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. Author manuscript; available in PMC: 2026 Apr 11.
Published in final edited form as: Biol Res Nurs. 2025 Oct 14;28(2):187–194. doi: 10.1177/10998004251387628

DNA Methylation of SLC6A4 and NR3C1 in Term and Preterm Infants: A Pilot Study of NICU-Associated Stress

Kathryn J Malin 1, Yvette Conley 2, Aleigha Barry 3, Rosemary White-Traut 4
PMCID: PMC13068098  NIHMSID: NIHMS2157323  PMID: 41744048

Abstract

Background:

Preterm infants requiring prolonged hospitalization in the neonatal intensive care unit (NICU) are often exposed to early-life stress, which may contribute to long-term health consequences. Epigenetic markers, particularly DNA methylation (DNAm) of stress-related genes such as SLC6A4 and NR3C1, have been identified as potential biomarkers of early-life stress. Limited research has compared DNAm patterns between term and preterm infants or examined associations between NICU-related stress and DNAm in preterm populations.

Objectives:

This pilot study aimed to (1) compare DNAm patterns between preterm and term infants, (2) examine relationships between NICU-related stress and DNAm alterations in preterm infants, and (3) explore DNAm changes over time in preterm infants.

Methods:

Buccal swabs were collected from 10 term and 23 preterm infants within the first week of life. Eleven preterm infants returned for a repeat sampling three months post discharge. The Neonatal Infant Stressor Scale (NISS) was used to calculate early-life stress. DNAm at specific CpG sites were analyzed using Mann-Whitney U tests and Spearman’s rho correlations.

Results:

Preterm infants exhibited significantly different DNAm patterns at select CpG sites compared to term infants. Among preterm infants, higher NISS scores during hospitalization correlated with altered DNAm patterns in both genes. At three months post-discharge, new and persistent DNAm alterations were identified; although, total average DNAm did not significantly differ over time.

Conclusion:

These findings suggest that NICU-related stress is associated with DNAm alterations in preterm infants, supporting the need for further investigation of DNAm as a biomarker of early-life stress.

Keywords: preterm infants, NICU, DNA methylation, SLC6A4, NR3C1, early-life stress

Introduction

Preterm infants born prior to 37 weeks gestation account for 10.4% of infants born each year in the United States (CDC, 2025). Infants born preterm are at higher short and long-term adverse health and developmental outcomes (Hee Chung et al., 2020; Morniroli et al., 2023). These risks may be secondary to the early-life stress associated with prematurity and prolonged hospitalization (Hee Chung et al., 2020; Pavlyshyn et al., 2024). Long-term hospitalization may be necessary depending on gestational age, illness, and level of NICU care. On average, infants born at 32 weeks’ gestation spend about 24 days in the NICU. Extended NICU stays have been associated with increased risk for infections, disruptions in development of parental-infant bonding, and increased healthcare costs (Ismail et al., 2023). Premature birth and the necessary NICU hospitalization has been linked to poor neurodevelopmental outcomes and increased risk for psychiatric problems such as attention deficit disorder and anxiety (Cheong et al., 2019; El-Metwally & Medina, 2020). Despite the growing evidence linking early life stress in preterm infants to adverse health and development, measurement of the early-life toxic stress and the mechanism of action remains understudied and poorly understood.

Epigenetic measures are a promising biomarker of stress and offer a novel approach to describing and quantifying the stress experienced by preterm infants. Epigenetics is a growing field of study that focuses on phenotypic changes that do not include alterations in DNA sequences. These changes occur through chemical alterations to the DNA and result in variations in gene expression. Epigenetic alterations are sensitive to environmental exposures and influences and are associated with meaningful differences in health and illness (Cavalli & Heard, 2019). Epigenetic alterations of the Solute Carrier Family 6, member 4 gene, SLC6A4, and the glucocorticoid receptor gene, NR3C1, are associated with poorer neurodevelopmental outcomes Malin et al., 2023. Specifically, alterations in DNA methylation (DNAm) of the SLC6A4 and NR3C1 genes have been described in preterm infants (Malin, Kruschel, et al., 2023; Provenzi et al., 2018). SLC6A4 is located on chromosome 17 and is responsible for encoding the membrane protein that transports the neurotransmitter serotonin and is responsible for terminating and recycling serotonin (SLC6A4 Gene - Solute Carrier Family 6 Member 4, n.d). Diseases associated with the SLC6A4 gene include obsessive-compulsive disorder, anxiety, and major depressive disorder (SLC6A4 Gene - Solute Carrier Family 6 Member 4, n.d; Stelzer et al., 2016). Differences in methylation of the promotor regions of SLC6A4 leads to variations in serotonin transporter availability via changes in gene expression (Provenzi et al., 2016). Increased methylation on CPG sites 5 and 6 have been reported in infants who experienced high amounts of pain stimuli while in the NICU (Provenzi et al., 2015). Increased methylation of SLC6A4 at age 7 in children born prematurely has been linked with excessive painful experiences while in the NICU (Chau et al., 2014). Similarly, alteration in methylation patterns of NR3C1, which is located on chromosome 5 and is responsible for encoding glucocorticoid receptors, has been associated with glucocorticoid resistance (NR3C1 Gene-Nuclear Receptor Subfamily 3 Group C Member 1, 2025; Stelzer et al., 2016).

