To expand the knowledge about the consecutive expression of genes involved in the immune system development in preterm neonates and to verify if the environment changes the gene expression after birth we conducted a prospective study that included three cohorts: (A) extremely (gestational age (GA): 23–26 weeks;
According to the current statistics, invasive neonatal infections are responsible for about 36% of the estimated 4 million neonatal deaths annually [
The development of the fetal immune system begins at 4.5–6 weeks of gestation. Throughout gestation two major systems of fetus defense gradually develop: the nonspecific innate immune mechanism and the adaptive immune system [
Adaptive immune response is relatively immature at birth due to limitations of exposure to antigens in utero and due to the impaired functions of B and T cells. Therefore, the protection of the neonate against infection mainly depends upon passively acquired antibodies transferred from the mother and components of the innate immune system [
The ontogenesis of the immune system correlates with the developmental age of the fetus, but little is known when each of the respective aspects of the immune system matures normally in utero and what the consequences of the premature birth on these processes are.
It is known from studies conducted so far that the premature neonates show both qualitative and quantitative deficits in comparison with an adult or term neonate immune response. These studies were based on quantitative evaluation of the elements (cells, receptors) [
The introduction of the microarray technique into clinical studies was one of the most important turning points responsible for the dramatic progress in the field of human genetics during the last decade.
The aim of the study was to evaluate the consecutive expressions of genes involved in the immune system development in preterm neonates and to verify if the environment changes the gene expression after birth.
A prospective study was conducted between September 1, 2008, and November 30, 2010. The entry criteria were (a) preterm birth <32 weeks gestational age, (b) birth weight ≤1500 g, and (c) the need for respiratory support. All patients were outborn in local hospitals and transported to the Polish-American Children’s Hospital, which is a tertiary care unit for the region.
The majority of patients are referred from first-level neonatal care hospitals, which provide mainly for rural areas. Detailed perinatal history (birth weight, gestational age, and Apgar score at 1 and 5 minutes after birth) and history of treatment in the referral hospital (mechanical ventilation, oxygen therapy, surfactant treatment, and diagnoses) were taken on admission. Maternal fever/infection was used as surrogate for “clinical diagnosis of chorioamnionitis.” Data on histological chorioamnionitis were unavailable in most cases. Ureaplasma infection was defined as positive tracheal aspirate culture for
One hundred and twenty newborns were included in the study. The mean birth weight was 1029 g (SD: 290), and the mean gestational age was 27.8 weeks (SD: 2.5). The majority of the pregnancies were terminated by abrupt deliveries. Seven newborns died before the end of the first month of life; therefore, 113 children were included in the final analysis.
Forty-one infants were included in the extremely preterm cohort, 39 infants in the very preterm cohort, and 33 infants in the moderately preterm cohort. The clinical characteristics of the studied cohorts were presented in Table
Comparison of selected demographic data in the studied cohorts.
Extremely preterm (23–26 weeks) ( |
Very preterm (27–29 weeks) ( |
Moderately preterm (30–32 weeks) ( |
| |
---|---|---|---|---|
Birth weight, g ( |
|
|
|
<0.001a |
Gestational age (weeks) ( |
|
|
|
<0.001a |
Male gender | 24 (58%) | 20 (51%) | 17 (52%) | 0.76b |
Vaginal delivery/Cesarean section | 20/21 | 15/24 | 8/25 | 0.01b |
Multiple pregnancy | 4 (10%) | 8 (20%) | 6 (18%) | 0.56b |
Small-for-gestational-age infant | 4 (10%) | 1 (2%) | 5 (15%) | 0.1b |
Maternal hypertension | 8 (19%) | 7 (18%) | 2 (8%) | 0.74b |
Maternal diabetes | 3 (7%) | 2 (5%) | 0 | 0.79b |
Maternal fever/infection prior to delivery | 5 (12%) | 6 (15%) | 4 (13%) | 0.8b |
1st minute Apgar score (Me; 25–75th percentile) | 2 (1–4) | 5 (4–6) | 5 (2–6) | 0.01c |
5th minute Apgar score (Me; 25–75th percentile) | 6 (4–6) | 7 (6-7) | 7 (6–8) | 0.001c |
Delivery room intubation | 28 (69%) | 16 (40%) | 11 (33%) | 0.01b |
Surfactant therapy | 33 (81%) | 29 (73%) | 14 (43%) | 0.003b |
Initial a/A ratio |
|
|
|
0.01a |
Prenatal steroids | 10 (25%) | 12 (29%) | 22 (66%) | 0.01b |
Ureaplasma infection | 12 (29%) | 8 (21%) | 6 (18%) | 0.28b |
Pharmacological closure of patent ductus arteriosus | 22 (54%) | 23 (59%) | 11 (33%) | 0.08b |
Surgical closure of patent ductus arteriosus | 18 (44%) | 4 (10%) | 2 (6%) | <0.001b |
Length of mechanical ventilation (Me; 25–75th percentile) | 42 (25–52) | 3 (1–10) | 2 (0–6) | <0.001c |
Bronchopulmonary dysplasia | 40 (98%) | 24 (62%) | 6 (18%) | <0.001b |
After obtaining written informed consent from the parents, blood samples (0.3 mL) were drawn from all the study participants on the 5th and 28th day of life (DOL) for the assessment of whole genome expression in peripheral blood leukocytes. Subsequently, Ficoll isopaque gradient centrifugation (30 min, 2100 rpm, RT), two times wash in 1x PBS (12 min, 1600 rpm, 40°C), and finally RiboPure Blood Kit (Ambion, Life Technologies, Carlsbad, USA) were used for total RNA extraction. RNA concentration was measured with the use of NanoDrop Spectrophotometer (NanoDrop ND-1000; Thermoscientific, Waltham, USA), and RNA quality was determined by 2100 Bioanalyzer (Agilent, Santa Clara, USA).
