influence longevity through other pathways than those involved in blood pressure regulation. To further investigate the role of the 5q33.3 locus in relation to longevity and survival, the Christiansen group explored the association between the variant and functional capacity, i.e. self-reported diseases and age-related phenotypes, in nearly 1,600 oldest-old Danes (mean age 93.1 years). Preliminary results indicate an association between the minor allele of the variant and a decreased risk of heart attack, a decreased risk of wet lungs (a potential indicator of heart failure), a decreased risk of gout (linked to cardiovascular problems), and an increased ADL strength score which results are currently being investigated in additional cohorts.

The Slagboom group investigated the possibility that a long non-coding RNA (lncRNA) at the longevity locus is involved in the mortality protecting effect but did not succeed as yet to reveal the functional genomic effect of this locus. Associations of the variant with all phenotypes that were available in large GWAS and/or the Leiden Longevity Study were explored. As indicated, the variant has previously been associated with systolic and diastolic blood pressure. In addition it has been associated with infant head circumference, LDL and total cholesterol change under the influence of statins and with neuroticism (P = 0.011) according to GRASP ( Interestingly, the associations with blood pressure seemed to be dependent on BMI (The International Consortium for Blood Pressure Genome-Wide Association Studies et al. 2011 Nature) and age (Simino et al. 2014 AJHG). In addition, we observed a nominal significant association of the SNP with fructosamin and the metabolite GlycA in the Leiden Longevity Study.

An interesting effect is also that genetic variation at the early B-cell Factor 1 (EBF1) gene at 5q33.3 associated with cardiovascular disease risk factors such as central obesity and type 2 diabetes in interaction with psychosocial stress (Singh et al., Eur J Hum Genet (2015) 23, 854-862). For IDEAL we investigated whether genetic variation at 5q33.3 was associated with early developmental parameters. Genetic variation at EBF1 indeed is associated with birth weight (GRASP ( We also found that the genetic variation at 5q33.3 that associated with longevity also associates with methylation state of some sites in blood of which one associates with EBF1 expression in adipose tissue. The chromosome 5q33.3 locus harbours two putative enhancers of which we indeed showed enhancer activity in osteosarcoma cell lines. We have not yet been able to link any of this knowledge to explain the difference between carriers and non-carriers of the longevity associated genetic variant.

We also identified four regions in the genome that harbour longevity loci by performing a linkage analysis thereby finalizing the GEHA study (Beekman et al., 2013). Other genome wide studies in IDEAL included those of the Christiansen group in the oldest-olds indicating that the genome-wide burden of structural copy number variations associates with higher mortality in long-lived individuals

In addition to genome wide studies the IDEAL cohorts were explored for associations of longevity and ageing-associated phenotypes with genetic variants in candidate genes. Various positive associations were found of which the robustness must generally still be tested in other cohorts worldwide. The findings included the insulin signalling, DNA repair and pro/antioxidant pathway genes (Soerensen et al., 2012; Nygaard et al., 2014; Dato et al., 2014), the KOTHO locus (Mengelfrom et al., 2015), the syndecan-4 locus (Rose et al. 2016) and the nitrate oxide synthase genes (Crocco et al., 2013).

