Original Article
Association Between Maternal-Perceived Psychological Stress and Fetal Telomere Length Hamisu M. Salihu, MD, PhD, Lindsey M. King, MPH, CHES, CCRP, CTTS, Chiaka Nwoga, MPH, CPH, CCRP, Arnut Paothong, PhD, Anupam Pradhan, PhD, Phillip J. Marty, PhD, Rana Daas, BS, and Valerie E. Whiteman, MD Objective: Our study aimed to investigate the association between maternal-perceived psychological stress and fetal telomere length. Methods: We recruited women in labor upon hospital delivery admission. Based on responses to the Perceived Stress Scale, we categorized participants as having “high,” “normal,” or “low” perceived stress. We collected umbilical cord blood samples (N = 71) and isolated genomic DNA from cord blood leukocytes using quantitative polymerase chain reaction. We used a ratio of relative telomere length derived by telomere-to-single-copy gene ratio (T/S ratio). We applied analysis of variance and bootstrapping statistical procedures. Results: Sixteen (22.5%) women were classified as having low perceived stress, 42 (59.2%) were classified as having normal perceived stress, and 13 (18.3%) were classified as having high perceived stress. Fetal telomere length differed significantly across the three stress groups in a dose–response pattern (T/S ratio of those with low perceived stress was greater than those with normal perceived stress, which was greater than those with high perceived stress [P < 0.05]). Conclusions: Our findings support our hypothesis that maternalperceived psychological stress during pregnancy is associated with shorter fetal telomere length and suggest maternal stress as a possible marker for early intrauterine programming for accelerated chromosomal aging.
From the Department of Family and Community Medicine, Baylor College of Medicine, Houston, Texas, the Departments of Epidemiology and Biostatistics and Global Health, University of South Florida College of Public Health, and the Department of Obstetrics and Gynecology, Division of MaternalFetal Medicine, University of South Florida, Tampa. Correspondence to Dr Hamisu M. Salihu, Department of Family and Community Medicine, Baylor College of Medicine, 3701 Kirby Dr, Suite 600, Houston, TX 77098. E-mail:
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[email protected]. This work was supported by the James and Esther King Biomedical Research Program, Florida Department of Health (grant no. 4 KB03 and 1KG14-33987) and the University of South Florida Neuroscience Collaborative-Seed Grant Program and the University of South Florida College of Public Health Interdisciplinary Research Development Grant. The funders had no involvement in the study design or the collection, analysis, and interpretation of data. The authors did not report any financial relationships or conflicts of interest. Accepted July 11, 2016. Copyright © 2016 by The Southern Medical Association 0038-4348/0–2000/109-767 DOI: 10.14423/SMJ.0000000000000567
Key Words: bootstrapping statistical methodology, fetal telomere length, maternal psychological stress, neonatal umbilical cord blood
M
aternal psychological stress is highly prevalent and leads to a multitude of gestational and infant-correlated complications.1,2 Unmanaged high levels of stress create potential risks for the mother and fetus.3–5 Further evidence suggests that maternal exposure to stress can lead to adverse neurodevelopmental outcomes6 through a process of “fetal programming.”7 Telomeres, DNA-based regions that cap the ends of chromosomes to protect them from deterioration during replication, play a critical role in cellular division. Telomerase, a ribonucleoprotein enzyme, elongates chromosomes by adding TTAGGG sequences (highly conserved repetitive DNA sequences) to the ends of existing chromosomes in all vertebrates. Telomeres progressively shorten and cells become inherently more susceptible to degradation as the shortening occurs, which is the result of low telomerase levels.8 Chromosomes with critically shortened telomeres are genetically unstable and lead to apoptosis.9 Shortened telomeres are therefore a biomarker of cellular and biological aging, longevity, and disease.10,11 Stress is associated with indicators of accelerated cellular aging, including oxidative stress, shortened telomere length, and lessened telomerase activity in healthy adults.12 Furthermore, childhood stress has been linked to significantly greater telomere erosion.13,14 Despite well-established evidence of stress and telomere length degradation in adults and children, there is a paucity of published research regarding maternal stress and fetal telomere length.15 The objective of our study was to evaluate whether perceived stress is associated with fetal telomere length.
Key Points • We investigated the association between maternal-perceived psychological stress and fetal telomere length using quantitative polymerase chain reaction in 71 umbilical cord blood samples. • Fetal telomere length differed significantly across the three maternal stress groups in a dose–response pattern. • Our findings support the hypothesis that maternal-perceived psychological stress during pregnancy is associated with shorter fetal telomere length at birth.