100 ng of total RNA was used for the single microarray experiment. GeneChip Human Gene 1.0 ST Arrays (Affymetrix, Santa Clara, USA) were used. Whole transcript microarray experiment was performed. Details of microarray experiment were published previously [
To validate the results obtained by the microarray analysis real-time PCR technique was used. A total of 30 cDNA samples remaining after microarray analysis (30 patients) were used for the validation procedure. Samples were randomly selected from the studied groups.
From each sample 100 ng of cDNA was used for the single TaqMan Gene Expression Assay, and the expression level of 14 randomly selected genes was determined. Amplification reaction was performed with the use of TaqMan Universal PCR Master Mix and appropriate TaqMan probes (Life Technologies, Carlsbad, USA). Each sample was analyzed in duplicate.
Average between the expressions of endogenous controls (housekeeping genes: GAPDH and Actin-B) was used for determination of the relative expression levels with the use of
Following TaqMan Gene Expression Assays were used: Hs00900055_m1 (VEGF gene), Hs00952786_m1 (AK5 gene), Hs00217864_m1 (OLAH gene), Hs01030384_m1 (ILR2 gene), Hs00187022_m1 (ADAM23 gene), Hs00736937_m1 (DAAM2 gene), Hs00360669_m1 (CD177 gene), Hs00255338_m1 (KLRC4 gene), Hs00171191_m1 (FBN1 gene), Hs00539582_s1 (LRRN3 gene), Hs00196254_m1 (NELL2 gene), Hs00924296_m1 (MPO gene), Hs00541549_m1 (ABCA13 gene), Hs00197437_m1 (OLFM4 gene), GAPDH (Hs02758991_g1), and Actin-B (Hs99999903_m1) (Life Technologies, Carlsbad, USA).
Based on gestational age the studied group was divided into three cohorts: (a) extremely preterm infants (born below 27 weeks of gestation), (b) very preterm infants (born between 27 and 29 weeks of gestation), and (c) moderately preterm infants—born between 30 and 32 weeks of gestation. Gene expression profile was compared between the cohorts on the 5th and 28th DOL. Moreover, the changes between expression values between 5th and 28th DOL of whole study group were analyzed, too. The final step of analysis included comparison between the results of gene expression recorded on the 28th DOL in the group of extremely preterm infants and the results of gene expression recorded on the 5th DOL in the group of very preterm infants. The last comparison provided opportunity to compare both groups in the similar postmenstrual age.
Basic demographic data were compared using the one-way analysis of variance or Kruskal-Wallis analysis of variance as appropriate. Qualitative values were compared using the chi-square test.