In addition to genome wide and candidate based association studies by using single nucleotide polymorphisms (SNPs) as variants, several groups applied next gen sequencing techniques in the genetic longevity studies. The Passarino group performed the complete sequencing of GEHA samples (an EU project of Genetics of Healthy Ageing concerning nonagenarian siblings and younger controls) showing that mutations falling in different regions of mtDNA can interact with each other and influence the susceptibility to the longevity phenotype (Raule et al., 2014). In particular, mutations in subunits of OXPHOS complex I had a beneficial effect on longevity, while the simultaneous presence of mutations in complex I and III was found to be detrimental. The group provided many new data on the correlation between genes involved in energy metabolism and age related phenotypes such as the variability of Uncoupling proteins and Alzheimer Diseases. The Slagboom group performed whole genome sequencing on 220 nonagenarians with a positive family history of longevity and a control group. They could not discriminate germline variants that contribute to longevity but found accumulation of somatic mutations in TET2 and DNMT3A genes. The same mutations have frequently been found in leukemia patients and in published studies of middle age subjects the mutations are associated with mortality. Among octo- and nonagenarians however carriers of these mutations do not have any increased mortality risk (van den Akker et al., 2016). These results may be relevant for the medical diagnostics field. The twin study also supports an increasing prevalence of somatic mosaic copy number variants with age (Nygaard et al., 2016).
6.2 Biomarkers of biological and healthy ageing
First of all IDEAL groups jointly published a paper with other members in FP7 EU project MIMOMICS in which research that may lead to identification of biomarkers was discussed (Deelen et al., 2013). The subsequent steps that may prove a marker indicating any phenotype (in serum, other tissues or bodily functions) to be a biomarker of biological ageing would include a change as function of chronological age, would preferably show differences between long-lived families and normative ageing controls and would have to predict morbidity and/or mortality in prospective studies. This latter characteristics would ultimately have to be predictive of the absolute morbidity or mortality risk of individuals for any biomarker to be used in the clinic. The IDEAL research was focused on creating a range of potential biomarkers that can now be tested for their value in absolute risk assessment in repeated samples of longitudinal studies. At the molecular level IDEAL groups studied gene expression (Passtoors et al., 2013; 2015; van den Akker et al., 2014), DNA methylation of nuclear and mitochondrial genes, nuclear (Nygaard et al., 2015) and mitochondrial gene copy-number, glycosylation, metabolomics profiles, lymphocyte characteristics, and lymphocyte telomere length (Deelen et al., 2014) as potential biomarkers.
The Passarino group monitored biochemical and clinical data on the aging population of Calabria in Southern Italy, revealing many biomarkers of frailty. In particular those regarding the renal function are of particular interest (Berardelli et al., 2013; Montesanto et al., 2014). Of note, they showed that the renal clearance, is a good biomarker of frailty until the age of 85-90 years, but has no value after this age, due to the different metabolism of proteins. They pioneered studies into the involvement of mitochondrial DNA variation in the longevity phenotype. Within IDEAL they aimed to investigate genetic variability in mitochondrial DNA (mtDNA) as driver or biomarkers of aging and to analyse the epigenetic changes involving the nuclear genes involved in mitochondrial function and epigenetic changes at the mtDNA level, which was considered impossible at the beginning of IDEAL. This work led to important new insights currently considered seminal. Mitochondrial DNA variability affects the epigenetic state of nuclear DNA. Also, in contrast to what was maintained by many authors, mitochondrial DNA showed epigenetic modifications some of which were correlated with aging and frailty. Furthermore the group showed in human and mouse tissues differential methylation of the promoters of nuclear genes involved in ribosomal function and mitochondrial biogenesis and function of age (Bellizi et al., 2013; D’aquila et al., 2015). The Slagboom and Christiansen groups showed that mitochondrial DNA copy number declines with age from approximately 50 years and onwards (van Leeuwen et al., 2014; Mengelform et al., 2014). Lower DNA copy number in twins was associated with poorer outcomes in terms of cognitive performance, physical strength, self-rated health, and higher all-cause mortality. Genome wide gene expression patterns in blood of middle-aged twins pointed to an upregulation of several pathways, with an over-representation of particularly immune-related pathways, with both cognitive level and decline. Bioinformatic analysis of the blood transcriptome with chronological age (van den Akker et al. 2014 ) used large published datasets to show that modules of co-expressed genes with age included genes involved in T-cell activation, ribosome activation and a novel module that associated also to mortality risk including the ASF-1 gene that previously associated to familial longevity and mortality (Passtoors 2013).
The Slagboom, Franceschi, Passarino, and Christiansen groups studied the characteristics of longevity families in the past 10 years. In multiple subsequent generations of such families, many members survive to very, and sometimes exceptionally, high ages. What is especially remarkable is that longevity families have an intrinsically healthy metabolism with an average lifestyle and body composition. We found gene expression of the mTOR pathway in particular to be associated with age and familial longevity in these cohorts (Passtoors et al., 2013) This nutrient sensing pathway is known from mutant studies in animal models to be mediating lifespan extension upon calorie restriction. The group was the first to find indications that also in humans the pathway is involved in longevity. The longevity families in which this was observed are not in any way calorie restricted, the association rather points to an intrinsically healthy metabolism. The immune metabolic health of such families and more generally for healthy ageing was demonstrated by the four groups also in collaboration with the Pawelec group using the most up to date analyses in the field of molecular epidemiology. Metabolic health of longevity families was shown by lipidomics profiles (Gonzalez-Covarrubias et al., 2013) , metabolomics (Collino et al., 2013; Montoliu et al., 2014), glycomics (Dall’Olio et al., 2013) and immune profiling. On these topics the groups collaborated with other EU projects such as HIGH-GLYCAN, MIMOMICS and BBMRI

Finally the Christiansen group collaborated with the second SME in the IDEAL consortium Nordic Bioscience on measurements of TAU as a biomarker for cognitive decline in the elderly. In this study the level of tau was measured in plasma from 48 twin subjects (73-89 years of age) using two tau-targeted assays. We further extended the study to include an additional 124 twin subjects (73-95 years of age) to examine the tau level by age and cognitive decline. As for now, no indication of association with age, cognitive level or cognitive decline and tau was found. The variation of mean level of tau, on samples collected under similar conditions, showed large technical variation between the pilot and extended analysis, which could not be explained by demographic factors. Adjusting for the technical variation did not alter the conclusion, that tau level in plasma thusfar did not associate with age or cognition in healthy elder Danish twins.

7. Data integration and mathematical theories to link early development and ageing (WP7).
7.1 Mathematical theories to explain the link between early development and ageing.
The aim of the Uller group was to develop a conceptual framework that can explain why it is that individuals show a responsiveness to early environmental conditions that can manifest itself later in life as ill health or an increased rate of senescence, and the role of epigenetic mechanisms in this process. We further aimed to provide a series of mathematical models that could generate directional predictions to be tested empirically.
The Uller group showed that an information-theoretic perspective on inheritance and developmental plasticity provides a coherent and unified approach to understanding when environmental responsiveness is adaptive (English et al. 2015). Applying this framework we have identified conditions under which the environment experienced early in life (in particular nutrition) will have long-term consequences for lifespan and other important life history characters (English et al. 2016). We have also shown how environmental uncertainty can select for maternal and other transgenerational effects, including incomplete epigenetic resetting (Uller et al. 2015). These predictions have been evaluated empirically (Uller et al. 2013; English & Uller in review). Finally, we have generated novel predictions regarding the underlying processes of long-term effects of maternal nutrition on epigenetic variation in her offspring. Using a combination of mathematical models and genome-wide data from the Dutch Hunger Winter cohort we have demonstrated the value of this perspective, which questions the explanatory validity of the standard evolutionary model within the field of Developmental Origins of Health and Disease research as applied to humans (Tobi et al. in preparation).
7.2 Integration of epigenetic IDEAL results
Information and leads have continually been exchanged throughout the work within IDEAL. With a large amount of data now being generated, the ultimate task of establishing the robustness of major longevity determinants has begun. Multiple partners investigated the relation between DNA methylation and chronological age and to what extent epigenetic changes underlie the biological age together comprising a comprehensive catalogue of genomic regions that display ageing-related changes in regulatory control in humans. Comparison of methylation sets between studies showed that various designs are highly complementary in uncovering additional ageing-related epigenetic changes.

