At whole body level

Mortality: Accelerated ageing ultimately leads to a shorter lifespan. Animal experiments have shown that diet restriction during pregnancy and shortly after birth shortens the lifespan in the offspring.2 ,18 Small size at birth and at 1 year of age, as indirect measures of a poor start in life, have also been associated with a decreased lifespan.19 We have previously investigated mortality in the Dutch Famine Birth Cohort up to the ages of 50 and 57, but could not demonstrate an effect of prenatal famine exposure on mortality.20 ,21 However, this was possibly due to the relatively young age of the cohort at these times, because recent data suggest that, up to the age of 62 years, women exposed to famine in early gestation had a higher overall adult, cardiovascular, cancer and breast cancer mortality risk.14 In the current proposed study, we aim to study all-cause mortality and cause-specific mortality up to the age of 68 for the whole cohort. Mortality data and causes of death for these people will be retrieved by linking the cohort to Statistics Netherlands.

Age-related diseases: In previous Dutch famine studies, it has been shown that famine exposure during early gestation is associated with increased risk for coronary heart disease, type 2 diabetes, an atherosclerotic lipid profile, altered clotting and breast cancer, all age-associated diseases.10 In the present study, we will assess the presence of other clearly age-associated diseases, including stroke, fractures and cataract. Stroke is a highly prevalent age-related brain disease and a common cause for disability and invalidism in the elderly. Two studies investigating low-birth weight as a proxy for a poor-prenatal environment have shown adverse associations between low-birth weight and the incidence of stroke at older age.22 ,23 Ageing is associated with bone loss and osteoporosis, a disease that leads to an increased risk of fracture particularly affecting older people. The Helsinki Birth Cohort Study has shown that shortness at birth was associated with increased hip fracture risk.24 Visual acuity generally declines with age and several eye diseases, such as cataract, may emerge. Cataract is a cloudy area in a part or the whole of the lens. There is some indirect evidence that a poor start early in life leads to increased symptoms of ageing in the eye. Lower weight at 1 year of age is an indicator of poor early growth. In men and women who were born in Hertfordshire (UK), lower weight at 1 year of age was associated with an increased incidence of cataract at the age of 64–74 years.8 We will study the incidence of stroke, fractures and cataract operations in the entire cohort by linkage to hospital discharge diagnoses requested from the National Medical Registry.

Measurements at organ level

All the measurements described below will be performed in the subsample of 150 participants.

Brain: One of the major brain-associated ageing symptoms is that of cognitive decline. Evidence from animal experiments shows that adverse early-life circumstances affect cognitive and behavioural function in older age. Rats that were undernourished during the perinatal period showed slowness in learning a maze, behavioural rigidity and high emotionality resembling the characteristics of rats at old age.25 Associated with cognitive decline in older age are white matter hyperintensities and cerebral microbleeds in the brain. A very small human study by Hulshoff-Pol et al26 suggested an increased incidence of white matter hyperintensities after prenatal famine exposure. They took 18 schizophrenic patients of whom half had been exposed to the Dutch famine during early gestation and matched them to 18 non-schizophrenic people of whom again half had been exposed to famine during early gestation. Five of the 18 famine-exposed participants showed white matter hyperintensities compared with one in the unexposed group. This finding is highly interesting and requires further investigation in a larger sample. We will measure cognitive decline and function. To measure cognitive decline over a period of about 10 years, we will repeat the cognitive tasks we performed between 2002 and 2004. At that time, we assessed a number of cognitive abilities. Two cognitive tasks, a Stroop-like colour-word conflict task and a mirror drawing task, were performed as part of a psychological stress protocol. Two other tasks, a story repeating task and the Alice Heim 4 test, were applied to measure memory and general intelligence. Data suggested that performance on the Stroop task measuring selective attention was worse in those exposed to famine during early gestation.12 We will run these tests again and measure the difference in performance. Additional tests that will be included are: Trail making A and B (measuring processing speed, attention and mental flexibility) and the 15 Words test (measuring memory). Incidence of white matter hyperintensities and cerebral microbleeds will be examined with standardised MRI of the brain. The total number and volume of white matter signal hyperintensities and total number of microbleeds will be assessed as well as the total cortical volume, hippocampal volume and lacunar volume.

Bone: As reported above, small size at birth was associated with hip fracture risk.24 Frailty may thus have its origins before birth. A questionnaire will be used to determine whether the participant has ever been diagnosed with osteoporosis, whether there is a history of fracture(s) and, if so, at what age the fractures occurred. In addition, we will study the incidence of fractures in the entire cohort by using hospital discharge diagnoses linked to the National Medical Registry.

