Background
The developmental origin of the health and disease hypothesis is based on the premise that many chronic diseases have their roots in fetal development. Specifically, maternal stress during pregnancy is associated with altered fetal development and many adverse long-term health outcomes. Although the mechanisms underlying this effect are currently unclear, at the cellular level 1 possible mediator is the regulation of telomere length. Telomere dynamics appear to play a role in disease progression, and an adverse intrauterine environment may contribute in the establishment of short telomeres in newborns. In accordance with this, it was recently reported that prenatal stress is significantly associated with shorter mean newborn telomere length. However, this finding has yet to be replicated, and currently we know nothing about whether different size classes of telomeres within the telomere length distribution are differentially affected by prenatal stress. Examining telomere length frequency distributions is important, because the shortest telomeres in the distribution appear to be the most indicative of telomere dysfunction and thus the best predictors of mortality and morbidity in humans.
Objective
We investigated the effects of intrauterine exposure to maternal stress over the whole course of gestation on newborn mean telomere length and telomere length frequency distributions.
Study Design
We conducted a prospective cohort study of 24 mother–newborn dyads at an urban teaching hospital. Pregnant women with nonanomalous, uncomplicated pregnancies were recruited and assessed in the third trimester of gestation. Maternal psychosocial stress was quantified using the Holmes and Rahe Stress Scale and categorized as high stress (≥300 points) or low stress (≤299 points) exposure. Newborn telomere length was measured from cord blood at delivery using the Telomere Restriction Fragment assay.
Results
We found a significant negative association between maternal stress and newborn telomere length (β = −0.463, P = 0.04). Newborns whose mothers experienced a high level of stress during pregnancy had significantly shorter telomere length (6.98 ± 0.41 kb) compared to newborns of mothers with low stress (8.74 ± 0.24 kb; t = −3.99, P = .003). Moreover, the difference in newborn telomere length between high-stress and low-stress mothers was due to a shift in the telomere length distribution, with the high-stress group showing an underrepresentation of longer telomeres and an over-representation of shorter telomeres.
Conclusion
Our findings replicate those of other recent studies and also show, for the first time, that the prenatal stress−associated difference in newborn mean telomere length is due to a shift in the overall telomere distribution.
Introduction
Converging evidence from epidemiological, clinical, and experimental studies suggests that exposure to suboptimal conditions in early life can produce long-term effects on health and disease susceptibility. The developing fetus is especially sensitive to intrauterine perturbations, and this has led to the developmental origins of the health and disease hypothesis, which posits that an individual’s long-term risk of disease is dependent, in part, on the quality of the intrauterine environment. Although a number of intrauterine factors may contribute to the long-term risk of disease, fetal exposure to maternal stress appears to represent a particularly salient insult. The mechanisms that link prenatal stress exposure to disease decades later are still unclear, but recent work suggests that at the cellular level, telomere dynamics may represent 1 such underlying mechanism.
Telomeres are protective complexes of noncoding, repetitive DNA, and shelterin proteins that cap chromosomes and promote chromosomal stability. As cells divide, telomeres shorten both because of incomplete replication and through oxidative damage. Telomere length (TL) varies between chromosomes within each cell and among cells within a tissue because of different replicative histories and past levels of cellular damage. This results in a distribution of TLs within the tissue of an individual. Although there are mechanisms in place to rebuild the shortest telomeres, such as the enzyme telomerase, if a telomere has undergone a critical degree of shortening, it can lead to cellular senescence. Senescent cells lose the ability to replicate and cease to divide or undergo apoptosis, contributing to the aging phenotype and susceptibility to disease. Based on this, measuring the average TL of the TL distribution has gained traction as a biomarker for cellular function and aging, and can serve as an early predictor of disease onset for cardiovascular disease, stroke, Alzheimer’s disease, diabetes mellitus, childhood autism, as well as overall mortality risk.
