NR3C1 gene methylation and cortisol levels in preterm and healthy full-term infants in the first 3 months of life

The first 1000 days of life after conception represent a period in which the developing brain is more subject to external influences, impacting learning, behavior and physical and mental health [1]. Exposure to intense and prolonged adversities in this period can unbalance the hypothalamic–pituitary–adrenal (HPA) axis, the main mechanism for stress regulation [2,3], and can be harmful to neurological and behavioral development [4–6].

Epigenetic alterations in NR3C1, responsible for glucocorticoid receptor transcription, seem to play an essential role in the HPA axis disarrangement. Methylation of NR3C1 decreases its expression and impairs the negative feedback loop’s sensitivity, further enhancing cortisol levels and subsequently predisposing individuals to disease [7]. Several studies describe associations between early stress in life (e.g., childhood adversities) and NR3C1 gene methylation in different tissues, including the brain [8], peripheral blood [9,10] and saliva [11,12]. NR3C1 gene methylation is related to the type and chronicity of adverse experiences in childhood [13]. Maternal stress during pregnancy has also been associated with methylation of NR3C1 in the cord blood and placenta [14–19]. Most studies have reported increased methylation of NR3C1 related to traumatic childhood experiences [2]. The 1_F region of the NR3C1 gene, which corresponds to region 1₇ in rodents, has been the most investigated after a pioneering study by Weaver et al. demonstrated an association between greater maternal care in rats and a lower HPA axis response to stress, with reduced methylation in the NR3C1 gene in pups [20].

One of human life’s earliest potential adversities is prematurity (with extremely or very low birthweight), which is always associated with the need for prolonged care in a neonatal intensive care unit (NICU) [21]. Premature birth itself may reflect prenatal stress [22]. Although essential for the survival of very low-birthweight infants, prolonged NICU admission is associated with intense stress due to excessive sound and light stimulation, frequent venipunctures, ventilatory support, infections and other sources of suffering, with reduced interaction with the mother and family [23,24]. Repeated activation of the stress response in a period of great sensitivity to external stimuli for brain remodeling can have severe consequences for the newborn [25].

Chronic and intense activation of the stress response system can cause an excessive and prolonged release of mediators, leading to chronic hyperactivation or hypoactivation of the HPA axis [6]. In the first week of life, a proportion of extremely preterm infants are transiently unable to produce cortisol at adequate levels to maintain homeostasis, with an adaptation of the HPA axis around 14 days of life [26]. However, late preterm and critically ill term infants also have an inadequate hormonal response to stress, keeping adrenocorticotropin and cortisol levels low [27]. The adrenal gland undergoes complex adaptive changes in the neonatal period, causing transient hypoadrenalism and a decline in cortisol concentration [28,29], both by gland immaturity and transitory enzyme deficiencies [26]. As the placenta produces large amounts of corticotropin-releasing hormone, the loss of stimulus at birth may compromise the ability to respond to HPA axis disease in the immediate neonatal period, causing relative adrenal insufficiency [30]. External factors, such as antenatal corticosteroid use, also reduce cortisol levels immediately after delivery [28].

A few studies have evaluated NR3C1 gene methylation in preterm infants admitted to the NICU [31–33] but showed contradictory results. Hypermethylation [31,32] and hypomethylation [33] were associated with more severe conditions. The heterogeneity of studies due to different methods of methylation analysis and different CpG sites analyzed makes it challenging to interpret the results. Many gaps remain regarding the impact of prematurity and NICU admission on the methylation status of the 1_F region of NR3C1. We hypothesized that preterm infants have hypermethylation of the 1_F region of NR3C1 at birth, reflecting prenatal stress, compared with healthy full-term infants. Another hypothesis is that the methylation status of this region in premature infants increases during NICU stay compared with the methylation status of healthy full-term newborns during the first 3 months of life.

This study aims to describe the methylation pattern of the 1_F region of the NR3C1 gene and cortisol levels from birth to hospital discharge in premature newborns during their stay in the NICU compared with healthy full-term newborns not hospitalized in the first 3 months of life.

