Telomere Length in Small for Gestational Age Babies

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Open Access

Peer-reviewed

Research Article

Preterm infants have significantly longer telomeres than their term built-in counterparts

• Vimal Vasu,
• Kara J. Turner,
• Shermi George,
• John Greenall,
• Predrag Slijepcevic,
• Darren K. Griffin

ten

• Published: June 28, 2017
• https://doi.org/10.1371/journal.pone.0180082

Figures

Abstract

At that place are well-established morbidities associated with preterm nativity including respiratory, neurocognitive and developmental disorders. Even so several others accept recently emerged that characterise an 'aged' phenotype in the preterm infant by term-equivalent age. These include hypertension, insulin resistance and altered body fat distribution. Evidence shows that these morbidities persist into adult life, posing a pregnant public health concern. In this study, we measured relative telomere length in leukocytes as an indicator of biological ageing in 25 preterm infants at term equivalent age. Comparing our measurements with those from 22 preterm infants sampled at birth and from 31 term-born infants, we tested the hypothesis that by term equivalent historic period, preterm infants accept significantly shorter telomeres (thus suggesting that they are prematurely aged). Our results demonstrate that relative telomere length is highly variable in newborn infants and is significantly negatively correlated with gestational age and birth weight in preterm infants. Further, longitudinal assessment in preterm infants who had telomere length measurements available at both birth and term age (n = 5) suggests that telomere attrition rate is negatively correlated with increasing gestational historic period. Contrary to our initial hypothesis however, relative telomere length was significantly shortest in the term built-in control group compared to both preterm groups and longest in the preterm at birth grouping. In addition, telomere lengths were not significantly dissimilar between preterm infants sampled at birth and those sampled at term equivalent historic period. These results indicate that other, as yet undetermined, factors may influence telomere length in the preterm born infant and raise the intriguing hypothesis that equally preterm gestation declines, telomere compunction rate increases.

Citation: Vasu V, Turner KJ, George S, Greenall J, Slijepcevic P, Griffin DK (2017) Preterm infants accept significantly longer telomeres than their term born counterparts. PLoS I 12(6): e0180082. https://doi.org/10.1371/periodical.pone.0180082

Editor: Gabriele Saretzki, University of Newcastle, UNITED KINGDOM

Received: March 21, 2016; Accepted: June 9, 2017; Published: June 28, 2017

Copyright: © 2017 Vasu et al. This is an open access article distributed nether the terms of the Creative Commons Attribution License, which permits unrestricted apply, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: All relevant data are within the paper and its Supporting Information files.

Funding: This work was supported from the Medical Research Council (GB) (case studentship sponsored by Digital Scientific UK) and via an Due east Kent Hospitals University NHS Foundation Trust internal project grant scheme award is acknowledged. The funders had no role in study design, data collection and analysis, conclusion to publish, or preparation of the manuscript.

Competing interests: The authors take declared that no competing interests exist.

Introduction

Preterm nativity, defined by the World Health Organisation every bit birth at less than 37 weeks completed gestation, is estimated to business relationship for 14.9 million or eleven.1% of all births worldwide. Although survival rates are improving, contempo data suggests that preterm birth is a risk factor in half of all neonatal deaths and that 1 million deaths each year are a direct result of complications associated with preterm birth [1, 2]. Furthermore, immediate and long term morbidity rates in those that survive have remained high and largely correlate with the degree of prematurity [ane, 3–6].

Past term age, preterm babies display a phenotype that is different from that of the term built-in infant. Specifically, these babies have altered adipose tissue partitioning [7, eight], ectopic fat deposition as intrahepatocellular lipid [9], hypertension [ten–12] and insulin resistance [xiii, 14]. Moreover, there is preliminary prove indicating that many of these morbidities persist into early adult life and as such, may represent a meaning public health outcome [15–18]. As the morbidities described are associated with ageing in adults, the observed phenotype of the preterm infant may exist indicative of premature ageing. Attenuation or early recognition of this phenotype would therefore be desirable to reduce morbidity and to appropriately manage long-term health in these individuals.