In broader populations, significant alterations in methylation patterns of SLC6A4 and NR3C1 have also been linked to stress associated with the COVID-19 pandemic lockdown. These results further support the hypothesis that epigenetic alterations in these two genes are salient biological markers of stress (Nazzari et al., 2022). Yet, despite these early research findings, there remains a paucity in data describing differences in term and preterm infant DNAm patterns of SLC6A4 and NR3C1. Furthermore, there is a need to identify dysregulation in methylation patterns for preterm infants, and whether they are associated with known stressful experiences while in the NICU. To address these questions, we conducted this pilot study to (1) compare DNAm in preterm and term infants (2) examine relationships between NICU-related stress and DNAm alterations in preterm infants, and (3) explore changes in DNAm over time in preterm infants.

Materials and Methods

Ethical approval was obtained from the study hospital system (IRB NUMBER 00044310) and informed consent was obtained from participants’ legal guardians prior to any data collection.

Sample

Using a cross-sectional study design, 10 term infants and 23 preterm infants were enrolled during their hospitalization in the postpartum unit or NICU. Inclusion criteria for the term infant group included birth ≥37 weeks completed gestation, mother over the age of 18, mother did not receive prenatal betamethasone, infant delivered vaginally without instrumentation, infant to be discharged with the biologic mother, and the infant is less than seven days old. Inclusion for the preterm group included birth ≤32 weeks gestation, mother over the age of 18, infant does not have a known genetic defect, infant to be discharged with the biologic mother, and infant is less than seven days old. The criteria of discharge home with biologic mother were included to allow for the timely consent and data collection required for the study. Data for the study was collected from the study hospital which is a level 4 Midwestern NICU. The average census in the study hospital NICU is 61 infants and is connected to a large Midwestern postpartum unit that serves over 3,000 postpartum mothers per year.

Data Collection

Once informed consent was obtained, data were collected from both the term and preterm infant participants within the first week of life. Three months after discharge from the NICU, preterm infants then returned to the study hospital for repeat data collection. Sample collection included oral buccal swabs obtained at the bedside. Using an Isohelix swab kit, two samples were collected at each point in time to ensure an accurate result. The average of the two samples obtained at each data collection session were used for analysis. Swabs were inserted into the infant’s mouth and rubbed firmly against the inside of the cheek for a minimum of 20 s. Swabs were then stored in a −80°C freezer until shipment for analysis. Review of the electronic health record (EHR) was completed at the time of enrollment and again at the time of discharge for the preterm infant group. The protocol for this study was modeled after a previous feasibility study, indicating high likelihood of successful participation and data collection (Malin, Kruschel, et al., 2023).

Measures

Demographic and Infant Medical Characteristics.

Demographic and infant medical characteristics were collected from the EHR. These data included the infant’s gestational age, race/ethnicity, birth weight, primary diagnosis while in the NICU, Apgar scores, and length of hospitalization.

DNA Methylation.

DNA was extracted from buccal cells, bisulfite-treated, and amplified using a two-stage PCR protocol with barcoded primers. Libraries were pooled and sequenced on an Illumina MiniSeq with a 20% phiX spike-in. Raw sequence data were processed in CLC Genomics Workbench, mapped to a bisulfite-converted reference, and analyzed to calculate percent methylation at CpG sites. All laboratory and sequencing procedures were performed at the Genomic and Microbiome Core Facility at Rush University and previously have been described in further detail (Griffith et al., 2025; Malin, Kruschel, et al., 2023; Naqib et al., 2018). The percent methylation for each CpG site was reported based on the C residues (derived from 5-methyl-cytosine) to C + T residues at any site within the DNA sequence of SLC6A4 and NR3C1 promotor regions. Build GRCh38 was used in the DNAm analysis and CpG numbering systems for both SLC6A4 and NR3C1 have been previously described and are displayed in Figure 1 (Griffith et al., 2024; Provenzi et al., 2016).