Figure 5. Overlap in CpG sites that display ageing-related changes in DNA methylation across IDEAL studies in humans using the same DNA methylation profiling platform.

In order to integrate datasets across species and tissues, an analysis of genes showing age-related changes at the level of the transcriptome (mouse) or methylome (mouse, human) revealed a significant overlap in the genes involved across mammalian species suggesting that an evolutionary conserved mechanism may be the target of ageing-related loss of epigenetic control.

Figure 6. Overlap between genes (orthologues) displaying age-related changes in epigenetic control or transcriptional activity in mouse and human.

Potential Impact:
1. Summary of the main message
In IDEAL we investigated molecular and physiological consequences of adverse developmental conditions on the health phenotype and how this affects and interacts with the ageing process. It has become clear from the epidemiological studies in human populations that diverse parental conditions such as age, fertility and obesity affect the health of the offspring and when the offspring becomes parents in turn this can potentially lead to a vicious trans-generational cycle of health problems. Although numerous molecular changes are observed following exposure to adverse early developmental conditions which can have health consequences, we believe that in the population at large there is the potential that the effects of early life exposures can be counteracted by the responses of our physiological systems. Birth weight discordant twins, for example, did not reveal major disconcordancies in adult health illustrating that natural variation in early life circumstances does not necessarily link to age-related morbidity. Hence our life course research documented the plasticity of physiological systems despite adverse early environments. This message has the potential to contribute to changing the attitude towards a more active treatment of elderly people. Especially our intervention studies showed plasticity of physiological system across species. For instance, many of the physiological and genomic consequences of a high fat diet during mouse development and early adult life could be reversed in middle age by lower fat diet. Also in fruit-flies the nutritional environment during development had a large impact on gene expression in the first day of adult life, but this effect was largely erased during the rest of adult life and under the influence of nutrition. Also for human elderly, a lifestyle intervention changed metabolic health along with parameters of well-being. Molecular profiles that indicate ill health can be remarkably reversible in adulthood.
On the other hand our studies have definitely shown persistent physiological and epigenetic changes throughout life that are introduced by harsh early-life environments. We found especially the period around conception sensitive to such epigenetic changes, demonstrated for example by the study of individuals perinatally exposed to famine in the uterus during the Dutch Hungerwinter. We also showed that the application of assisted reproductive technology (ART) can have persistent adverse health effects and the analysis of epigenetic changes associated to in vitro fertilization revealed tens of loci sensitive to ART. The link of such epigenetic changes to health effects is not completely clear yet. For instance, although we found a gene expression signature of development in very old flies, only a small fraction of these genes correlated with the lifespan phenotype. Therefore, in addition to the reversibility of the molecular effects of early life exposure, most of these effects in fruit-flies are likely to reflect “the ghost of development past”. Crucially, IDEAL has created reference datasets of genes and methylated loci sensitive to early exposures that may be used by the science community for increasing biological understanding of affected pathways and for future classification of potentially vulnerable groups of individuals that need (preventive) treatment.

The study of animal models revealed the physiological and molecular consequences in adulthood and late life of infections, nutritional conditions, and stress during development. In mice we showed that prenatal infection affects the development of the brain and behaviour and even metabolism later in life. We demonstrated that prenatal infections during development cause neurological disorders, a link that was previously observed by epidemiological studies in humans but could only be proven in animal studies. Such studies support the deep concern for infections by the ZIKA virus for pregnant women, for example. It became clear that in mice the physiological consequences of prenatal infection transmitted across generations and altered the expression of genes relevant for neurodevelopment and DNA methylation status of loci in the vicinity of such genes. We also showed in mice that physiological and epigenetic, and phenotypic consequences of high fat diets during development are transmitted to subsequent generations. It became clear that skeletal development in mice is extremely sensitive to epigenetic regulation of HOX genes and thyroid hormone. Skeletal development of Xenopus was strongly affected by exposure to stress hormone in combination with thyroid hormone. This related to the findings in humans that thyroid signalling altered by genetic and epigenetic variation, affects endochondral ossification during development of the skeleton and the risk of osteoarthritis later in life.
In IDEAL we also focused on gender effects given that many ageing features have a sex-specific pattern. In the human studies we found significant effects on the health of mothers depending on reproduction and developmental features of offspring. Sex determination in worms and flies during development was shown to affect age-related pathology especially involving the gut microbiome in females and the female specific effects in the lifespan extension obtained by dietary restriction. Mouse genes involved in steroidogenesis have been shown to affect postnatal uterine maturation and could help to understand certain forms of female subfertility.

IDEAL research revealed multiple interesting longevity loci of which some were associated with age-related physiological phenotypes in middle age and also some associate with birth weight providing a link between early life features and healthy ageing. Age-changes were observed for the nuclear genome (the epigenome, transcriptome), the mitochondrial genome, the gut microbiome, the glycome and metabolome providing the bases for the development of potential biomarkers that associate with morbidity and mortality. Changes in the human immune system associate with (un)healthy aging. Our mouse studies on this topic indicate that these changes might originate from age-affected migration of stem-cells as the epigenetic status of the genes involved in this progress change with ageing. The range of potential biomarkers we have identified should be tested as indicators of absolute risk of disease and death, as monitoring tools in intervention studies or as classification tools in clinical research. Indeed, IDEAL scientists are leading on-going studies that focus on improvement of the classification of the heterogeneous elderly population and for monitoring the effects of interventions. The IDEAL results serve as a reference for these human and animal studies, especially those listing phenotypic consequences, methylation labile loci and genes whose expression is responsive to early life conditions and ageing.