Materials & methods

Study design, study population & ethics

This is an observational, longitudinal, prospective study. Preterm infants ≤1500 g and appropriate for gestational age [34], who were those with an expected prolonged NICU stay, were included consecutively from April 2018 to May 2019. Healthy full-term infants born at ≥37 weeks and appropriate for gestational age who were born in the same period were also included [35]. Data from patients who died or did not return for follow-up after entering the study were considered up to the point at which they participated. The research ethics committees of the institutions involved approved the study. All parents or legal guardians signed written informed consent agreeing to their children’s participation in the study and to the inclusion of their data, which have been anonymized.

Collection of sociodemographic, clinical & laboratory data

Sociodemographic and clinical variables of neonates and pregnant women were extracted from medical records. Gestational age was determined by first-trimester ultrasound, supplemented by the date of the last menstrual period. In the absence of these data, the neonatologist used the newborn’s physical examination (Ballard and/or Capurro methods). For preterm infants, severity scores (Clinical Risk Index for Babies [36] and Score for Neonatal Acute Physiology Perinatal Extension [37]) were collected on admission to the NICU, and the Neonatal Therapeutic Intervention Scoring System score [38] was recorded daily.

Umbilical cord blood samples were collected from all infants at birth (day 0: D0) to analyze NR3C1 gene methylation. Peripheral blood samples were collected for testing of NR3C1 methylation and cortisol levels on day 5 of life (D5) in all infants (preterm and term newborns) and on day 30 of life (D30) and day of hospital discharge (Dd) in preterm infants. Term newborns had buccal swabs collected for both exams (NR3C1 methylation and cortisol levels) on D30 and D90 of life.

For NR3C1 methylation testing, blood samples were collected in tubes containing potassium ethylenediaminetetraacetic acid as an anticoagulant and initially stored at 2–8°C at the maternity hospital for up to 72 h before being transported to the epigenetic laboratory, where they were aliquoted and held at -80°C until the subsequent processing steps were carried out. Buccal swabs (Oracollect-DNA, OCR-100, DNA GENOTEK Inc, Ottawa, Canada) were collected by placing the swab where the gum touched the cheek mucosa. When positioned, the swab was gently rubbed ten-times in this region and on the opposite side of the mouth, with repetitive forward and backward movements. Then the swab was placed in the tube containing the collection liquid, which was closed and vigorously inverted for 15 s. The tubes with the biological material were stored at laboratory room temperature (20–25°C) until being analyzed up to 3 months after collection [39].

For cortisol analysis, preterm newborns had blood samples collected in a gel tube on D5, D30 and D90, and term newborns on D5. After clot retraction, they were centrifuged for 12 min at 3200 r.p.m. Salivary samples were collected from term newborns on D30 and D90 using a tube with a V-bottom (Salivette, Sarstedt, São Paulo, Brazil), which was kept in the mouth for 3 min or as long as necessary to become saturated with saliva. Then they were centrifuged for 5 min at 4000 r.p.m. Serum and salivary cortisol samples were always collected at 8 am and stored at 2–8°C in the maternity hospital for up to 72 h until they were transported to the epigenetic laboratory.

A neonatologist of the NICU team collected all samples.

Methylation analysis of NR3C1

Genomic DNA was extracted from blood mononuclear cells or buccal cells using a DNeasy Blood & Tissue Kit (#69506, Qiagen, Hilden, Germany). The quantity and purity of DNA were determined using a NanoDrop™ spectrophotometer 2000c (ThermoFisher Scientific, MA, USA). The amount of 1000 ng of genomic DNA was bisulfite-converted using the EZ-96 DNA Methylation Kit (#D5002, Zymo Research, CA, USA). According to the manufacturer’s instructions, the converted DNA was eluted with 24 μl of the M-elution buffer. Next, 2 μl of bisulfite-converted DNA was amplified by PCR on the Veriti™ 96-well thermal cycler (Applied Biosystems, CA, USA) using a PyroMark PCR kit (#978703, Qiagen) in a total volume of 50 μl containing 0.2 μM of primers and PyroMark PCR Master Mix; 1% agarose gel electrophoresis was used to confirm DNA amplification. Biotinylated PCR products in a total volume of 40 μl were immobilized on streptavidin-coated sepharose beads (GE Healthcare, IL, USA). Subsequently, pyrosequencing was performed using PyroGold PyroMark Q96 reagents on the PyroMark Q96 ID (both from Qiagen). The methylation percentage of each CpG site was generated automatically using the PyroMark Q96 software (v. 2.5.8) with standard quality control settings. PCR primers and pyrosequencing primers were designed with PyroMark Assay Design SW2.0 (Qiagen) to target 40 CpG sites of the NR3C1 1_F region (8–47, according to standard numbering sites revised by Chalfun et al. [40]) (Figure 1). A detailed protocol with the steps for methylation analysis is provided in Supplementary Material 1.