Neonatal data used for the study was recorded daily during hospitalization in NICU in a prospective manner and stored in computer databases. For the purpose of the study the following data was used: sex, birth weight, gestational age, intrauterine growth parameters, Apgar score, incidence of preeclampsia, maternal diabetes, preterm rupture of membranes, chorioamnionitis, delivery type, delivery room management, presence of respiratory distress syndrome (RDS), length of mechanical ventilation, surfactant administration, use of ibuprofen for patent ductus arteriosus (PDA), PDA ligation, early- and late-onset septic episodes, ureaplasma infection, prevalence of intraventricular hemorrhage (IVH), periventricular leukomalacia (PVL), weight gain during NICU stay, and length of hospitalization. The microarray data were preprocessed using the R/Bioconductor package aroma.affymetrix [
Moderated
Multiple testing corrections, using the Benjamini-Hochberg procedure, were applied to control the false discovery rate (FDR) [
DAVID annotation tools were used to explore which predefined gene sets were significantly enriched in one group compared to another [
The mean fold change values representing differences in expression between the groups with regard to each of the 14 genes selected for validation were compared between the microarray and the TaqMan Gene Expression experiments. Student’s
All data were collected and analyzed in the adherence to the Minimal Information about a Microarray Experiment guidelines. All primary microarray data were submitted to GEO public repository and are accessible through GEO Series accession number GSE32472 (
One hundred and thirty-eight genes presented a monotone trend on the 5th and 308 on the 28th day of life. A summary of the number of differentially expressed genes between the groups is presented in Table
Summary of the number of differentially expressed genes between the studied groups.
Comparison |
|
| |
---|---|---|---|
5th DOL | Genes presented a positive monotone trend (expression in extremely preterm is lower than that in very preterm; expression in very preterm is lower than that in moderately preterm) | 56 | |
Genes presented a negative monotone trend | 82 | ||
|
|||
28th DOL | Genes presented a positive monotone trend | 253 | |
Genes presented a negative monotone trend | 55 | ||
|
|||
5th DOL versus 28th DOL (paired |
Genes whose expressions were significantly higher on the 28th DOL compared to those on the 5th DOL | 1522 | 150 |
Genes whose expressions were significantly lower on the 28th DOL compared to those on the 5th DOL | 2909 | 337 | |
|
|||
Extremely preterm infants measurement on the 28th DOL versus very preterm infants measurements on the 5th DOL | Genes whose expressions were significantly higher in the group of extremely preterm infants compared to those in the group of very preterm infants | 176 | 29 |
Genes whose expressions were significantly lower in the group of extremely preterm infants compared to those in the group of very preterm infants | 186 | 27 |
4431 genes were differentially expressed between 5th and 28th DOL (paired
When the samples collected on 28th DOL in extremely preterm infants and on 5th DOL in very preterm infants were compared, the expressions of 176 genes were significantly higher in the group of extremely preterm infants and the expressions of 186 gene genes were significantly lower. The difference in expression measured as a fold change of 56 genes was greater than 1.5.
Differentially expressed genes (with
A summary of the analysis is presented in Table
Summary of the pathway analysis for the differentially expressed genes between the groups. Pathways with FDR value less than 15% are shown.
Input to pathway analysis | Pathway name | FDR (%) |
---|---|---|
Genes presented a positive monotone trend on the 5th DOL, adjusted for multiple comparison |
T-cell receptor signaling pathway |
0.6 |
|
||
Genes presented a negative monotone trend on the 5th DOL, adjusted for multiple comparison |
No pathway | |
|
||
Genes presented a positive monotone trend on the 28th DOL, adjusted for multiple comparison |
Primary immunodeficiency |
0.01 |
|
||
Genes presented a negative monotone trend on the 28th DOL, adjusted for multiple comparison |
No pathway | |
|
||
Genes whose expressions were significantly higher on the 28th DOL compared to those on the 5th DOL (paired |
Graft-versus-host disease |
<0.001 |
|
||
Genes whose expressions were significantly lower on the 28th DOL compared to those on the 5th DOL (paired |
Cell cycle |
0.27 |
|
||
Genes whose expressions were significantly higher in the group of extremely preterm infants (measurements on the 28th DOL) compared to those in the group of very preterm infants (measurements on the 5th DOL); |
Graft-versus-host disease |
0.01 |
|
||
Genes whose expressions were significantly lower in the group of extremely preterm infants (measurements on the 28th DOL) compared to those in the group of very preterm infants (measurements on the 5th DOL); |
No pathway |
The differentially expressed genes between 5th and 28th DOL studies belonged to 21 pathways; 12 pathways were upregulated and 9 pathways were downregulated. Among 12 pathways whose expression was higher on 28th DOL than on 5th DOL were T-cell receptor signaling, hematopoietic cell lineage, and intestinal immune network for IgA production pathways.
When the expression of genes on the 28th DOL in the group of extremely preterm infants was compared to expression on the 5th DOL in the group of very preterm infants, only 3 pathways were differentially upregulated. Surprisingly, there was no pathway downregulated.