Given the role that telomere dynamics may play in disease progression, current efforts have turned to identifying the mechanisms that cause variation in TL. TL is a function of both the initial setting of TL and TL attrition over time. Little is currently known about the causes of variation in the initial setting of TL, but 1 possibility is that the intrauterine environment plays an important role in the establishment of initial (newborn) TL. In accordance with this hypothesis, recent work by Entringer et al showed that prenatal stress was significantly associated with shorter mean newborn leukocyte TL. However, this finding has yet to be replicated. Moreover, although average TL is the most commonly used biomarker to describe TL variation among individuals in a population, it is not the mean TL but rather the shortest telomeres that lose function and initiate cellular senescence. Therefore, characterizing the entire distribution of TL within a sample from an individual may provide particularly valuable information about the relative increases in the number of short telomeres, which is more indicative of telomere dysfunction than measures of mean TL.
In this study, we investigated the effects of intrauterine exposure to maternal stress over the whole course of gestation on newborn mean TL and TL frequency distributions. Thus, our study builds on previous work exploring prenatal stress and TL to replicate and expand on these previous studies. The notable additional contributions of our study involve the characterization of maternal stress over the entirety of gestation and also its effect on the entire frequency distribution of telomeres. The use of TL frequency distributions provides a better and more meaningful resolution on putative prenatal stress effects, because the shortest telomeres are the most indicative of telomere dysfunction and the best predictors of mortality and morbidity in humans.
Materials and Methods
Subject characteristics
The study cohort of pregnant women was enrolled at an urban teaching hospital in Philadelphia, PA, between April and August 2013. Eligible women were between the ages of 18 and 35 years and delivered a single, viable, nonanomalous infant. Women were excluded if the pregnancy was complicated by medical conditions that could affect fetal growth, such as hypertension, diabetes, or smoking; if they developed any condition that required steroid therapy; or if they developed chorioamnionitis. Medical records were reviewed to obtain sociodemographic characteristics and medical history, and to determine eligibility pertaining to maternal age at birth, maternal prepregnancy weight, obstetric complications during pregnancy, length of gestation, weight, height, and birthweight adjusted for gestational age. Financial incentive or compensation was not offered. The result was a sample of 24 mother–newborn dyads. The sociodemographic and newborn characteristics of this population are displayed in Table 1 . The study was approved by Drexel University Institutional Review Board and informed, written consent was obtained from all participants.
| Maternal characteristics | Mother–newborn dyads, N = 24 |
|---|---|
| Sociodemographic | |
| Age, y, mean ± SD | 25.1 ± 4.0 |
| Body mass index | 29.5 ± 8.5 |
| Race/ethnicity | |
| Non-Hispanic white | 13% (n = 3) |
| African American | 75% (n = 18) |
| Hispanic | 13% (n = 3) |
| Newborn characteristics | |
| Gestational age at birth, wk, mean ± SD | 40.0 ± 1.1 |
| Birthweight, g, mean ± SD | 3298 ± 510 |
Measures
Maternal stress
When the mother presented at the hospital for delivery, maternal psychosocial stress was assessed using the Holmes and Rahe Stress Scale questionnaire (also referred to as the Social Readjustment Rating Scale). This measurement scale has been called the gold standard for stress assessment and has been shown to correlate with health outcomes in a variety of studies. The 43-item dichotomous survey objectively ascertained whether the mothers had experienced life events within the past year that were likely to initiate a physiological stress response (see Supplemental material ). Each survey event is associated with a numerical value, and this scale was used to assess the cumulative stress the mother experienced over the course of her pregnancy. A score of ≥300 indicates the mother is at high risk of stress-induced illness, while scores ≤299 correlate with lower risk levels of stress-induced illness.