Analysis of cortisol

Serum cortisol was measured using an enzymatic chemiluminescence assay with a Cobas 8000 analyzer series e602 (Roche Diagnostics, São Paulo, Brazil), and salivary cortisol was measured using liquid chromatography–tandem mass spectrometry, according to the manufacturer’s instructions (Waters/Micromass, Manchester, UK).

Statistical analysis

Depending on the required analyses, methylation percentages were reported as mean and standard deviation or median and interquartile range (IQR). Cortisol levels were reported as z-scores due to different reference levels for blood and saliva samples. The medians between groups were compared using the Mann–Whitney U test. Using generalized linear models, longitudinal analyses of the percentage of methylation were performed for each CpG site and the 40 CpG sites in conjunction (total methylation). The analysis was performed using the PROC GENMOD procedure in SAS OnDemand for Academics (SAS Institute, Inc., NC, USA), with a log-link function and γ-distribution due to the asymmetric distribution of the outcome variable. We fitted models to our data, including the variables time, group, and the time*group interaction. Autoregressive, exchangeable, independent and unstructured covariance matrix structures were tested, and the appropriate structure was selected for each site based on the ‘quasi-likelihood under the independence model criterion’ parameter. Longitudinal analysis of the z-scores of cortisol levels was performed using a linear mixed-effects model (PROC MIXED procedure in SAS) with an unstructured covariance matrix. The trend of changes in the percentage of methylation and cortisol levels over time (increase or decrease) in each group and the comparison between groups of the course of methylation percentage and cortisol levels over time were evaluated.

Results

Study data, demographic & clinical features

A total of 46 premature babies and 49 full-term babies were included in the study. In the preterm group, 34 babies completed the study with all samples collected; five died between D5 and D10 but had samples from D0 and D5 analyzed, and seven had no Dd sample. In the group of full-term newborns, 35 completed the study until D90, one returned only on D5, and 13 did not return for follow-up.

In premature infants, the median gestational age was 28.5 weeks (IQR: 27–30), and the median birth weight was 1075 g (IQR: 860–1345); 76% were born by cesarean section, with a 5-min median Apgar score of 8 (IQR: 8–9). The median length of stay in the NICU was 61 days (IQR: 38–74). The mean Dd of the preterm infants who had samples collected on this day was widely variable (69.5 ± 32 days); the median was 62 days (IQR: 45–75). The median Neonatal Therapeutic Intervention Scoring System score was 19 (IQR: 16–25.8). The most frequent clinical conditions in this group were metabolic disorders (56.5%), patent ductus arteriosus (47.8%), hyaline membrane disease (43.5%) and bronchopulmonary dysplasia (41.3%). Confirmed sepsis occurred in 19.6%. Approximately 80% used nasal continuous positive airway pressure, 53% underwent invasive mechanical ventilation, and 37% used inhaled corticosteroids at the time of hospital discharge. Term infants had a median gestational age of 39 weeks (IQR: 38–40) and median birth weight of 3320 g (IQR: 3150–3575); 45% were born by cesarian section, with a 5-min median Apgar score of 9 (IQR: 9–9). These and other demographic and clinical features are shown in Tables 1 & 2.