Figure 1.

Figure 1.

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Location of CpG Sites: Figure one Displays the Location of CpG Sites Along the NR3C1 and SLC6A4 Genes

Stress Associated with NICU Hospitalization.

Early-life stress for infants in the preterm group was quantified using the Neonatal Infant Stressor Scale (NISS; Newnham et al., 2009). The NISS is a validated instrument specifically developed to capture the range of stressful experiences that infants encounter during hospitalization in the NICU. It consists of 44 acute stressful events (e.g., diaper changes, x-ray, suctioning) and 24 chronic stressful NICU events (e.g., presence of sepsis, invasive ventilation). Each stressor is weighted and recorded over a 24-h period, allowing for a comprehensive assessment of daily stress exposure. The total NISS is derived from summing the acute and chronic stressor scores, with higher scores reflecting greater stress associated with the care and medical interventions required during NICU hospitalization. For this study, NISS scores were obtained for the preterm infants on the same day of life that the buccal swab was collected. Reliability and construct validity of the NISS have been reported (Malin, Kruschel, et al., 2023; Pourkaviani et al., 2019).

Statistical Analysis

Descriptive statistics were used to summarize sample characteristics, early life stress, and DNA methylation of SLC6A4 and NR3C1. Mann-Whitney U tests and Spearman’s rho correlations were computed to assess relationships among infant characteristics, early life stress, and percent methylation of CpG sites of SLC6A4 and NR3C1.

Results

After informed consent was obtained, a total of 10 term infants and 23 preterm infants were enrolled in the study. Eleven preterm infants completed data collection 3 months after NICU discharge. Significant differences in gestational age, birth weight, Apgar’s, and length of hospital stay were identified between the term and preterm groups. Descriptive data are displayed in Table 1.

Table 1.

Infant Characteristics Providing Mean (SD) or Frequency (%)

Infant characteristics
Term infants (n = 10) Preterm infants (n = 23) p-value
Gestational age (weeks) 39.2 (1.19) 29.1 (1.97) <.001
Birth weight (grams) 3,327 (369) 1,167 (356) <.001
Apgar score at 1 min 8 (0) 4 (2.3) <.001
Apgar score at 5 min 9 (0) 6.9 (1.8) <.001
Length of hospitalization in days 2 (0) 80.4 (41.5) <.001
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Term and Preterm Infant DNAm

Significant differences were observed on three specific CpG sites of each gene (Table 2). SLC6A4 CpG sites were more likely to demonstrate increased methylation in the preterm infant group as compared to the term infant group. Average total percent methylation of SLC6A4 was not significantly different between the term (median = 35%) and preterm infant (median = 36%) groups (U = 117, z = −0.059, p = .950). Similarly, the average total percent methylation of NR3C1 did not significantly differ between the term and preterm infant groups term (median = 22%) and preterm infants (median = 25%) groups (U = 141, z = −0.99, p = .320).

Table 2.

Term and Preterm Infant Methylation on Significant CPG Sites of SLC6A4 and NR3C1

Methylation in term infants
 Mean methylation in preterm infants
M (SD)
 M (SD)
n = 10 n = 23 p-value
SLC6A4
 CPG site 2 0.19 (0.1) 0.39 (0.29) .030
 CPG site 15 0.17 (0.06) 0.40 (0.3) <.010
 CPG site 20 0.14 (0.10) 0.22 (0.15) .010
NR3C1
 CPG site 9 0.13 (0.02) 0.26 (0.25) .040
 CPG site 10 0.39 (0.37) 0.17 (0.12) <.010
 CPG site 14 0.22 (0.26) 0.26 (0.20) .020
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Note. Details regarding CpG site locations for SLC6A4 can be found at Provenzi et al. (2016); details regarding CpG site locations for NR3C1 can be found at Griffith et al. (2024).

Preterm Infant DNAm and Stress

Methylation of SLC6A4.

In the preterm infant group, significant correlations between percent methylation of SLC6A4 and elevated NISS scores were identified. Specifically, during the first week of life, acute NISS scores were associated with increased methylation of CpG site 1 (r (21) = 0.42, p = .046) and CpG site 12 (r (21) = 0.45, p = .030). Chronic NISS scores were significantly associated with increased DNAm during the first week of life on CpG site 13 (r (21) = 0.44, p = .030) and decreased methylation of CpG site 10 (r (21) = −0.40, p = .020) and CpG site 20 (r (21) = −00.43, p = .040). Three months after NICU discharge, the total average percent of DNAm was significantly associated with increased acute NISS scores (r (10) = 0.69, p = .010). Furthermore, significant relationships among DNAm of specific CpG sites were observed at three months after NICU discharge (Table 3). No significant relationship was observed between chronic NISS scores collected during the first week of life and DNAm at 3 months after discharge from the NICU.