Cord blood telomere length
Telomeres were measured with the Telomere Restriction Fragment (TRF) assay, and the procedure was carried out according to previous studies. Briefly, DNA was extracted from newborn umbilical cord blood using the Puregene Blood Core Kit B following the manufacturer’s specifications (Qiagen, Hilden, Germany). DNA integrity was assessed through the use of integrity gels, and then cord blood TL was measured using the telomere restriction fragment assay. A 10-μg quantity of DNA was digested using 1.0 μL of RsaI (New England Biolabs, Ipswich, MA, R0167L) and 0.2 μL of HinfI (New England Biolabs, R0155M) in CutSmart Buffer (New England Biolabs, B7204S) overnight at 37°C. The digested DNA was separated using pulsed field gel electrophoresis (3 V/cm, 0.5- to 7.0-second switch times, 14 °C) for 19 hours on a 0.8% nondenaturing agarose gel. The gel was then dried without heating and hybridized overnight with a 32 P-labeled oligo (5′-CCCTAA-3′) that binds to the 3′ overhang of telomeres. Hybridized gels were placed on a phosphorscreen (Amersham Biosciences, Buckinghamshire, UK), which was scanned on a Storm 540 Variable Mode Imager (Amersham Biosciences). We used densitometry (ImageQuant 5.03v and ImageJ 1.42q) to determine the position and strength of the radioactive signal in each of the lanes compared to the molecular marker (1 kb DNA Extension Ladder; Invitrogen, Carlsbad, CA).
A major advantage of the TRF assay is that it provides frequency distributions of telomere length for each sample. The resulting plots allow visualization of the relative abundances of TRFs at each molecular weight (MW), providing useful information on where differences in TL occur among groups. For each individual sample, the area under the optical density curve was calculated in 1-kb intervals from 1 to 20 kb, and each interval was divided by the area under the curve for the entire distribution. The background was fixed as the nadir of the low-MW region on the gel (<1 kb). Relative abundances of TRFs in each of the MW classes were log transformed and then fit by least-squares fourth-order polynomial regression with the MW classes (1–20 kb).
Statistical analysis
As determined by the Shapiro–Wilk test, neonatal cord blood TL was normally distributed (W = 0.99, P = .97). To examine the association between maternal psychosocial stress during pregnancy and newborn cord blood TL adjusted for the effects of other possible determinants, a linear regression model was used that included the effects of maternal stress, maternal age, gestational age at birth, and birthweight (adjusted for gestational age). To further explore how the magnitude of maternal stress affects newborn cord blood TL, the study population was divided into 2 groups based on their stress score (high, ≥300 points; low, ≤299 points). Demographics for the stress groups were compared using the χ 2 test for dichotomous variables. We used a t test accounting for unequal variances to compare cord blood TL between the stress groups. Means ± standard deviations are shown.
Frequency distributions of TL for the high-stress and low-stress groups were produced following Haussmann et al. Following Jemielity et al, we compared skewness and kurtosis values of the distribution that were not normally distributed and so were analyzed using a Wilcoxon–Kruskal–Wallis test. All statistical analyses were performed in JMP (v11.1.1).
Materials and Methods
Subject characteristics
The study cohort of pregnant women was enrolled at an urban teaching hospital in Philadelphia, PA, between April and August 2013. Eligible women were between the ages of 18 and 35 years and delivered a single, viable, nonanomalous infant. Women were excluded if the pregnancy was complicated by medical conditions that could affect fetal growth, such as hypertension, diabetes, or smoking; if they developed any condition that required steroid therapy; or if they developed chorioamnionitis. Medical records were reviewed to obtain sociodemographic characteristics and medical history, and to determine eligibility pertaining to maternal age at birth, maternal prepregnancy weight, obstetric complications during pregnancy, length of gestation, weight, height, and birthweight adjusted for gestational age. Financial incentive or compensation was not offered. The result was a sample of 24 mother–newborn dyads. The sociodemographic and newborn characteristics of this population are displayed in Table 1 . The study was approved by Drexel University Institutional Review Board and informed, written consent was obtained from all participants.