Comparison of the magnitude of methylation between groups

At birth, we detected methylation (any percentage other than zero) at 32 sites in preterm infants and 26 in full-term infants (Table 3). Preterm newborns showed significantly higher percentages of methylation in CpG 12 (p = 0.029), 42 (p = 0.001) and 47 (p = 0.005) compared with full-term infants. On D5, the only significant difference between groups was a lower percentage of methylation at CpG 33 (p = 0.036) in preterm infants; on D30, methylation percentages in preterm infants were significantly lower in CpG 21 (p = 0.003), 36 (p = 0.004), 37 (p = 0.013), 38 (p = 0.030) and 47 (p = 0.046), and higher in CpG 42 (p = 0.001) and 45 (p = 0.043). On Dd in preterm infants, compared with D90 in full-term infants, the percentage of methylation was significantly lower in CpG 21 (p = 0.004) and 36 (p = 0.008) and higher in CpG 42 (p = 0.002) (Figures 2 & 3 & Supplementary Tables 1–4). Regarding the mean overall methylation percentage of sites 8–47 of exon 1_F, preterm newborns had a significantly higher percentage methylation at birth compared with full-term infants (0.54 ± 1.94 vs 0.40 ± 1.76; p = 0.001), but there was no difference between the groups on D5 (0.36 ± 1.64 vs 0.44 ± 1.90; p = 0.793), on D30 (0.27 ± 1.21 vs 0.59 ± 2.33; p = 0.760) or on the last day (0.28 ± 1.12 vs 0.51 ± 2.09; p = 0.450) (Table 3 & Supplementary Tables 1–4).

The trend of methylation over time in each group

In preterm infants, methylation percentages decreased over time at eight sites (CpG 12, 15, 16, 19, 30, 31, 34 and 41), increased at two sites (CpG 28 and 32) and remained undetectable at six sites (CpG 8, 10, 20, 25, 27 and 29). In 24 sites, the changes were not significant (Figures 2 & 3 & Supplementary Table 5). In full-term infants, methylation percentages decreased over time at 11 sites (CpG 18, 24, 26, 27, 30, 31, 33, 40, 43, 44 and 46), increased at eight sites (CpG 9, 10, 13, 21, 32, 36, 37 and 38) and remained undetectable at five sites (CpG 23, 25, 28, 3 and 41). In 16 sites, the changes were not significant (Figures 2 & 3 & Supplementary Table 5). It was not possible to analyze changes at CpG 25, as the percentage methylation remained undetectable on all occasions in both groups. The mean overall methylation percentage of sites 8–47 exhibited no significant change over time in the full-term infant group (p = 0.3116) but decreased in the preterm infant group (p = 0.0241) (Figure 4 & Supplementary Table 5).

Comparison of methylation changes over time between groups

Comparing the changes in the methylation percentage over time between groups showed significant differences at 13 sites (CpG 9, 10, 16, 19, 21, 24, 27, 28, 33, 36, 40, 41 and 47), nine of which (CpG 9, 10, 16, 19, 21, 27, 36, 47 and 33) showed smaller changes in methylation percentage in the preterm group compared with the full-term infants. It was impossible to compare changes at CpG 25 between groups, as the percentage of methylation remained undetectable on all occasions in both groups. A significant difference was also observed when comparing the changes in the mean overall methylation percentage of exon 1_F between groups (p = 0.0336) (Figure 4 & Supplementary Table 6).

Analysis of cortisol levels

On D5, cortisol levels were significantly higher in preterm infants than in full-term infants (p < 0.0001). On D30 and the day of the last measurement, there was no significant difference between the groups (p = 0.245 and p = 0.866, respectively) (Figure 5 & Supplementary Table 7). Between D5 and the last measurement, cortisol levels increased significantly in full-term infants (p = 0.0177) but showed a nonsignificant decrease in preterm infants (p = 0.6375) (Figure 5 & Supplementary Table 8). The comparison of changes in cortisol levels over time between the groups showed no difference (p = 0.0879) (Figure 5 & Supplementary Table 9). The intra- and inter-assay coefficients of variation (imprecision) for measured cortisol levels were: for serum cortisol, 6.7% (intra-assay) and 7.9% (inter-assay); for salivary cortisol, 3.52% (intra-assay) and 3.56% (inter-assay).