Telomeres are repetitive DNA sequences at the end of chromosomes that become shorter with each prison cell cycle [19, 20]. When telomeres have shortened beyond a disquisitional length, a Dna damage response is initiated, committing the jail cell to apoptosis or senescence [21, 22]. It is widely believed that an accumulation of senescent cells within a population leads to a loss of tissue office and ultimately organismal ageing [23]. Indeed, available data indicate a shut negative correlation between chronological age and telomere length [24–28]. Furthermore, evidence supports a link betwixt shortened telomeres and age-related morbidities in adults, many of which characterise the preterm infant phenotype described higher up [29–33].

Despite contempo academic focus on telomere biology in newborns [34–49], niggling is known about telomere length and regulation in preterm infants. In summary the evidence to date demonstrates a reduction in telomere length with advancing gestational historic period, especially in those built-in at less than 32 weeks completed gestation [l, 51]. This issue appears to be specific to life ex utero, since age matched foetuses exercise not conform to this tendency [52]. Furthermore, other physiological events during labour, such equally condition of membrane rupture has as well been shown to be relevant to telomere length in preterm infants [50, 53].

The ex utero environment of the neonatal intensive intendance unit differs fundamentally from both the in utero environment and the postnatal surroundings experienced by the term infant. For example, exposure of preterm infants to elevated levels of oxygen may potentially pb to increased reactive oxygen species and induce Dna impairment (to which the K-rich telomere sequence is particularly susceptible) [54, 55]. In addition, altered nutrition, slumber cycles and general routine intendance procedures may induce increased levels of stress. In adults, similar stresses are associated with telomere attrition [56–59]. Therefore these and other ex utero factors might act to modulate telomere length in the preterm baby, resulting in a phenotype reminiscent of premature ageing.

Therefore, the aim of this study was to deport a prospective observational report to compare telomere lengths of preterm infants sampled at birth and at term equivalent age with that of term infants. Although a minor number of studies take compared telomere lengths in preterm infants with that of those born at term, none have assessed telomere length in preterm infants at term equivalent age (i.e. 37–42 weeks). Given that the evidence available demonstrate a reduction in telomere length with advancing gestational maturity in preterm infants and that the ex utero environment may exist relevant, we sought to exam the hypothesis that by term equivalent age, telomere length is shortened in preterm infants in comparison to term born infants.

Materials and methods

With institutional research ethics commission approval (10/H1109/51) and informed parental consent we conducted a prospective observational report over a 4 year period (June 2011 to June 2015). A total of 47 preterm infants with a nascency gestational historic period of < 32 completed weeks gestation were recruited from the level 3 (regional) neonatal unit at the William Harvey Hospital in Ashford, Kent and the level 1 neonatal unit of measurement at the Queen Elizabeth the Queen Female parent Hospital in Margate, Kent. Nosotros chose to include preterm infants born < 32 weeks completed gestation in light of previous data, which showed a rapid and meaning decline in telomere length with advancing gestational historic period in infants born at < 32 weeks [51]. In improver, this gestational age cut off is oftentimes utilised in other studies of preterm nativity and allows for recruitment of a reasonable sample size inside a reasonable fourth dimension period. Of the 47 preterm infants sampled, 22 were sampled within 48 hours post-obit birth and 25 were sampled at term equivalent age. In addition, a full of 31 term born infants who required blood sampling in the first 48 hours following birth for reasons such as evaluation of neonatal jaundice or suspected sepsis were recruited from the postnatal wards at these hospitals equally a pragmatic comparator cohort. Babies were excluded from the written report if they had an antenatal or postnatal diagnosis of a severe built malformation or were unlikely to survive. Once recruited, several demographic factors were collected from each participant. These included baby gestational age at birth, babe weight at birth and maternal age in all three groups.