The validation procedure did not reveal significant differences between the results obtained with use of microarrays compared to real-time PCR technique [
The use of microarrays has provided a new opportunity for studying even 20000 human genes in a single experiment. The greatest advantage of this method is that it enables the assessment of a huge number of genetic factors (practically all human gene expression), while only a small amount of blood is necessary for testing, which is very important in preterm neonates.
In this study we assessed the whole genome expression in a cohort of preterm neonates in the 5th and 28th day of life. The cohort was subdivided into three groups depending on the gestational age (extremely preterm, very preterm, and moderately preterm), as described above. The genomic expression was compared between these three groups. To our knowledge this cohort is the first one designed to assess the whole genome expression among preterm neonates. The nature of the study was mainly exploratory.
Differential expression analysis revealed small subsets of genes that presented positive or negative monotone trends in both the 5th and 28th DOL in the three subgroups of patients, depending on the gestational age. On the contrary, we found a much higher number of genes that revealed positive or negative monotone trends when we compared gene expression between the 5th and 28th DOL in the whole cohort. In this comparison, significantly more genes were underexpressed in the 28th DOL rather than overexpressed. Finally we attempted to assess the impact of the extrauterine environment on genomic expression and compared the group of extremely preterm newborns on 28th DOL with very preterm newborns on 5th DOL. The analysis revealed a comparable number of genes that were over- and underexpressed.
Using pathway enrichment analysis, which allows us to identify network expression alterations that may be insignificant on the level of individual genes, we identified pathways that were differentially regulated with regard to gestational age. We found that most of the pathways that revealed a positive monotone trend on both the 5th and 28th DOL between all three age groups are involved in host immunity. Similarly we observed a significantly higher expression of pathways involved in immune response when we compared patients on 5th and 28th DOL, regardless of the gestational age. Both analyses revealed a relative increase in T-cell receptor signaling pathway and intestinal immune network for IgA production.
The neonatal immune system, both innate and adaptive, bears features of functional immaturity and therefore significantly contributes to neonatal morbidity and mortality [
Conversely, the adaptive immune response requires antigen exposure and develops gradually after birth. In newborns, cytokine production in response to stimuli exhibits polarization with Th-1 response downregulation [
In the current study we demonstrated for the first time a coherent trend of increasing expression of genes involved in T-cell receptor signaling with increasing gestational and chronological age. The functional changes of the developing immune system, including regulatory T-cell (Treg) maturation [
Intestinal immunity consists of two major components—innate defenses (gastric acid, protolithic enzymes, intestinal mucin, permeability, defensins, cathelicydins, and lectins (
Apart from genetic predisposition, disruption of the intestinal barrier secondary to hypoxic mucosal injury, bacterial colonization and formula feeding, immaturity of the gut mucosal immunity, and a relative deficiency of sIgA in the premature neonate have been implicated as a potential risk factor for NEC [
We know that birth initiates the conversion from the intrauterine sterile environment to the extrauterine confrontation with microbial and food antigens. This stimulus has a profound effect on the maturation of mucosa associated lymphatic tissues (e.g., Peyer’s patches) and other lymphoid tissues [
Our studied groups varied significantly with the percentage of children whose mothers received antenatal steroids. According to the literature, prenatal use of steroids may influence gene expression in children born preterm [
The effect of congenital infection, which was present in some of our patients, may influence gene expression.
All children included in our study were transferred from regional hospitals and the aim of the study was to assess the role of environmental factors occurring after birth. It should be noted that it was not feasible to directly assess the potential role of intrauterine factors, for example, by means of analysis of pattern of genome expression in lymphocytes obtained from cord blood samples. This situation might bias our results. Unfortunately, even assessment of cord blood samples would reflect only the very last phase of pregnancy, directly preceding the preterm delivery, which was triggered by various factors and not the physiological phenomena during pregnancy.
Last but not least, it should be noted that this investigation was performed in leukocytes, not, for example, in intestine tissues, which raises the question of whether gene expression of white blood cells reflects gene expression in the peripheral tissue. However, according to some researches, around 90% of transcripts are coexpressed in different tissues of the body, for example, in peripheral blood mononuclear cells and skeletal muscle cells [
Based on pathway enrichment analysis, we identified a few pathways which presented a positive monotone trend during the gestational age; most of them are involved in host immunity. We were not able to identify any pathway where expression of genes decreases with the gestational age. Despite differences in gestational age, patients with the same postconceptional age have a very similar expression of genes. The results are of sufficient interest to warrant further investigation on this subject.
The authors declare that there is no conflict of interests regarding the publication of this paper.
This study was supported by unrestricted Grants of Financial Mechanism of European Economic Area (PL0226) and the Polish Ministry of Science (E023/P01/2008/02/85).