Prematurity and early life programming

Preterm infants are at increased risk of cardiometabolic and neurodevelopmental disorders in later life. The typical postnatal growth pattern of failure to achieve the equivalent of a normal fetal growth rate, followed up by catch-up growth, altered adiposity and altered hypothalamic-pituitary-adrenal axis (HPA) activity may be predisposing factors. Potential mechanisms that may mediate such programmed effects include altered DNA methylation and faster telomere attrition. A prospective cohort of 46 very preterm (25+2 to 31+5 weeks’ gestation, mean 28.6) and 40 full term (38+3 to 42+2 weeks’ gestation, mean 40.2) infants was established to investigate potential mechanisms. Infants were studied at birth, term equivalent age, 3 months and 1 year corrected for prematurity. At all time points, linear growth and body composition (by densitometry) were measured and buccal (epithelial) cells was collected for measurement of DNA methylation (5mC) and relative telomere length. Compared with full term infants, preterm infants were lighter (p < 0.001) and had a smaller head circumference (p < 0.05) at all time-points and were shorter at term equivalent (p < 0.001) and 3 months corrected age (p = 0.002). Preterm infants also had greater percentage body fat at term equivalent age (mean difference = 5.5%, p < 0.001), which normalised by 3 months corrected (mean difference = 0.9%, p = 0.4). Preterm infants had a blunted salivary cortisol response (mean difference 0.4 μg/dL, p = 0.02) to a stressor (physical examination) at 3 months compared to term infants at this age, suggesting altered activity of the HPA axis. 5mC is fundamental in the control of expression of imprinted genes involved in fetal growth. Notably, a number of studies in humans exposed to an adverse environment in early life have demonstrated altered 5mC at the differentially methylated regions (DMRs) controlling the expression of the key fetal growth factor insulin like growth factor 2 (IGF2) and at the linked H19 imprinting control region (H19 ICR). At birth, preterm infants had a significant decrease in 5mC at DMR2 compared with term infants at birth (β = –11.5, p < 0.001) and compared with preterm infants at term equivalent age (mean difference = -7.4, p = 0.01). By term equivalent age, preterm infants had decreased 5mC at both DMR2 (β = –2.8, p = 0.01) and the H19 ICR (β = –2.3, p = 0.048) compared with term infants at birth, although this difference disappeared at 1 year corrected. Although research has suggested that catch up growth may confer an unfavourable metabolic phenotype, poor initial weight gain can associate with worse cognitive outcome. A pathway was established for obtaining advanced magnetic resonance images of the preterm brain. 5mC at H19 ICR and DMR2 in buccal DNA showed no association with measures of white matter microstructure or whole brain volumes. Term infants demonstrated telomere lengthening over the first year of life (mean difference = -0.3, p = 0.02). There was no significant change in telomere length over the first year of life in preterm infants (mean difference = 0.2, p = 0.34). However, as preterm infants at term equivalent age had longer telomeres compared to term infants at birth (β = 0.6, p < 0.001), ultimately there were no differences between the term and the preterm groups at 1 year corrected age (β = 0.3, p = 0.07). The DNA modification 5-hydroxymethylcytosine (5hmC) is a stable modification in its own right and is also thought to be an intermediate step in DNA demethylation. 5hmC is abundant in the placenta but has not been studied in the context of fetal programming. Additionally, previous research using methods such as bisulphite conversion would not have discriminated between 5mC and 5hmC and therefore the role of 5mC may not have been accurately measured. To study the relationship between 5mC, 5hmC and fetal growth, gene expression of candidate imprinted and non-imprinted genes in full term placental samples from the Edinburgh Reproductive Tissue BioBank was analysed. 5mC and 5hmC within the IGF2/H19 and KvDMR (controlling CDKN1C) loci was estimated using chemical capture and immunoprecipitation techniques that discriminate between modifications. Relationships between the expression of IGF2 (r = 0.3, p = 0.02) and CDKN1C (r = -0.3, p = 0.01) and birth weight across the normal range were found and in keeping with the known action of these genes. 5mC at IGF2 DMR0 (β = 0.3, p = 0.02) and KvDMR (β = 0.3, p = 0.02) and 5hmC at H19 gene body (β = 0.2, p = 0.04) associated with birth weight. Thus, DNA modifications at imprinted DMRs may modulate environmental influences on fetal growth across the normal range. DNA methylation at IGF2/H19 can be influenced by early life events. It remains to be seen whether any changes are present later in childhood and whether they associate with risk factors for the metabolic syndrome