Telomere length measurement

1ml of additional blood was collected by venepuncture in a paediatric lithium heparin canteen taken at the time of routine blood sampling in report recruits. In preterm infants, we aimed to collect a sample within 48 hours of life or at term equivalent historic period (between 37–42 weeks post menstrual historic period). Term born infants underwent blood sampling at the fourth dimension of routine blood sampling within the first 48 hours of life. Each blood sample was assigned a 2 digit numerical code and frozen at -eighty^°C. Samples were nerveless by a member of the research squad who was blinded to the group allocation of the blood sample and transported to the Schoolhouse of Biosciences, University of Kent under dry out ice. Samples were thawed and equilibrated to room temperature prior to genomic DNA extraction using a DNA isolation from mammalian claret kit (Roche). Isolated Dna was dissolved in Tris-EDTA pH8.0 and assessed for DNA concentration and purity using a Nanodrop spectrophotometer. All samples were stored at -xx^°C until telomere length analysis by a single observer, using multiplex quantitative real time polymerase chain reaction (qRT-PCR). Primer design for telomere and a unmarried copy reference gene (haemoglobin B) amplification was as previously described by Cawthon et al 2009 [sixty]. Simultaneous amplification of the telomere and single copy gene was achieved in a total reaction book of 25μl using SensiMix^TM SYBR No-ROX Kit (Bioline), 50nM each primer and 25ng of standard or unknown sample DNA. The reaction was carried out using a Rotor-gene Q 2 plex HRM platform under the following cycling conditions: 95^°C for 10 mins, ii cycles of 94^°C for 15 secs and 49^°C for 15 secs, 37 cycles 94^°C for 15 secs, 62^°C for x secs, 74^°C for 15 secs, 84^°C for ten secs and 88^°C for 10 secs. Each unknown and standard DNA sample was assayed in triplicate. Relative telomere length expressed as telomere to single re-create gene ratio (T/Southward ratio) was calculated using a standard comparative method (delta delta Ct) [61] (Equation one). Intra- and inter-assay variations were 1.05% and 0.41% respectively.

Equation i. Comparative method for the calculation of T/S ratio.

'Sample telCt' refers to the unknown sample telomere sequence amplification bicycle threshold, 'Sample scgCt' refers to the unknown sample single copy reference gene amplification cycle threshold, 'reference telCt' refers to the reference DNA telomere amplification cycle threshold and 'reference scgCt' is the reference Deoxyribonucleic acid single copy gene amplification bike threshold.

Statistical analysis

Data were tested for normality using the Shapiro-Wilk test and analysed using SPSS version 21 (IBM Corp. Released 2012. IBM SPSS Statistics for Windows, Version 21.0. Armonk, NY: IBM Corp). The primary effect, T/S, ratio was normally distributed and therefore parametric methods (univariate ANOVA) were used for analysis. Cohen's d was used to determine event size (small-scale, medium, or big) for mean T/Southward ratio between the groups.

Results

During the study period we obtained blood samples for telomere length analysis in 31 term built-in infants and 47 preterm infants (22 sampled within 48 hours of preterm nascence and 25 sampled at term equivalent historic period). The characteristics of recruited term and preterm infants recorded at the fourth dimension of sampling are displayed in Table 1. Within the term cohort, no infant was identified as having either blood culture positive sepsis or severe neonatal jaundice requiring neonatal exchange claret transfusion. In addition, the characteristics recorded at birth of preterm infants sampled at term equivalent age are shown in Table ii. As would exist expected, birth gestation and birth weight of preterm infants (both preterm infants sampled at birth and those sampled at term) were significantly different (p = <0.01) when compared to term infants (Tabular array i). Birth gestation and nascency weight in preterm infants sampled at birth (Tabular array 1) and preterm infants sampled at term equivalent age (Table 2) were not significantly different (p = 0.28 and 0.18 respectively). There were no meaning differences between the groups with respect to maternal age (Table 1). In each of the 3 cohorts, there were a greater number of male person infants than female infants, notwithstanding the proportion of each sex was like in each group (Fig 1.) The Preterm infants sampled at term age demonstrated satisfactory postnatal growth in comparing to published Uk data [62].

Table ane. Baseline characteristics at the time of sampling in term infants, preterm infants sampled at birth and preterm infants sampled at term equivalent age.