Table 3.

Correlations Between Increased Chronic NISS Scores and Methylation on Significant CPG Sites of SLC6A4 and NR3C1 3 Months after NICU Discharge

Spearman’s ρ n = 10 p-value
SLC6A4
 CpG 4 0.68 .020
 CpG 7 0.67 .020
 CpG 8 0.63 .030
 CpG 16 0.58 .050
 CpG 18 0.59 .040
 CpG 19 0.70 .010
NR3C1
 CpG 7 −0.66 .020
 CpG 20 −0.82 <.001
 CpG 24 −0.87 <.001
 CpG 31 −0.72 .007
 CpG 32 −0.69 .010
 CpG 33 −0.74 .005
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Note. *n* = 10 for all correlations (reported as r (10). Details regarding CpG site locations for SLC6A4 can be found at Provenzi et al. (2016); details regarding CpG site locations for NR3C1 can be found at Griffith et al. (2024).

Methylation of NR3C1.

Significant correlations between percent methylation of NR3C1 and elevated NISS scores were identified during the first week of life. Decreased DNAm of the total average percent methylation of NR3C1 and chronic NISS scores was observed (r (21) = −0.42, p = .047), as well as at specific CpG sites: CpG 6 (r (21) = −0.56, p = .005), CpG 8 (r (21) = −0.54, p = <.001), CpG 11 (r (21) = −0.47, p = .020), CpG 26 (r (21) = −0.63, p = <.001), CpG 29 (r (21) = −0.54, p = .008), and CpG 33 (r (21) = −0.43, p = .043). While no significant relationships were observed between percent DNAm and acute NISS scores during the first week of life, significant relationships were observed at three months. Specifically, increased acute NISS scores were associated with increased DNAm of the following CpG sites: CpG 6 (r (10) = 0.78, p = .003), CpG 16 (r (10) = 0.58, p = .050), CpG 17 (r (10) = 0.61, p = .030), CpG 18 (r (10) = 0.58, p = .45), CpG 19 (r (10) = 0.68, p = .020), and CpG 22 (r (10) = 0.72, p = .008). Increased chronic NISS scores were associated with also decreased DNAm (Table 3).

Changes in DNAm in Preterm Infants Overtime

Significant increases in DNAm of specific CpG sites on NR3C1 were observed though the total average DNAm of NR3C1 did not significantly change. The following CpG sites of NR3C1 demonstrated significant increases in methylation three months after NICU discharge as compared to the first week of life: CpG 8 (t (11) = 3.35, p = .006), CpG 14 (t (11) = 2.39, p = .035), CpG 17 (t (11) = 3.18, p = .009), CpG 18 (t (11) = 2.95, p = .013), CpG 20 (t (11) = 3.35, p = .006), and CpG 32 (t (11) = 3.09, p = .010). No significant changes in the total average DNAm of all the CpG sites on SLC6A4 were observed from the first week of life to 3 months after discharge in the preterm infant group.

Discussion

Our pilot study results demonstrate a significant difference between preterm infant and term infant’s DNAm of the SLC6A4 and NR3C1 genes. Significant relationships were identified between higher levels of stress associated with NICU hospitalization and alterations in DNAm of both the SLC6A4 and NR3C1 genes in preterm infants were also identified. Finally, changes in DNAm over time in preterm infants were identified in specific CpG sites of NR3C1.

These results provide new evidence linking the stress experienced by preterm infants to dysregulated methylation patterns of the SLC6A4 and NR3C1 genes. Furthermore, our data demonstrate that methylation patterns differ between preterm and term infants within the first week of life. These data suggest that methylation patterns for preterm infants may be important indicators of epigenetic differences and require further investigation to determine relationships with future health and development. These results are additive to the theoretical framework for preterm behavioral epigenetics described by Provenzi et al. (2018), positing that prenatal and postnatal adverse events impact the future health and development of preterm infants through epigenetic alterations which imprint on stress regulating genes.

When comparing the DNAm patterns between the preterm and term infant groups, significant differences were identified on CpG sites on both SLC6A4 and NR3C1. Preterm infants have previously demonstrated significantly different methylation patterns than their term counterparts when assessing atypical brain development (Wheater et al., 2022). Using epigenome-wide association studies (EWAS) data, Wheater et al. (2022) identified differences in over 8,000 CpG sites in saliva from preterm infants as compared to term infants as well as associated differences in white brain matter. Furthermore, genome-wide methylation differences between preterm and term infants have been described in both fetal brain and cord blood samples (Merid et al., 2020; Spiers et al., 2015). Our results support the hypothesis that gestational age is an important factor influencing methylation, linking health and developmental outcomes to gestational age at birth.