2. Joint Dissemination Activities
First of all, details on results obtained by IDEAL research have been made available at the IDEAL Web site ( The impact of our IDEAL results has been further enlarged in the extensive number of dissemination activities of all beneficiaries. IDEAL results have been disseminated and discussed in national and international medical and scientific meetings (National meetings in six EU countries, in the US: Cold Spring Harbor Meetings, Gordon Conferences, meetings at the National Institute of Health and National Intsitute of Aging, Ageing meetings in Brasil, Asia et cetera). Collaborations are on-going between many IDEAL labs and groups all over the world which networks were established thanks to the IDEAL project. Our research has been communicated by publications in scientific journals, presentations at scientific meetings/conferences, presentations for the civil society and policy makers, interviews and presentations in national and international media. IDEAL has trained and provided experience to numerous university and high school students and PhD students of which 10 have already finished their dissertation.
The results of the IDEAL consortium so far were not of a nature or robust enough in human populations to directly influence clinical practice. Nevertheless the interest of clinicians to learn about our sensitive genome has been demonstrated in two international conferences we organised in the last year. We aimed to create a platform that would bring IDEAL scientists in contact with clinicians involved in gerontology, geriatrics, obstetrics, gynaecology, paediatrics, and so on. to discuss the translational potential of the IDEAL results. This is quite a challenge as the different background makes it very difficult to attract clinicians to meetings on basic research in ageing. As, among others, the IDEAL coordinator is active in the medical/biomedical field being chair of the Medical Research Profile on Ageing at the Leiden University Medical Centre and by reserving a central position of clinical researchers in the organising committees successful meeting were organised in Leiden. Consequently, our results raised a lot of interest from the clinical professionals and in both dissemination meetings one of the main conclusions was that given the relevance of the current results, integration of the efforts of basic and clinical researchers should be high on the agenda of IDEAL stakeholders.

The first international dissemination ‘Connecting development and late life health’ was held in October 12-14th 2015 in Leiden, The Netherlands. We had renowned Keynote speakers (such as M. Hanson, committee member of the world health organisation), clinical scientists S. Scherjon and C. de Groot and G. Davey Smith) in addition to IDEAL speakers. The conference website ( provides information about the congress, a conference booklet, and a conference report with the presentations. Film fragments of lectures and discussions will also be placed on the website. Flyers, and posters were distributed to announce the meeting. Day Chair and discussion leader of the meeting was a journalist Eveline Brandt (
In total 111 people from 11 countries registered for the congress amongst them were gynecologists, pediatricians, obstetricians, orthopedicians, geriatricians, molecular biologists, twin researchers, evolutionary biologists, and epidemiologists (Figure 7). On average attendants rated the meeting as good to very good.

Figure 7. Distribution of field of expertise (Top panel) and nationality (Bottom panel) among the attendants of the IDEAL dissemination meeting, October 2015.

The second international meeting ‘Translational aging research: challenges and opportunities’ was a Lorentz conference, an expert meeting held in January 2016 and was attended by many IDEAL members. This meeting was aimed to discuss translational aspects of ageing research in general, with a clear focus on IDEAL research. The information produced at the meeting is currently being summarised in a scientific and journalist report (written by journalist Rebecca Miller) that will be placed on the website and will be presented at a third meeting on April 15 2016, the kick-off of the Dutch Society for Research on Ageing (see below; DuSRA or NVVVO in Dutch; position of the coordinator in the Leiden Medical Centre allowed her to attract many disciplines to participate in these discussions with the IDEAL community. The IDEAL network also allowed the coordinator to set up this DuSRA society. At the kick off meeting the coordinator will present the Ageing Research Agenda as emerged from the IDEAL and Lorentz meetings.

Scientific report Lorentz meeting January 6-8, 2016
Aim. The Lorentz workshop brought clinicians, epidemiologists, basic scientists and computational scientists together to focus on the translational potential of knowledge created in the field of ageing research. Although it became clear that research in elderly people to improve care, biomedical research to improve the health span, and basic science to unravel molecular and cellular ageing mechanisms are different scientific communities the aim of the meeting was to find innovative ways to improve communication and to seek common research questions. Moreover, we aimed to describe how the instruments and tools in model systems can be placed at the service of Population and Patient-based studies to deliver true translational research: the end result should be applicable in the clinic, in prevention, diagnosis or treatment.
Outcome. The main conclusion of the workshop was that to improve translational potential of the ageing field clinical researchers and basic scientists should jointly establish pipelines of connected research strategies focused on functional systems such as the musculoskeletal and neuro-cardiovascular system. Crucial for this is that navigators should be trained connecting clinical and basic research within these pipelines. Clinicians and biologists are equally focused on finding drivers and markers of biological ageing. Based on state-of-the-art knowledge, the field needs to prioritize the most informative and functional biomarker sets that indicate physiological ageing in human systems. This is vital to monitor health improvement in response to for instance interventions. At the same time, research in animal models should maximize the comparability of phenotypes and related pathways to match those relevant in human ageing. At the same time, animal-based studies into molecular mechanisms of ageing potentially reveal hubs in vital and conserved regulatory systems and will thus contribute to novel targets for intervention. This is important as drugs are often developed for their effect on surrogate endpoints, but due to the pleiotropic effects of treatment, drugs frequently fail to improve the clinical endpoint or even adversely affect health. The better the molecular networks of ageing humans are understood, assisted by animal research, the better the surrogate endpoints can be chosen.
Improve communication. For The Netherlands the conclusions of the Lorentz workshop will be presented at the kick-off meeting of the Dutch Society for Research on Ageing (DuSRA) that has recently been founded in 2015. Again the focus of that meeting is on how to connect clinical and basic research. All material of the Lorentz meeting will be available for IDEAL partners to disseminate in other EU scientific communities.
Stimulate Healthy Ageing. In humans a decline in function is detectable from the thirties onward, but with the gain of knowledge there are increasing opportunities to intervene. The functional decline is a highly heterogeneous process, not linear, and includes metabolic shifts at different points in the lifetime and stochastic changes that diversify the phenotypes of ageing individuals. To understand the driving forces behind this variation, research is needed from stem cell and genetic research through to the role of lifestyle and nutrition. Clinicians, nutritionists, basic and biomedical scientists together designed pipelines towards translational research in ageing for functional systems (neuro-endocrine, musculoskeletal). This awareness is timely; at the DuSRA kick-off meeting also the scientific director of the National Institutes of Ageing (NIA) USA will indicate how the NIA has made attempts to bring the right parties together for translational research strategies. The Dutch ageing research community is now primed to improve the situation in The Netherlands and to jointly approach policymakers and funding agencies to help setting our targets and work on the pipelines that are under development. This may serve as an opportunity to also internationally increase the attention for ageing research and the translational potential of this field.