Data are presented equally mean (95% CI).

https://doi.org/10.1371/journal.pone.0180082.t001

T/S Ratio

Hateful T/S ratio and T/South variability for preterm infants sampled at birth, preterm infants sampled at term and term born infants are shown in Fig 2. Mean T/S ratio and T/South variability of male person and female infants overall and inside each cohort are shown in Table 3. Finally, Fig 3A, 3B and 3C show correlation of T/S ratio with gestational age, birth weight and maternal age respectively in term infants and preterm infants sampled at birth.

Tabular array 3. Gender distribution and mean (95% CI) T/S ratios of male and female infants included in the written report overall and within each study cohort.

p value signifies results from univariate ANOVA analyses of T/South ratios in males compared to females overall and within each cohort. (*Gender information missing in two/31 term infants).

https://doi.org/x.1371/journal.pone.0180082.t003

We did not discover any difference in T/S ratios in males compared to female infants either overall, or within each cohort. However, our results show that both gestational historic period and nativity weight were negatively correlated with T/S ratio (p = 0.001, p = 0.006 respectively) and that maternal historic period was positively correlated with T/S ratio (p = 0.011). Moreover, our data demonstrate a gradient outcome with T/S ratios being highest in preterm infants sampled at birth and lowest in term infants, with term infant T/S ratios being significantly shorter than those of both preterm infants sampled at term (p = 0.04) and preterm infants sampled at nascency (p = 0.004) (Fig 2). Cohen's d analyses revealed a big event size when comparing T/S ratios between term and preterm infants sampled at birth (d = -0.85, r = -0.39) and a medium effect size when comparing T/Southward ratios in term infants versus preterm infants sampled at term equivalent age (d = -0.71, r = -0.33). Though there was a reduction in mean T/S ratio betwixt preterm infants sampled at nascence and preterm infants sampled at term equivalent age, this was not statistically significant (p = 0.42).

For reasons highlighted in the discussion section, we were simply able to obtain longitudinal telomere length samples (at the time of birth and at term equivalent age) in 5 preterm infants. A post hoc analysis of this longitudinal data enabled calculation of telomere attrition rate (Equation 2) between birth and term historic period.

Equation ii. Methodology for adding of telomere compunction charge per unit in five preterm infants sampled at nascence and at term equivalent age

Table 4 shows that in understanding with the cantankerous sectional data described above, a reduction in T/Southward ratio was observed between the time of nascence and term equivalent age in all five babies. However, the magnitude of this reduction was highly variable between these 5 babies. Paired t-test analysis revealed no difference between nascence sample T/S ratio and term sample T/South ratio (p = 0.07) for these 5 babies (in keeping with cantankerous sectional data presented in Fig 2. indicating no departure between nativity sample T/S ratio and term sample T/S ratio in preterm infants). Intriguingly, our results as well advise an inverse relationship between telomere attrition rate and both birth weight (Pearson Correlation -0.47) and gestational age (Pearson Correlation -0.44). Notwithstanding, this was non of statistical significance (p = 0.42 and p = 0.46 respectively). These information are shown in Fig 4A and 4B.

Discussion

In this report we assessed whole blood leukocyte telomere length in a accomplice of preterm and term born infants shortly subsequently nascency, and for the beginning time, we as well assessed telomere length in preterm infants at term equivalent historic period. Our data practise not back up our initial hypothesis that telomere length is significantly shortened by term equivalent historic period in preterm infants and in comparison to term infants. On the contrary, our results show that preterm infants at term equivalent historic period take significantly longer telomere lengths than term born infants. Withal, in keeping with other published data, our results point a significant reject in telomere length with advancing gestational historic period at birth [50–52]. Nosotros as well observed a positive correlation between maternal age and T/S ratio, suggesting that older mothers deliver babies with longer relative telomere lengths. The strengths of this study are that it represents the only study to have directly compared telomere length measurement in preterm infants, assessed at term age and term built-in infants using a well established methodology. Potential limitations include the express number of preterm infants in which we were able to obtain both birth and term historic period (longitudinal) telomere lengths and the observation that our 'healthy' comparator group of term infants cannot, past definition, be classified as entirely 'salubrious' by the observation that they required claret testing. Nonetheless, this latter betoken is only reflective of the fact that obtaining research blood samples in completely healthy babies is non ethically audio.