Characteristics of salivary telomere length shortening in preterm infants

ObjectiveTo examine the association between gestational age, telomere length (TL) and rate of shortening in newborns.Study designGenomic DNA was isolated from buccal samples of 39 term infants at birth and one year and 32 preterm infants at birth, term-adjusted age (40 weeks post-conception) and age one-year corrected for gestational duration. Telomere length was measured by quantitative real-time PCR. Demographic and clinical data were collected during clinic or research visits and from hospital records. Socioeconomic status was estimated using the deprivation category (DEPCAT) scores derived from the Carstairs score of the subject's postal code.ResultsAt birth, preterm infants had longer telomeres than infants born at term. However, there was no difference in telomere length between preterm infants and term infants at one year of age, implying that the rate of telomere shortening was greater in pre-term than term infants. Interestingly, TL at age 40 weeks post-conception in preterm infants was significantly longer than term infant TL at birth, suggesting that time since conception is not the only factor that affects rate of shortening. Several factors, including sex, fetal growth restriction, maternal age, maternal booking body mass index (BMI), mother education level and DEPCAT score, also differed between the preterm and term groups.ConclusionsPreterm infants have longer telomeres than term infants at birth. In the studied cohort, the rate of telomere shortening was greater in the premature group compared with the term infants. This finding agrees with previous studies using cord blood, suggesting that the longer TL in premature infants detected at birth do not persist and demonstrating that use of saliva DNA is acceptable for studies of telomere dynamics in infants. However, that the TL at age 40 weeks post-conception in preterm is longer than term infants at birth suggests that biological factors other than time since conception also affect rate of shortening

Association between preterm brain injury and exposure to chorioamnionitis during fetal life

Preterm infants are susceptible to inflammation-induced white matter injury but the exposures that lead to this are uncertain. Histologic chorioamnionitis (HCA) reflects intrauterine inflammation, can trigger a fetal inflammatory response, and is closely associated with premature birth. In a cohort of 90 preterm infants with detailed placental histology and neonatal brain magnetic resonance imaging (MRI) data at term equivalent age, we used Tract-based Spatial Statistics (TBSS) to perform voxel-wise statistical comparison of fractional anisotropy (FA) data and computational morphometry analysis to compute the volumes of whole brain, tissue compartments and cerebrospinal fluid, to test the hypothesis that HCA is an independent antenatal risk factor for preterm brain injury. Twenty-six (29%) infants had HCA and this was associated with decreased FA in the genu, cingulum cingulate gyri, centrum semiovale, inferior longitudinal fasciculi, limbs of the internal capsule, external capsule and cerebellum (p < 0.05, corrected), independent of degree of prematurity, bronchopulmonary dysplasia and postnatal sepsis. This suggests that diffuse white matter injury begins in utero for a significant proportion of preterm infants, which focuses attention on the development of methods for detecting fetuses and placentas at risk as a means of reducing preterm brain injury

Placental 5-methylcytosine and 5-hydroxymethylcytosine patterns associate with size at birth

Altered placental function as a consequence of aberrant imprinted gene expression may be one mechanism mediating the association between low birth weight and increased cardiometabolic disease risk. Imprinted gene expression is regulated by epigenetic mechanisms, particularly DNA methylation (5mC) at differentially methylated regions (DMRs). While 5-hydroxymethylcytosine (5hmC) is also present at DMRs, many techniques do not distinguish between 5mC and 5hmC. Using human placental samples, we show that the expression of the imprinted gene CDKN1C associates with birth weight. Using specific techniques to map 5mC and 5hmC at DMRs controlling the expression of CDKN1C and the imprinted gene IGF2, we show that 5mC enrichment at KvDMR and DMR0, and 5hmC enrichment within the H19 gene body, associate positively with birth weight. Importantly, the presence of 5hmC at imprinted DMRs may complicate the interpretation of DNA methylation studies in placenta; future studies should consider using techniques that distinguish between, and permit quantification of, both modifications