The third meeting to come is the DuSRA meeting to be held in Leiden at April 15 2016. Invited to the DuSRA kick-off meeting will be board members of the German and UK sister societies, the scientific director of the US National Institute on Aging, Dutch funding bodies, and IDEAL WP leaders.
The IDEAL dissemination activities of IDEAL Principle Investigators have also reached Asian research communities (with whom the IDEAL genetics groups have started to collaborate) and reached a research community in Sao Paulo of the Albert Einstein hospital, the University of Sao Paulo (USPI) and the Fiocruz Institute in Rio di Janeiro. The Brazilian society is confronted with rapidly growing populations of elderly and with early life exposures such as the ZIKA virus. As a result of het dissemination activities, the coordinator was asked to visit these institutes in September 2015 and March 2016. Representatives of the Albert Einstein Hospital will be present at the DuSRA kick-off meeting. Our IDEAL scientists and WP-leaders have equally presented IDEAL results worldwide.

3. Children’s book, high school students and the general public.
As part of disseminating knowledge to the public, Nyman has written a children’s book on IVF. It is published in both Swedish and English and available at the moment for sale at and Nyman started a Facebook page about the Children’s book on IVF:

Several groups paid special attention yearly to education to high school students to disseminate IDEAL knowledge. This effort has three purposes: a) to raise the interest of young students for the ageing field, given that the ageing population is growing fast, b) to raise the interest of young students for scientific research in general, using ageing as a principle topic on which to be trained in research skills and c) to raise the awareness of young students that healthy ageing starts very early in life (for example by not smoking, physical activity, and healthy nutrition). We highlight a selection of activities here.

The Passarino group organises ‘OpenLab’ for the diffusion of scientific knowledge, ( ) which meets high school students (usually 12 times per year) and spreads the knowledge on the research of the group and other themes. The LifeLab initiative in Southampton welcomed over 3300 students. LifeLab was shortlisted for the BBSRC Innovator Award and the Times Higher Education Award and featured in a range of print, radio and TV media
The Slagboom group constructed a training program for high school students on how to be creative in science using ageing research as basis. This is a ten step program to set up a research project. See: The training program on research into ageing was disseminated at multiple occasions both at the University and in the schools. We made a movie to attract students to this course, and we continue to improve the course and the written syllabus. We developed a film showing the interactions between professor Slagboom and students to match the syllabus and we recently obtained finances from BBMRI, the biobanking consortium to make an official teaching program using also anonymized biobank data in this teaching program.

Within the IDEAL project the Zwaan group developed a “Natural selection practical” for secondary schools. As Richard Dawkins suggests in ‘the selfish gene'(1976) there are parallels between what he terms ´memes´ in culture, and genes in nature. Memes can be seen as cultural analogues to genes in that they self-replicate, mutate and respond to selective pressures. In the workshop designed as learning material for secondary schools, we apply this concept to drawings (of a life form) as drawings can also be replicated (i.e. redrawn), show variation between students (new mutations occur when drawings are redrawn) and can be selected by another student to be redrawn. And so we expect a gradual change of drawings to happen when this process is repeated enough times. These drawings can be made in lessons to explain all the facets that are crucial for evolutionary to and thereby students learn what evolution is and how it works. Above an example is given of how drawings evolved in seven generations where above after 8 drawings were made the individual with the thickest cross was selected as surviving offspring, while below the individual with the thinnest cross was selected. Also, Zwaan was one of the editor and authors of a cahier explaining the ageing process and its impact on current and future societies (( of the “Stichting Biowetenschappen en Maatschappij” (