Muscle: Sarcopenia, the loss of skeletal muscle mass and strength with age, has serious consequences for the quality of life in older people. Several cohort studies have shown that grip strength at a later age is reduced in those who were born small at birth or at 1 year of age. Cohorts included older men and women born in Hertfordshire, UK, and in Finland and middle-aged British men and women.8 ,27 ,28 These results suggest that sarcopenia may have its origins before birth. Further evidence for this notion comes from a study in which it was shown that not only grip strength but also muscle mass was decreased in the elderly with a low-birth weight.29 Physical performance declines with age and loss of muscle mass and strength. A study performed in the UK in older men who were small at birth or at 1 year of age showed an association with poorer standing balance.30 This suggests that a decline in physical performance may be another aspect of the ageing process affected by poor early-life circumstances. As an indicator of muscle strength, grip strength of the dominant hand (maximum of three measurements) will be measured by means of a Jamar handgrip dynamometer. We will also measure physical performance with a number of tests, including a 3 min walk, chair rises and standing balance, as well as with a physical performance questionnaire.

Eye: As has already been referred to above, visual acuity generally declines with age. Besides studying the incidence of cataract operations in the entire cohort, we will measure visual acuity with standard Snellen charts in the subsample.

Measurements at cell level

All the measurements described below will be performed in the subsample of 150 participants.

Telomere length: Telomeres are DNA repeat sequences that cap the ends of chromosomes. Telomeres decrease in length by a predictable amount with each cell division. Recently, it has also been shown that telomere length declines in response to oxidative damage (see below). When telomere length becomes critically short, the cell is no longer able to replicate and enters cellular senescence. Telomere length has been postulated as either a mechanism of or a marker for cellular ageing and longevity regulation. It has been associated with many diseases related to ageing as well as to the lifespan itself.31 Experiments in rats showed that feeding rat mothers a protein-restricted diet leads to reduced telomere length in the kidney, aortic tissue and pancreatic islet tissue in the offspring, possibly explaining the shortened lifespan in these animals.32–34 We will investigate telomere length in DNA extracted from isolated white blood cells.

Oxidative stress: Oxidative stress occurs when there is an imbalance between the production of reactive oxygen species (ROS), a natural by-product of the normal metabolism of oxygen in the body, and the ability of the body to neutralise ROS. Oxidative stress can result in oxidative damage to the cells, tissues or organs and can also cause telomere shortening independently of cell division. Oxidative stress has been implicated in ageing and many age-associated diseases, such as cancer, diabetes, cardiovascular disease and neurodegenerative diseases. It has also been suggested as one of the causes of cellular ageing.35 Studies in the progeny of rats who received a protein-restricted diet in utero showed an increased production of ROS and a decreased oxidative defence resulting in increased oxidative stress.33 ,34 Prepubertal children who were born either small or large for gestational age were shown to have increased oxidative stress quantified by impaired oxidant/antioxidant status.36 We will measure oxidative stress quantified by two oxidative stress markers. As a first marker, urinary F2-IsoP, a biomarker for systemic, whole body oxidative stress over time, will be assessed. As a second marker, a single cell gel electrophoresis assay (comet assay) will be applied. This assay measures DNA damage at the level of a single cell. We will use white blood cells for this.

Chronic basal inflammation: There is increasing evidence that chronic upregulation of pro-inflammatory mediators are implicated in the ageing process and diverse age-related diseases including cardiovascular disease, diabetes and dementia.37 Experimental evidence suggests that this chronic upregulation may be programmed by undernutrition in utero. Desai et al5 showed that a 50% food restriction of the maternal diet led to increased basal inflammation but decreased inflammation responses to inflammatory provoking stimuli in the offspring. Several indices of reduced fetal growth have been associated with increased levels of basal C reactive protein (CRP), a major inflammatory factor, in adulthood.38 ,39 For example, a large population-based study showed that a 1 kg decrease in birthweight was associated with an 11% increase in CRP in adults aged 30–59 years.39 The primary cell effectors of inflammation, leucocytes, have also been shown to be associated with small size at birth.40 In a study of over 5000 people, total leukocyte count at the age of 31 years was higher in those with lower birth weights. We will measure basal inflammation status by assessment of two important indicators of inflammation status: CRP and total white cell count.

Measurements at gene expression level

We aim to investigate gene expression levels of several genes associated with ageing. As this part of the study is planned to be performed at the end of the entire study, we will let the choice of genes to be assessed depend on the discovery of novel genes that will be implicated in the ageing process. Currently, SIRT1 and p16, both implicated in accelerated ageing in mice after protein restriction during fetal life and subsequent overfeeding in postnatal life, are strong candidates.34 ,41