Conceived and designed the experiments: AAL HBB. Performed the experiments: HY HBB AAL. Analyzed the data: AAL LWJ HBB. Contributed reagents/materials/analysis tools: AAL LAW HBB. Wrote the paper: AAL LWJ HBB HY. Consented patients: LAW.
The authors have declared that no competing interests exist.
Preterm parturition is characterized by innate immune activation and increased proinflammatory cytokine levels. This well established association leads us to hypothesize that preterm delivery is also associated with neonatal T lymphocyte activation and maturation.
Cord blood samples were obtained following term, preterm, and deliveries complicated by clinical chorioamnionitis. Activation marker expression was quantitated by flow cytometric analysis. Infants born following preterm delivery demonstrated enhanced CD4+ T lymphocyte activation, as determined by CD25 (Term 9.72% vs. Preterm 17.67%, p = 0.0001), HLA-DR (Term 0.91% vs. Preterm 1.92%, p = 0.0012), and CD69 expression (Term 0.38% vs. Preterm 1.20%, p = 0.0003). Neonates delivered following clinical chorioamnionitis also demonstrated increased T cell activation. Preterm neonates had an increased frequency of CD45RO+ T cells.
Preterm parturition is associated with neonatal CD4+ T cell activation, and an increased frequency of CD45RO+ T cells. These findings support the concept that activation of the fetal adaptive immune system in utero is closely associated with preterm labor.
Preterm birth impacts up to 12% of all deliveries, is the leading cause of neonatal morbidity and mortality, and accounts for approximately one half of long-term neurologic morbidity in children. While advances in medicine have improved the survival of premature infants, the rate of preterm birth in the United States has not decreased
Several lines of evidence support the association of infection with preterm labor. Intrauterine infection or the systemic administration of lipopolysaccharide (LPS) to pregnant animals can result in preterm labor and delivery
There is a well-described association between fetal inflammation and preterm birth. The fetal inflammatory response syndrome (FIRS) was originally described by Gomez, et al in 1998, characterized by increased cord blood IL-6 levels, and associated with a high rate of severe neonatal morbidity including respiratory distress syndrome, neonatal sepsis, bronchopulmonary dysplasia, intraventricular hemorrhage, periventricular leukomalacia and necrotizing enterocolitis
T cell activation can be assessed by the presence of activation markers on the cell surface, furthermore T cell activation in the presence of cognate antigen leads to the development of memory CD45RO+ T cells
T lymphocytes were identified in cord blood samples based on cell size (forward scatter) and cellular complexity or granularity (side scatter), and CD3 expression. To evaluate the effect of preterm delivery on neonatal T cells, whole blood was stained
Cord blood samples obtained simultaneously from an uncomplicated term delivery (control) and a delivery complicated by preterm labor were stained with fluorochrome labeled antibodies for cell surface activation markers (CD25, HLA-DR, and CD69, y axis of each dot plot) and subjected to three-color flow cytometric analysis. Cells were gated based on size and CD3 expression, quadrants were set based on isotype controls, and samples were compensated electronically for overlap in fluorescent emission. The percentage of cells in each quadrant is indicated. These data are representative of 34 term infants and 12 preterm infants.