4. Impact of IDEAL findings, message and dissemination per topic.

4.1 Consequences of developmental conditions; human studies into parental factors.
The impact of the Cnattingius group’s study of Swedish populations has been disseminated in the clinical domain. The results of the research focusing on reproductive factors and mother’s breast cancer risk have been presented to clinicians working with breast cancer patients. The results focusing on prognosis of very preterm infants and of early term infants have been presented to pediatricians, aiming at improving the outcomes of these vulnerable groups. The following relevant conclusions were drawn. a) Presence of pregnancy complications may serve as an indicator of mother’s subsequent risk of cardiovascular diseases; b) Very preterm infants may be followed with respect to future cardiovascular diseases; c) As early terms births are at increased risk of infant mortality, elective early term delivery should be minimized, d) Birth weight is positively associated with obesity in adulthood, e) To stop the vicious circle of obesity across generations, it may be especially important to prevent overweight and obesity in high birth weight infants f) Offspring birth weight should not be regarded as a prognostic factor with respect to risk of premenopausal breast cancer.
The Nyman group studied consequences of parental fertility deficits. In registers short and long term effects of infertility and fertility treatments on children’s’ and couple’s health were studied. The group found that IVF treatments are associated with higher mammographic breast density in the mother, and that a diagnosis of depression/anxiety is associated with lower rates of pregnancy and live births. As for potentially adverse consequences of assisted reproductive technology (ART) treatment, an increased risk for autism, mental retardation and cancer in the ART offspring was found. Results of the Uppstart study will be presented by the group leader at the European Society of Contraception. Nyman will present the results from the Upstart study in a conference May 2016 in Basel and presented at the main dissemination IDEAL congress in Leiden in October 2015. Nyman presented on many occasions about “Exposure to ART, phenotypic and epigenetic effects”. Among others at the EU society of human reproduction and embryology (2014, 2015) and other internationally renowned meetings on reproduction in Sweden, Italy and the US, Nyman and colleagues have organised, held and taught a highly rated course in Reproductive Epidemiology (2014) with amongst others, Prof Allen Wilcox as a guest teacher. Numerous courses were given at undergraduate level on scientific development and courses in epidemiology and biostatistics and genetic epidemiology were given at the graduate level.

Another type of early exposure was investigated by the Heijmans group. They accumulated crucial evidence for the first weeks of pregnancy being a key sensitive period during which the embryo is prone to undergo persistent epigenetic changes with long-term health consequences. This is the primary societal implication of the research in the Dutch Famine study. The findings have been the focus of various lectures to general audiences and policy makers including the UK-based Progress Educational Trust and the European Trade Union Institute. Policy makers are interested in defining sensitive periods in pregnancy (e.g. relevant for the work place) and if epigenetics is a source of biomarkers to monitor adverse exposures before these are expressed as adverse health outcomes.
The Christiansen group showed that late adverse consequences are not generally observed in relation to early life conditions. The group studied identical twins during a long follow up period. They especially investigated identical twins with a highly different (discordant, but still within the normal range) birth weight and found that these did not differ extensively with respect to highly relevant age-related features of late life health and only to a small extent with epigenetic changes. So, birth weight is not necessarily a good indicator of an environment so compromised that late life adverse consequences can be predicted. These results raised interesting discussions in the field that gained attention especially by David Barker in the nineties showing that low birth weight associated with increased risk of cardiovascular disease late in life. Dissemination of the Christiansen group included contributions in subsequent years to a course on aging for a lay audience and meetings of the Danish Pension Fund, in town halls, libraries and schools: Health and wellbeing late in life – when are you too old and : What does is take to live to 100 years? Also Lectures were given at conferences on the increasing, active healthy aging population: What information do we need to help make policies? The group gave Radio, (national) Television and newspaper interviews. An interview was given in “Nature”: Centenarians: Great expectations.

4.2 Consequences of Developmental exposure; animal models
Skeletal development. The research performed by the Sachs group provides molecular determinants and candidate genes to analyse the mechanisms leading to early life adverse epigenetic modifications or DNA mutations that can affect late life. They provide a number of novel genes, which have not been linked to pathologies and represent putative new markers or therapeutic targets. Their further analysis will provide information for the medical community to use the novel leads for diagnosis or target them in the context of new therapies. Every year during the national science festivals in Paris IDEAL presentations on thyroid hormones and amphibian metamorphosis were used to communicate the importance of early life for good health in aging to the general public. The presentation was accessible to the public from 8 years onwards.

Infection and Brain development. The antenatal period is highly sensitive to the damaging effects induced by environmental insults such as infections, and thus considerable efforts have been made by human epidemiological studies to delineate the role of prenatal infection in neuropsychiatric and neurological disorders with developmental components. The Meyer group studied the consequences of inducing prenatal immune challenges on altered brain and behavioral development in animal models. Their results strongly support a causal role of immune-mediated neurodevelopmental abnormalities in major psychiatric and neurological disorders and transmission of these effects and epigenetic consequences across generations. One of the remarkable findings includes the identification of the synergistic neuropathological effects of low-intensity prenatal immune challenges and exposure to traumatizing experiences during peripubertal development. So the adverse effects induced by prenatal infection may reflect an early entry into developmental brain disorders, but the specificity of subsequent disease or symptoms is likely to be influenced by the genetic and environmental context in which the prenatal infectious process occurs. Some of this research provided scientific advice towards the identification (and possible prevention) of the neuropathological consequences in offspring of mothers who were infected with Zika virus during pregnancy. These advise have been made public internationally through media releases (e.g. New York Times:; HEALTHAIM:

Nutrition. For humans the Calorie Restricted (CR) diet is very difficult to adhere to. The study in mice performed by the Steegenga group in which three groups of mice were exposed to either CR, medium fat (MF) or intermittent (IM: weekly alternation of MF and CR) showed that weekly calorie restriction strongly improved healthy aging like continuous CR. Although at old age the effects were not as strong as observed for the CR diet. The possible impact for human healthy ageing could be that an alternating diet may be easier to adhere to and can still be very effective to improve healthy aging. In a human intervention study linked to the IDEAL program it became clear how metabolically beneficial a lifestyle change between 60 and 70 years can indeed be (van de Rest et al., 2016), be it that fasting is not advised at this age. The fact that genes related to hepatocellular carcinoma in the mouse study were found to be irreversible affected by 12M exposure to a MF diet it seems that in humans MRIs of the liver should be made on a regular basis after weight loss of obese individuals. The IDEAL mouse aging study was extensively used for various educational purposes such as for the course “Interventions for Healthy Ageing in Humans and Model Species” to teach students how to conduct a life-long dietary intervention in aging mice and to what the effects of the different diets are; The data were presented during the PhD course: “Epigenesis and Epigenetics”; MSC students to learn to do aging-research during their master thesis.
The Burdge/Lillicrop groups studied the potential vicious cycle of overweight mothers and children mentioned above in more mechanistic detail in female mice. They investigated to what extent fat in the diet (four conditions of increasing fat content) changes the physiology and regulation of the genome across four generations. They observed that at young age a range of different amounts of fat in the diet is tolerated but in adult mice the high fat diet results in increased serum glucose and high blood pressure. Investigation of the expression of the genome, especially the function of genes in their liver, central in controlling serum glucose, showed that mice fed a high fat diet aged more rapidly than those fed a low fat diet and that mice fed the high fat diet aged progressively over several generations. Similarly, a key process in gene regulation, DNA methylation, was altered across generations and that this effect was greatest in mice fed a high fat diet. A number of engagement activities to academics, the general public and policy makers both within and beyond Europe and have been recorded via CORDIS. Burdge provided input to the BBSRC strategic workshop “Crops and human nutrition”. Setting the UK research agenda on food and health in 2016 and two times to the Annual Course on the Impact of Genomics on Public Health Course – Clinicians, healthcare workers and policy makers (NHS Wales), 2014, 2015. Hanson made input to Inputs made to the UN Global Strategy for Women’s, Children’s and Adolescent Health (2015); to the Chief Medical Officer for Britain’s Annual Report (2015); to the WHO Global Report on Ageing (2015); to the FIGO recommendations on Adolescent, Preconception and Maternal Health (2015).

Reproduction. The Levi group investigated reproductive capacities in IDEAL. Their findings might find direct applications for several human conditions including: Premature Ovarian Insufficiency (POF), reduced fertility, endometriosis and osteoporosis. Presentation of results to several medical and scientific meetings were given, collaborations of the group with two clinical departments were initiated (Hôpital de la Pitié-Salpêtrière, Paris, and Ospedale San Raffaele, Genova, Italy) to study the role of DLX5/6 in endometriosis and POF. Three lectures for open public were organized, available online. Conference: La vie de l’embryon: de la fécondation á la naissance. Conference: l’age adulte: le maintien de l’integrite a la naissance. Conference: le vieillissement et la mort: le cycle du vivent; un processus reversible?
Sex determination. Developmental and reproductive processes from early life may extend into adulthood and may determine pathology in later life. Thus, differences in pathology between the sexes may reflect sex differences in developmental and reproductive processes. The Gems group has carefully documented massive sex differences in the overall pattern of age-related pathology in the nematode. Further research into sex determination in Drosophila melanogaster by the Partridge group indicated substantial sex differences in ageing gut pathology driven by stem cell activity. These sex differences underpin sex-biased responses to diet, where gut tumour formation is alleviated by dietary restriction and lifespan is extended, in females and in genetically feminized males. Despite better maintenance of gut barrier function, ageing males have higher systemic inflammation and a poor response to oral infection, compared to females. Canterbury Science Festival (2014) Public lecture, Invited talk at Youth Forum (Imperial College, London, 2014) Lecture to students from around the world, Invited Talk: ‘Immunity and ageing: why is life deadlier for the male?’, UCL Science Centre (London, 2013) Open public lecture. ‘Healthy Ageing’ Exhibition - Thai National Science and Technology Fair (Bangkok, 2012). Invited debate presentation at Darwin’s Birthday Party, Natural History Museum, (London, 2012).

4.3 Plasticity of the living system and evolutionary perspectives
Why should conditions experienced during fetal life affect health in adulthood and maybe even lifespan? Evolutionary biology such as performed by the Zwaan and Uller groups provides a unified perspective on how natural selection shapes individual responses to the environment throughout life. Our research demonstrates that adjusting one’s life trajectory following early exposure to a stressful environment can be adaptive. It can even be adaptive to allow earlier generations to shape development through some form of non-genetic inheritance. There is no doubt that environments experienced early in life can have long-term effects on individual health and ageing. However, our work casts doubt on the explanatory validity of the standard evolutionary model within the field of Developmental Origins of Health and Disease as it is often applied to humans. This can have consequences for when we expect early conditions to have negative effects on health late in life and hence the application of, for example, preventive medical interventions. We show that, in at least some cases, early environmental effects and non-genetic inheritance is expected for reasons that are not part of a mechanism evolved to allow us to adapt to changing conditions. The theoretical modelling in conjuncture with the work on relevant animal models such as the fruit-fly and a polyphenic butterfly clearly indicated that “arm chair” adaptive evolutionary explanations are unlikely to be true and that the default explanation should be that many of the molecular marks of developmental exposures reflect the “ghost of development past” rather than causally underpinning the health and ageing phenotypes.
After the publication of a study where we exposed fruit-flies to sustained variable nutrition conditions we made a movie together with the company “Beeldmixer”, to explain what we have done in the study. Initially this was done to accompany a message on the website of the Wageningen University that this paper was published, but worked very well to communicate to laymen people what we do in the lab. It starts the discussion about what is and can be done in science. Link to movie:

4.4 The ageing process.
Genetics of longevity. Together all the human studies in IDEAL contributed to identification of novel longevity loci by EU and worldwide collaborations. The genes at these loci in the genome may explain the large differences in middle age between healthy ageing and physically disabled people. We have listed which beneficial effects the discovered genomic loci have across the lifespan. Carriers have highly relevant ‘bonus’ features such as low blood pressure, resistance to CMV infections, sensitive to insulin etc. We showed that partly such features can be attained even in middle age by healthy lifestyles. Dissemination of the Slagboom group on heathy ageing, genetics and behaviour included 17 interviews in local and national newspapers and journals and many lectures for lay public and policymakers and Film fragments: KRO “EUREKA Hoe word ik 100” 17 oktober 2013 ( ), NCRV “Altijd wat” – 7 augustus 2012; ( ), RTL4 “Lang Zullen We Leven”- 7 januari 2012.
Potential biomarkers. For monitoring the ageing process we investigated a range of novel technologies and potential biomarkers by which large human populations can be investigated and jointly analysed: a) immune parameters can be measured and the composition of the gut microbiome b) metabolites in blood can be measured as profiles (metabolomics); c) epigenetic studies measuring the DNA methylation profile of the whole genome; d) transcriptome studies measuring all transcribed genes in the genome, e) copy number changes in the genome and in mitochondrial DNA. A point of discussion which was explained to the field by Slagboom and Franceschi groups (Bioassays 2014) is that markers may change as a function of chronological age (as a clock), but informative markers one aims to use in the clinic to predict, classify or through which to monitor development of morbidity and mortality in elderly need to report on biological age. Since that paper IDEAL provided many potential biomarkers by testing biomarkers for the relation with chronological age, with morbidity and/or mortality.
The change in immune parameters as a result of the process of ageing. In the last years there has been a true explosion in the literature of the use of the term “inflammaging” which was originally proposed by Claudio Franceschi, also workpackage leader of WP2. Inflammaging stands for the age-associated increase in the circulating levels of pro-inflammatory compounds, indicating a state of chronic, sterile inflammation that characterises old age. Inflammaging is thought to be a perfect example of the consequences of antagonistic pleiotropy, meaning that inflammation is good at young age and turns to be detrimental in old age. Within the course of IDEAL we further added to this knowledge, by investigating for the first time the role of the gut microbiome (GM) in inflammaging and immunosenescence more in general. The role of GM is indeed emerging as more important than previously thought in modulating a series of physiological responses, including inflammation. Our studies pave the way for future interventions on the maintenance of GM composition in old and very old age as a possible strategy to attain longevity and avoid or postpone a number of diseases with inflammatory pathogenesis, including colorectal cancer, and neurodegenerative conditions such as Parkinson’ Disease.
These insights have been presented to several international medical and scientific meetings (Cold Spring Harbor Meetings, Gordon Conferences, NIH and NIA meetings in the US, etc.) and collaborations are ongoing between the Franceschi lab and many groups all over the world many of which have been established thanks to the IDEAL network. The Franceschi group members participated to many dissemination initiatives to the general audience, including Italian broadcast television RAI3 (TV show “Elisir” on health, medicine and wellbeing), EXPO 2015 Milan ( ), and Italian newspapers such as Repubblica and Il Corriere della Sera.
Inflammaging features and also the relevance for CMV infection for ageing was studied by the Pawelec group. A presentation was given by Pawelec to start-up industry at the “BioRheinNeckar cluster conference, September 2013, advising on immunosenescence (BioRN is one of the major biomedical clusters in Europe and together with partners in Cambridge/UK and Leuven/Belgium part of the Health Axis Europe Alliance see also
Pawelec participated in the Italia Longeva meeting in Rome, November, 2014, on the occasion of the Italian EU Presidency, to advise on European ageing research and he moderated a roundtable on the occasion of the Biology of Aging conference in Singapore, October 2015, to advise the Singapore government on ageing research.
The change in the epigenome as a result of the process of ageing. Another large source of dissemination focused on the study of epigenetic (mainly DNA methylation) changes with ageing. Here a major discussion is ongoing about which epigenetic changes are informative of the biology of ageing. The Franceschi group showed that the change in some epigenetic marks associate with chronological age (i.e. the level of methylation of ELOVL2 gene). Franceschi and Slagboom showed that this clock locus, though associated linearly with chronological age from the moment of birth in all tissues, did not associate with any tested health markers in a two generational human study. Prof Steve Horvath developed a DNA methylation clock that predicts the chronological age of individuals and associates with mortality. During the IDEAL annual meeting in 2014, Horvath explained his mathematical algorithm to the IDEAL community in a course. Heijmans and Slagboom showed the relevance for ageing especially of loci that increase divergence from the chronological ageing clock affecting genome loci fundamental for the ageing processes. This renders the methylome as highly promising starting point to translate ageing mechanisms identified in model organisms to humans. The Meulenbelt group showed that genetic variation and epigenetic modification of genes in the thyroid pathway contribute to the risk of osteoarthritis (OA) by affecting the mature arrested state of chondrocytes in OA cartilage. The Beck group showed that genetic variation and mouse bone marrow stem cells undergoe epigenetic changes with age in gene involved in migration and adherence which may be highly relevant given the function of immune cells in blood. The Beck group organised 450k workshops in 2012 and 2013.

The change in the mitochondrial genome and function as a result of the process of ageing. Research of the Passarino group into the relevance of mitochondrial information for ageing and biomarkers in the study of centenarians has attracted a lot of attention in Southern Italy. The conclusions were that universal standards in the clinical approaches to treatment are not suitable for the highly aged. The mitochondrion involved in the production of energy, has a key role in ageing and the group found new aspects of their biology (such as the epigenetic of mitochondrial DNA). The socio economic impact of this research on the southern Italian society may be of great interest. At the professional level many contacts were established with local geriatricians, who have refined their approach to their patients, based on IDEAL results and are willing to interact with the local government in order to better and systematically approach the aging patients. In this region the aging population is growing even faster than elsewhere, and where the aging population has special features (such as the prevalence of males) which need to be addressed specifically. The group is studying spin off potential towards a personalized medicine. The Passarino lab and research has been featured by National Geographic (May 2013), translated in numerous languages. The group promotes healthier policy and healthier lifestyles. In particular, in the meeting organized in Milan in the frame of the World Expo ( ), dedicated to Nutrition on the 29th September 2015 on new approaches to diets. Among several interviews one was with RAI3, the national TV on longevity and healthy lifestyle

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