| Maternal characteristics | Mother–newborn dyads, N = 24 |
|---|---|
| Sociodemographic | |
| Age, y, mean ± SD | 25.1 ± 4.0 |
| Body mass index | 29.5 ± 8.5 |
| Race/ethnicity | |
| Non-Hispanic white | 13% (n = 3) |
| African American | 75% (n = 18) |
| Hispanic | 13% (n = 3) |
| Newborn characteristics | |
| Gestational age at birth, wk, mean ± SD | 40.0 ± 1.1 |
| Birthweight, g, mean ± SD | 3298 ± 510 |
Measures
Maternal stress
When the mother presented at the hospital for delivery, maternal psychosocial stress was assessed using the Holmes and Rahe Stress Scale questionnaire (also referred to as the Social Readjustment Rating Scale). This measurement scale has been called the gold standard for stress assessment and has been shown to correlate with health outcomes in a variety of studies. The 43-item dichotomous survey objectively ascertained whether the mothers had experienced life events within the past year that were likely to initiate a physiological stress response (see Supplemental material ). Each survey event is associated with a numerical value, and this scale was used to assess the cumulative stress the mother experienced over the course of her pregnancy. A score of ≥300 indicates the mother is at high risk of stress-induced illness, while scores ≤299 correlate with lower risk levels of stress-induced illness.
Cord blood telomere length
Telomeres were measured with the Telomere Restriction Fragment (TRF) assay, and the procedure was carried out according to previous studies. Briefly, DNA was extracted from newborn umbilical cord blood using the Puregene Blood Core Kit B following the manufacturer’s specifications (Qiagen, Hilden, Germany). DNA integrity was assessed through the use of integrity gels, and then cord blood TL was measured using the telomere restriction fragment assay. A 10-μg quantity of DNA was digested using 1.0 μL of RsaI (New England Biolabs, Ipswich, MA, R0167L) and 0.2 μL of HinfI (New England Biolabs, R0155M) in CutSmart Buffer (New England Biolabs, B7204S) overnight at 37°C. The digested DNA was separated using pulsed field gel electrophoresis (3 V/cm, 0.5- to 7.0-second switch times, 14 °C) for 19 hours on a 0.8% nondenaturing agarose gel. The gel was then dried without heating and hybridized overnight with a 32 P-labeled oligo (5′-CCCTAA-3′) that binds to the 3′ overhang of telomeres. Hybridized gels were placed on a phosphorscreen (Amersham Biosciences, Buckinghamshire, UK), which was scanned on a Storm 540 Variable Mode Imager (Amersham Biosciences). We used densitometry (ImageQuant 5.03v and ImageJ 1.42q) to determine the position and strength of the radioactive signal in each of the lanes compared to the molecular marker (1 kb DNA Extension Ladder; Invitrogen, Carlsbad, CA).
A major advantage of the TRF assay is that it provides frequency distributions of telomere length for each sample. The resulting plots allow visualization of the relative abundances of TRFs at each molecular weight (MW), providing useful information on where differences in TL occur among groups. For each individual sample, the area under the optical density curve was calculated in 1-kb intervals from 1 to 20 kb, and each interval was divided by the area under the curve for the entire distribution. The background was fixed as the nadir of the low-MW region on the gel (<1 kb). Relative abundances of TRFs in each of the MW classes were log transformed and then fit by least-squares fourth-order polynomial regression with the MW classes (1–20 kb).
Statistical analysis
As determined by the Shapiro–Wilk test, neonatal cord blood TL was normally distributed (W = 0.99, P = .97). To examine the association between maternal psychosocial stress during pregnancy and newborn cord blood TL adjusted for the effects of other possible determinants, a linear regression model was used that included the effects of maternal stress, maternal age, gestational age at birth, and birthweight (adjusted for gestational age). To further explore how the magnitude of maternal stress affects newborn cord blood TL, the study population was divided into 2 groups based on their stress score (high, ≥300 points; low, ≤299 points). Demographics for the stress groups were compared using the χ 2 test for dichotomous variables. We used a t test accounting for unequal variances to compare cord blood TL between the stress groups. Means ± standard deviations are shown.
Frequency distributions of TL for the high-stress and low-stress groups were produced following Haussmann et al. Following Jemielity et al, we compared skewness and kurtosis values of the distribution that were not normally distributed and so were analyzed using a Wilcoxon–Kruskal–Wallis test. All statistical analyses were performed in JMP (v11.1.1).
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