Our findings also demonstrate associations between elevated stress associated with NICU hospitalization and alterations in DNAm patterns of preterm infants. Similar to previous research, our findings indicate that elevated NICU stress is associated with dysregulation in methylation patterns of SLC6A4 and NR3C1 in preterm infants (Lester et al., 2015; Provenzi et al., 2015). Increased methylation of CpG sites 5 and 6 on the SLC6A4 gene was described in preterm infants who experienced high levels of pain related stress as compared to preterm infants with low levels of pain related stress while in the NICU (Provenzi et al., 2015). Similar to our results, hypomethylation of NR3C1 gene has been reported in preterm infants with high levels of stress (Giarraputo et al., 2017). Specifically, hypomethylation of CpG site 1 of the NR3C1 gene in preterm infants considered “high risk” based on their medical morbidity. These data also demonstrate the value of evaluating the methylation of individual CpG sites and not total/average DNAm across a gene. The relationship between DNAm and gene expression is complex and remains an area of active investigation. Existing research suggests that demethylation of entire genes or specific CpG sites is generally associated with increased gene expression. Conversely, hypermethylation is linked to decreased gene expression. Importantly, the functional impact of methylation depends on the location of the CpG sites, tissue specificity, and timing of exposure, all which are critical contextual variables when interpreting DNAm results (Aristizabal et al., 2020). Further explication of the DNAm patterns and relationships among early-life stress in the NICU and dysregulation of DNAm is needed before application of DNAm as a biomarker of early life stress. Despite this, our results provide early data to support future research describing phenotypical preterm infants who experience high levels of early life stress.

Additionally, the results from our pilot study builds on previous research linking DNAm alterations in preterm infants with suboptimal growth and development. Further, these results provide justification for further exploration of epigenetic alterations in other genes known to be associated with stress and development. For example, inflammatory factors have been found to be associated with DNAm of the BDNF and NRKBIA genes and early motor performance in preterm infants (Nist et al., 2024). Associations between DNAm alterations in preterm infants and suboptimal development include elevated DNAm of NR3C1 of HSD11B2 and slower oral feeding skill development have also recently been described (Griffith et al., 2025). As the science describing the relationships among early life stress in preterm infants and DNAm evolves, there is a need to connect these biologic processes with longitudinal measures of neurodevelopment to develop targeted stress reducing interventions in this population.

The results of our study must be considered in light of several limitations. First, the small sample size resulting from the pilot design makes it impossible to execute more sophisticated analysis to assess for the possibility of biological and technical variations in our sample. Data for the term infants was only collected at one point in time and did not include measures of infant stress since they were not admitted to the NICU. This limits the comparison of the two groups beyond the first week of life data. The corrected gestational age (CGA) at the time of data collection for the preterm infant group at the 3 month follow up appointment differed among subjects secondary to differing times of discharge from the NICU, leading to the possibility that differences identified may be a function of gestational age and not stress related to NICU hospitalization. Finally, despite being one of the most frequently utilized instruments to measure stress in the NICU, limitations of the NISS include poor concurrent validity with other stress measures, reliance on measurement of clinical events and not infant response, and the need for complete and accurate charting by the bedside nurse (Costa et al., 2025; Nist et al., 2024).

Despite these limitations, our pilot study offers a compelling foundation on which to build further research exploring the epigenetic embedding of early-life stress in preterm infants. As research continues to identify causes of altered health and development in preterm infants, it is important to consider the influence of stress on the genome as the preterm infants’ phenotype continues to be described. Identifying salient times in preterm infant development most susceptible to epigenetic alterations would allow for more precision in the management of these infants while also providing a life course framework in which to study the health and development of preterm infants.

Acknowledgement

We thank the Genomics and Microbiome Core Facility at Rush University for genetic data analysis and biostatistical support.

Funding

The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: Research reported in this publication was supported by the National Institute of Nursing Research of the National Institutes of Health under Award Number K23NR021043. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Other funding includes Children’s Wisconsin Peter Ruud Gift, Building Bridges to Research Based Nursing.

Footnotes

Ethical Approval

This study was reviewed and approved by The Medical College of Wisconsin’s Institutional Review Board (protocol number 00044310).

Declaration of Conflicting Interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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