Telomere length in preterm infants is a largely understudied area and to the all-time of our cognition, but three other studies take assessed telomere length in preterm infants in comparing to term born babies: Friedrich et al. found no significant departure between the two groups when they assessed string claret leukocyte telomere length [51], however Menon et al. and Ferrari et al. constitute significantly shortened leukocyte and placental telomere lengths respectively in term born infants compared to preterm infants born with intact membranes [fifty, 51, 53]. Interestingly, telomere length in preterm infants built-in following preterm pre-labour rupture of membranes was not different to term born infants and was also significantly shorter than preterm infants built-in with intact membranes. Furthermore, Ferrari et al. provided data to back up the hypothesis that unexplained stillbirths are associated with placental telomere compunction by demonstrating a reduction in placental telomere length between stillbirths and term born infants [53]. Moreover, the placenta is known to comprise sub-populations of karyotypically aberrant trophoblasts, which may have pregnant ramifications on telomere length measured in the Ferrari paper [63]. Indeed Ferrari et al. noted failed karyotype analysis in 15/42 stillborn cases.

While these findings (summarised in Table 5) offer interesting and novel insights into the physiological relevance of the events associated with telomere attrition that may pb to normal labour, preterm labour or unexplained stillbirth, none provide information on telomere length in the critical aberrant period of development that preterm born infants are exposed to in the neonatal intensive care unit during the weeks following preterm birth.

Our own data contradict the findings of Friedrich and Menon and are fundamentally unlike to those presented past Ferrari, who examined placental telomere length. Nosotros propose that our finding that telomere length is longer in preterm infants sampled at birth compared to term born infants might be explained past a period of high jail cell turnover and replicative stress during a menstruum of growth in the final weeks of pregnancy in the term infant that does not occur in those born preterm. As such, we suggest that the nigh prominent factors influencing telomere length in neonates are gestational maturity and birth weight (which are intrinsically linked except for where fetal growth restriction has occurred). However, given the high variability in telomere lengths shown past our ain and others' information [26, 27, 48, 49, 64, 65], we suggest that in that location may also exist other as notwithstanding undiscovered determinants of telomere length in newborns, which might include both genetic and epigenetic factors. Indeed other studies indicate heritability of telomere length and significant correlations between offspring telomere length and parental age [66–73]. Our own results and those of others point that this pattern is nowadays at birth [64].

In light of previous observations by Holmes et al. who found a meaning shortening of telomeres in the weeks following preterm birth [52], the finding that telomere length is not shortened at the time of term equivalent age in the preterm baby was unexpected. However, Holmes' study only examined 5 preterm infants and thus may have been underpowered. We advise that there may be at least 2 possible explanations for this finding. Firstly, it is possible that the results from our written report reverberate a tedious telomere compunction rate in the preterm infant during the initial weeks after birth, arising from slow replication and prison cell turnover. Indeed, many preterm infants undergo an initial phase of slow early on growth (in comparison to fetal growth rates) [74, 75]. Alternatively, an opposing view arises from observations by others, who have noted that following birth, lymphocyte expansion rate occurs independently of gestational age [76–78]. In order to sustain rapid expansion of immature lymphocytes in the commencement few weeks of life, whilst evading telomere loss and subsequent entry into jail cell cycle abort, it is possible that telomeres may be lengthened. In support of this, previous work has demonstrated up-regulation of telomerase and diffuse telomeres in response to stimulated expansion of naïve B lymphocytes isolated from adults and young children [79]. Should telomerase expression elicit a like effect during the expansion of immature lymphocytes in the neonatal period, 1 might expect a greater population of cells with longer telomeres in the preterm group that were sampled some weeks afterwards nativity (at term equivalent age) in comparison to term born controls sampled within 48 hours of nativity. Therefore it is also possible that our results can be explained past differences in the deportment of telomere maintenance mechanisms betwixt the study cohorts.