Discussion

In this study we observed a similar number of methylated sites (except on the day of birth) in the 1_F promoter region of NR3C1 in both preterm and full-term newborns during the first 3 months of life. Hypermethylation was present in a few sites in the preterm infants at birth, but hypomethylation predominated later. Preterm infants had fewer sites with changes in the percentage methylation over time (decrease or increase), but the predominant change in specific sites in both cohorts was a decrease. On the other hand, the mean overall methylation percentage of the studied sites of exon 1_F showed no significant change over time in full-term infants, but decreased in preterm infants. The clinical meaning of these observations is still obscure and difficult to interpret given that we expected to see an overall increase in methylation percentages in the preterm group during the NICU stay. Comparing the changes in methylation over time between groups, significant differences were observed in one-third of the CpG sites. In parallel, cortisol levels on D5 were higher in preterm than in full-term infants. While cortisol levels did not significantly change over time in preterm infants, they increased in full-term infants. Still, no significant difference between groups was observed when comparing these changes over time. The higher cortisol levels observed in preterm infants on the fifth day of life compared with full-term newborns may reflect the clinical severity of preterm infants included in this study.

Some studies have assessed factors such as prematurity and NICU admission and compared the methylation status of different genes in preterm and term newborns [41–44]; however, few have evaluated the NR3C1 gene [31–33], which is crucial in regulating the HPA axis.

Our results were intriguing and the opposite of what we expected. Our hypothesis – based on theories about HPA dysregulation associated with stress-induced glucocorticoid receptor gene methylation [40,45–47] – was that prematurity and adverse experiences during NICU admission would result in an increased percentage of methylation in exon 1_F of NR3C1 and increased cortisol levels in preterm infants from birth to hospital discharge compared with healthy full-term newborns discharged home and evaluated in the same time window. Studies have demonstrated that prolonged and repeated exposure to painful procedures in the neonatal period affects brain organization and neurodevelopment and is associated with increased methylation in stress-related genes [23,48]. This evidence supports our hypothesis. However, we observed that, except at birth, the predominant difference between groups was hypomethylation in preterm infants compared with full-term infants. It is possible that the inadequate intrauterine environment, culminating in preterm labor, represents a more intense source of stress than the subsequent adversities experienced in the NICU after birth. The first days of hospitalization, in which the premature infant is submitted to invasive and painful procedures and exposed to an excess of light and sound stimuli, are the most critical. As the premature infant improves, these negative stimuli decrease and are replaced by a more appropriate environment, including increasing time with parents. Currently, more humanized strategies are employed, with the encouragement of the kangaroo method, skin-to-skin contact with the mother in the first hour of life when possible, greater inclusion of the family in care [49–51], precautions regarding noise and light, and measures that provide some comfort and better organization to the newborns [25].

However, the findings of our study contrast with the results obtained by Kantake et al., who conducted the only study comparing exon 1_F methylation of NR3C1 in preterm and full-term infants [31]. In that study, CpG sites 1–33 were analyzed only at birth and on the fourth day of life, and preterm infants had higher weight and gestational age than our patients. The authors observed higher methylation percentages than ours and those reported in similar studies [11,14,15,19,33,52,53]. At birth, Kantake et al. reported a higher methylation rate in preterm infants only at CpG 4, and a lower percentage in CpGs 1, 5 and 8 compared with full-term infants [31]. On the fourth day, preterm infants had a higher methylation rate at several sites, including 15, 16, 21, 25, 26, 27 and 28. Between birth and the fourth day of life, these authors found an increase in methylation percentage in 11 sites in preterm infants (CpG 1, 2, 8, 9, 10, 14, 16, 25, 26, 28 and 29) and stability in term newborns. In our study, changes in methylation over time showed a predominance of decrease versus increase in both cohorts. Differences in methylation course between the two cohorts were present in 13 of the 40 sites studied. There are no studies in the literature with a similar longitudinal analysis. Despite the differences in the population of preterm infants and the follow-up time between our study and that of Kantake et al. [31], the results of the two studies seem contradictory. It is possible that the simplified information of a single gene obtained through these studies has captured different moments of a highly dynamic process and does not reflect the complexity of the interactions between the different genes and transcription factors involved. According to studies in animals and humans, epigenetic processes can be dynamic, with more remarkable plasticity in the first years of life, a fundamental phase for the growth and development of the brain and other organs, and less intense, but also possible in more advanced stages of life, such as adolescence and adulthood [25,54,55].