Southern Medical Journal • Volume 109, Number 12, December 2016
Copyright © 2016 The Southern Medical Association. Unauthorized reproduction of this article is prohibited.
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Salihu et al • Maternal Perceived Psychological Stress and Fetal Telomere Length
We hypothesized that increased psychological stress during pregnancy would be associated with telomere shortening in the fetus.
Methods Women in labor were recruited into our study upon delivery admission at Tampa General Hospital (TGH) in Florida. The University of South Florida Morsani College of Medicine faculty and resident physicians provide patient care at TGH. The majority of patients admitted for delivery at TGH are socioeconomically disadvantaged, racially/ethnically diverse, and primarily uninsured or insured through Medicaid. Eligible women were those 18 years or older who were capable of speaking English or Spanish and were delivering full-term single live births with no congenital malformations. A trained member of the study team consented women in English or Spanish before delivery. We used additional safety measures during the consenting process to mitigate physical discomfort and emotional duress. As such, we did not enroll any women in labor who were unable to offer their full attention to the explanation of the research procedures. We obtained approval from the University of South Florida institutional review board and ethics committee and conducted all of the research in accordance with the Code of Ethics and the 1975 Declaration of Helsinki. This study was a nested study as part of a larger study. Each participant was provided a questionnaire packet to complete before or after delivery but in advance of hospital discharge. We also abstracted material sociodemographic data and newborn measurements from electronic medical records. Stress exposure outcomes for this study were measured using the Perceived Stress Scale (PSS). The PSS is a 14-item scale that is used to quantify cumulative perceived stress within the previous month.16 The PSS evaluates the extent to which respondents believe that their lives are stressful, unpredictable, and uncontrollable, issues that have been found consistently to be components of stress.16 The PSS consists of easy-to-understand items and response options.16 All PSS responses are rated on a five-point Likert scale. Total scores range from 0 to 56; a higher score indicates greater perceived stress.16 For our study, we classified participants into three categories: PSS-14: “low” perceived stress, if the PSS-14 rank was ≤20th percentile score; PSS-14: “normal” perceived stress, if the PSS-14 rank was between the 20th and 80th percentile score, and PSS-14: “high” perceived stress, if the PSS-14 rank was >80th percentile score. Seventy-one maternal–fetal dyads were enrolled in the study and umbilical cord blood samples were collected at delivery using an Umbilicup (DeRoyal, Powell, TN). An Umbilicup is a cord blood collection device that allows for a large volume of blood to be safely collected without the exposure of a needle. Cord blood samples were processed within 48 hours of collection. All of the laboratory staff were blinded to medical and demographic identifiers. Genomic DNA was processed at the University of South Florida Global Health Infectious Disease Research Laboratory using a 200-μL buffy-coat layer as instructed
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by the manufacturer (DNeasy Blood and Tissue Kit, QIAGEN, Hilden, Germany) with slight adjustments. Approximately 20 ng DNA was used to determine the relative telomere lengths for all reactions (telomere repeat copy number [T/S] ratio). Using quantitative polymerase chain reaction (qPCR) cycle threshold (ΔCt) values, we applied the validated method of Cawthon et al17 to ascertain the relative telomere lengths (T/S ratio) using GoTaq qPCR Master Mix reagent (Promega Corporation, Madison, WI).17 The telomere primer concentrations were tel1, 270 nM, and tel2, 900 nM. The final 36B4 (single-copy gene) primer concentrations were 36B4u, 300 nM, and 36B4d, 500 nM. The thermal cycling profile started at 95°C incubation for 2 minutes for hot start activation, followed by 40 cycles of 15-second denaturation at 95°C, and 2 minutes at 54°C for annealing. We obtained the relative quantities of telomere repeats and the single-copy gene 36B4 by recording the individual qPCR generated ΔCt values in the standard curve acquired from an arbitrary human DNA ΔCt. Telomere was divided by absolute copy of the single-copy gene 36B4, T/S ratio, to devise the absolute copies of replications. Telomere length variations also can be determined using the Cawthon ratio ([1910.5 log T/S ratio] + 4157) and the relative T/S ratio or RTS (a log derivation of the ΔCt values using the intercept of the standard curve). Because these three variables are highly correlated, for this study we chose to use the T/S ratio because it is the most-often reported measure of telomere length based on the literature.18,19 For comparison among the sample descriptive statistics and the three groups, we used analysis of variance (ANOVA) continuous variables. The χ2 test was used for categorical variables and the Fisher exact test was applied where the expected cell size was