To further investigate these areas of uncertainty, it would take been beneficial to assess preterm babe telomere length longitudinally in all preterm babies recruited to our ain report. This would additionally unmask whatever potential hidden furnishings owing to a large spread in information every bit a upshot of variable genetic and epigenetic influences. Despite this being our original intention, obtaining samples for telomere length analysis at birth and at term equivalent age proved hard every bit a number of preterm babies were either discharged from infirmary or transferred dorsum to their local neonatal unit prior to term age. From the five preterm infants in whom nosotros were able to collect longitudinal information (Table iv), we demonstrated a reduction in telomere length betwixt birth and term age. Intriguingly our data additionally suggests an inverse relationship between telomere attrition rate and both nascence weight and gestational age (Fig 4A and 4B). Though we acknowledge that the sample size is pocket-size and at that place is no statistical significance, these preliminary data raise, for the first time, the biologically plausible hypothesis that telomere attrition rate in preterm infants peradventure associated with the degree of prematurity, with the nearly preterm infants manifesting college telomere attrition rates. Naturally, these information require confirmation in larger studies evaluating longitudinal telomere measurements in preterm infants. Our data also suggest that advanced maternal historic period is associated with increased relative telomere length in newborn infants. Okuda et al demonstrated a like association, albeit using a telomere restriction fragment (TRF) methodology to measure newborn telomere length [64]. This finding is of involvement and warrants replication in light of the increase in maternal historic period at fourth dimension of first pregnancy that has been observed over contempo years [80]. Nonetheless, the relationship we depict may be a confounding variable given the relationship between maternal telomere length and newborn telomere length observed by others [81, 82]. The increment in vitro fertilisation methods over the past two decades may also be a relevant factor merely in our own accomplice, the bulk of preterm births (93%) were as a result of a natural pregnancy.

It may be that analysis of average telomere length in the preterm infant at term equivalent age cannot act every bit a suitable marker of the aged phenotype observed. Mayhap rather than measuring telomere length at term equivalent historic period in preterm infants, it may exist more prudent to measure them further downstream eastward.chiliad. during later childhood or adolescence. This has proved to be informative in previous studies where shortened telomere length was shown to exist associated with respiratory morbidity in the ex-preterm infant [34, 83]. However, the disadvantage of doing this would be the introduction of known and unknown confounding variables (i.due east. lifestyle and epigenetic changes) which may influence telomere length [84]. Alternatively, in that location are a number of other putative markers of cellular senescence that may have more than relevance to the detection of senescence associated with preterm birth that require further investigation. A plausible alternative candidate is SIRT 1, which is known to be downwards-regulated in association with insulin resistance, cardiovascular disease and metabolic disease. Moreover, SIRT 1 down-regulation is known to be associated with accelerated string claret endothelial progenitor cell senescence in preterm infants [85]. Likewise, cell cycle regulators CDKN2A and CDK1A are known to be linked with ageing in adults and therefore warrant investigation in the newborn population [86–88].

In conclusion, our data point that preterm infants assessed at term equivalent historic period manifest longer telomere lengths than term born infants. In addition, our data and that of other groups [44, 64, 84, 89] show considerable variability in telomere length in preterm and term infants, suggesting that other mechanisms may be alongside gestational maturity that are as yet unexplored determinants of telomere length. This high level of variability leads to a degree of overlap between the data in each of the cohorts assessed here and equally such, we suggest that our information should be replicated by other groups and that hereafter piece of work in this area should evolve to examine a panel of markers of cellular senescence longitudinally. Furthermore, our findings lead us to speculate whether, as a response to preterm birth, at that place are mechanisms equally even so not understood that serve to up regulate telomerase. These should be the focus of future research.

Supporting data

Acknowledgments

Support via an internal project grant scheme award from East Kent Hospitals University NHS Foundation Trust is best-selling.

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