Numerous transcription factors bind to sites included in our study, such as NGFI-A, AP1 and SP1 [56]. Differences in the methylation status of specific CpG sites interfere with transcription factor binding and gene expression. For example, CpG sites 16–21, 37 and 38 are canonical binding sites for NGFI-A, while CpG sites 12, 13 and 30–32 are noncanonical binding sites [8]. NGFI-A has essential regulatory functions, including the growth and differentiation of nerve cells in the central and peripheral nervous systems [57]. NGFI-A binding sites have been more widely studied since Weaver et al. demonstrated alterations in methylation in the equivalent promoter region in rodents, according to maternal behavior [20]. Human studies indicated an association between early stress in the perinatal period and childhood and increased NR3C1 methylation at exon 1_F in CpG 37 [9,14,58]. Lester et al. observed increased methylation at this site in preterm infants at higher neurobehavioral risk [53]. Raffetti et al. reported an association between methylation at this site and drug and alcohol use in adolescents [59]. We observed an increase in methylation over time at some NGFI-A binding sites in full-term newborns, while in preterm infants, changes occurred in both directions, but decreases occurred in more NGF1-A binding sites than increases. Giarraputo et al. observed hypomethylation at site CpG 35 before hospital discharge (mean of 99 days) in more severe compared with less severe preterm infants [33]. We observed lower methylation percentages at sites 21 and 36 in preterm infants before hospital discharge compared with full-term infants. Compensatory epigenetic mechanisms may be triggered in premature newborns to increase the activity of the NR3C1 gene in situations of great adversity, such as admission to the NICU.

Another unexpected result was related to cortisol levels. Several studies have reported low serum cortisol levels in the neonatal period due to relative adrenal insufficiency, mainly in premature and critically ill infants [27–30,60]. Grunau et al. observed lower production of cortisol in extremely preterm infants exposed to painful procedures during their NICU stay [60]. Our study observed higher cortisol levels in preterm infants on the fifth day of life compared with full-term newborns. Some factors that denote greater severity, such as confirmed sepsis (19.6%), hyaline membrane disease (43.5%) and the need for mechanical ventilation (53.3%) in preterm infants included in our study, may be related to this exacerbated cortisol response after birth. Other factors, such as low Apgar score and vaginal delivery, should also be considered [28]. We did not observe differences in cortisol levels between preterm and full-term newborns on the 30th day of life and before hospital discharge. There was also no difference in the changes in cortisol levels between the two groups over time. However, premature newborns showed a decreasing trend of cortisol levels (though not significant) and decreasing mean percentage methylation over time. In contrast, term newborns showed increasing cortisol levels and mean methylation percentage over time. These findings support a relationship between cortisol and epigenetic changes in NR3C1 in the perinatal period. They are consistent with the premise that methylation of NR3C1 decreases gene expression and impairs the negative feedback loop’s sensitivity, which further enhances cortisol levels. However, data relating corticosteroid levels to NR3C1 gene methylation are inconsistent, showing both positive and negative associations [46]. Mediators other than cortisol may be involved in gene methylation changes in response to stress. Several neurochemical systems (e.g., protein complexes, activators and suppressors at the transcriptional level) are influenced by adverse experiences and interact, generating different outcomes [61].

Finally, in the present study, the percentage of methylation found at several sites was low and, in a significant number, was zero. The highest mean percentage in each site was around 4%, and considering all sites together, the mean percentage was below 1%. Would low methylation levels in peripheral tissues be enough to alter gene expression? Other studies that have evaluated perinatal and childhood adversity and methylation in the 1_F exon of NR3C1 in different tissues and cell types found similar results in the cord blood of newborns [14,16], in buccal swabs of infants [33,53], in the peripheral blood of newborns and infants [32] and in the saliva of preschool children [11]. In a systematic review of the association between NR3C1 gene methylation and perinatal stress experiences, Palma-Gudiel et al. reported less than 5% methylation in the included studies [46]. According to Leenen et al., minor methylation variations in the NR3C1 gene CpG sites result in the reorganization of transcription and production of protein isoforms in tissues, causing diseases such as Type 2 diabetes, depression, schizophrenia, arterial hypertension and cardiovascular diseases [62].

This study has strengths and limitations. It is the first study to perform a longitudinal analysis of methylation of the 1_F region of the NR3C1 gene and cortisol levels in very low-birthweight infants and full-term infants on four different occasions in the first 3 months of life. Our aim was exclusively descriptive, because healthy full-term infants are not an ideal control group for very low-birthweight infants who remain in the NICU; full-term infants were included only as a reference for the expected methylation pattern in supposedly less stressful conditions. The only similar study (Kantake et al.) evaluated methylation in both cohorts at birth and on the fourth day of life [31]. The other few studies assessed the associations of clinical and demographic variables with the magnitude of NR3C1 methylation only in preterm infants [32,33]. As a first limitation, a convenient sample was used based primarily on budget restrictions. Also, we did not find literature data on the expected difference in methylation rate between the study groups to calculate the ideal sample size. The only study comparing methylation rates between preterm and full-term newborns reported statistical significance but not the magnitude of the difference [31]. It is possible that due to insufficient sample size, significant differences at sites other than those described in this study may not have been detected. A second limitation was using different tissues (cord blood, peripheral blood and saliva) to analyze NR3C1 gene methylation. However, although tissue methylation patterns are tissue-specific [63,64], there is a high degree of similarity between subjects [65], and intraindividual DNA methylation can be more variable than interindividual DNA methylation [66]. Studies show that there is a correlation between CpG island methylation levels in cord blood [67], peripheral blood [68,69], buccal cells [64,70], saliva [71] and brain tissue. Given the impossibility of using brain tissue in clinical studies, peripheral tissues are used to assess epigenetic patterns in newborns, children and adults exposed to adversities. They are considered representative of the global methylation profile [64,67–70]. A third limitation of the study was the collection of a single blood or saliva sample for cortisol analysis at each time frame and the changes in the laboratory cortisol reference values. However, premature and term infants do not show the same patterns of the circadian cortisol cycle as are observed in adults [72]; the cycle is generally established around 2–4 months of age [73,74]. Even so, all the samples were collected at 8 a.m, and the variability in cortisol reference values due to blood or saliva collection was minimized using the z-score. A fourth limitation was the asynchrony between the Dd time point of the preterm group, which took place at an average of 69.5 days, and the D90 time point of the full-term group. While these time points may not be comparable, they show the direction of the methylation course over time in both groups. Finally, we did not control for several possible confounding variables that could influence the NR3C1 gene methylation course or the cortisol levels, and the results of this study should be interpreted with caution. The impact of demographic, clinical and social covariates on methylation and cortisol levels will be the subject of a later study.

Conclusion

This study showed that NR3C1 exon 1F hypomethylation was predominant in preterm newborns who remained in the NICU compared with non-hospitalized term infants in the same time window. Higher methylation levels at birth, associated with higher cortisol levels on the fifth day of life in preterm infants and with subsequent decline, suggest that the prenatal stress and unfavorable conditions of prematurity may impact the epigenome and that postnatal factors may modify these acquired epigenetic changes in a dynamic process. More studies on preterm infants are essential, because the perinatal period represents a critical phase for modulating brain architecture and functioning.

Future perspective

Many gaps remain to be elucidated in future research about the relationship between early stress and epigenetic changes. We are examining a single gene (NR3C1), but several other genes, such as NFKBIA, FKBP5, BDNF, GILZ and SLC6A4, are related to endocrine regulation (HPA axis and stress response) [3,44]. Some of these genes can influence each other, interacting in situations of adversity. Methylome-wide association studies examine thousands of genes but have reduced coverage for CpG sites in each gene and may not identify genes of interest [75,76]. The combination of methylome-wide studies and studies focused on a single gene may contribute to elucidating the complexity of epigenetic interactions. In addition, assessments of the association between methylation status and levels of mRNA and transcription factors are needed to provide more direct evidence on the role of epigenetic changes in gene expression [8], allowing us to make correlations between methylation patterns and phenotypic profiles [56].