Cord blood samples from idiopathic preterm and uncomplicated term deliveries were stained with labeled antibodies for cell surface activation markers (CD25, CD69, and HLA-DR) and subjected to flow cytometric analysis. Median CD25 expression in term infants (n = 34) was 9.72% (Interquartile range (IQR): 8.18, 11.30), 17.67% (IQR: 13.40, 21.02) in preterm infants (n = 12), and 16.21 (IQR: 10.91, 17.26) in infants with chorioamnionitis (n = 8). Median CD69 expression was as follows: 0.38% (IQR: 0.29, 0.70) in term infants, 1.20% (IQR: 0.67, 2.12) in preterm infants, and 1.58% (IQR: 0.56, 2.42) in infants with chorioamnionitis, Median HLA-DR expression was as follows: 0.91% (IQR: 0.40, 1.50) in term infants, 1.92% (IQR: 1.36, 2.36) within preterm infants, and 3.21% (IQR: 2.11, 5.13) within infants with chorioamnionitis (* represents p-value <0.05, ** represents
After finding significant neonatal T cell activation following preterm delivery, we assessed T cell activation in infants delivered to mothers with a diagnosis of clinical chorioamnionitis, defined as the presence of maternal fever, tachycardia, and uterine tenderness and a decision by the treating physician to institute intrapartum parenteral antibiotics. This group included both term and preterm infants. We found that infants delivered in the setting of chorioamnionitis also demonstrated significant T cell activation, with increased CD25 (P = 0.0149), CD69 (P = 0.0104), and HLA-DR expression (P = 0.0004), compared to term infants without clinical chorioamnionitis (
Antigen specific activation is involved in the formation of CD45RO+ T cells
Memory T cell frequency was compared between the term group (n = 23) and the preterm group (n = 15). Preterm neonates had an increased median percentage of memory CD4+ T cells (9.40%, IQR: 6.40, 28.10), CD45R0+/CD45RA-, compared to the median percentage of memory CD4+ T cells in term infants (6.10%, IQR: 2.37, 9.50) in term infants (
Preterm parturition is associated with fetal and neonatal inflammation. Several researchers have described increased neonatal serum cytokine levels following preterm delivery;
Following deliveries complicated by preterm labor, there was increased expression of the activation markers CD25, HLA-DR, and CD69 on CD4+ T cells within cord blood as measured by flow cytometry. For all activation markers assessed there was a statistically significant increase, when preterm neonates were compared with term neonates. Additionally, gestational age at the time of spontaneous birth was inversely correlated with T cell activation in the absence of clinical chorioamnionitis (data not shown). These findings suggest that early preterm delivery is associated with higher levels of immune activation which is in agreement with other studies that have shown that early preterm delivery is more likely to be secondary to an infectious etiology
Our findings support the concept that the fetal adaptive immune response and preterm labor are linked. As lymphocytes produce cytokines upon activation, activated T cells may be responsible, in part, for the increased fetal cytokine levels found in association with preterm birth. T cell activation in the setting of antigen or cytokine exposure can also lead to the development of memory T cells, characterized by the presence of CD45RO expression and the absence of CD45RA expression. As memory cells produce significantly more cytokines than do naïve T cells, increased levels of memory T cells could further contribute to the increased and rapid neonatal serum cytokine levels seen in the setting of preterm delivery. Investigators have observed an increased frequency of CD45RO+ T cells following deliveries complicated by intrapartum infection
The present study extends the work of other investigators, who have described innate immune activation following preterm birth
Infants born to mothers with clinical chorioamnionitis defined as a temperature more than 100.4°F, tachycardia, and uterine tenderness requiring antepartum and/or intrapartum antibiotics, also demonstrated T cell activation. Interestingly, T cell activation within preterm neonates was similar to infants delivered following chorioamnionitis. Both of these conditions were associated with significantly more T cell activation, compared to neonates following normal, term birth. These findings demonstrate an association between fetal lymphocyte activation, chorioamnionitis, and preterm labor; and support the concept that preterm parturition and chorioamnionitis are associated with significant immune activation and inflammation, impacting both the innate and adaptive immune systems. Further studies are needed to better understand whether fetal/neonatal immune activation mediates preterm birth, or is a result of preterm labor.
The role of the maternal immune system in preterm parturition also warrants further study. Although cytokines clearly mediate immune activation, it is not clear whether they originate from the maternal or fetal compartment or how easily cytokines cross materno-fetal boundaries. Moreover, little is known about maternal immune activation in the setting of preterm parturition. Identifying the source of cytokines associated with preterm birth, and their influence on both maternal and neonatal immune function, will increase our understanding of the immunopathogenetic processes associated with preterm birth.
Cord blood samples were obtained from patients delivering at University Hospitals of Cleveland/MacDonald's Women Hospital and The Johns Hopkins Hospital after obtaining written informed consent. The study was approved by the Institutional Review Boards of both institutions. Samples were obtained from term infants born without maternal complications (gestational age equal to or greater than 37 weeks, n = 34), preterm infants without clinical chorioamnionitis delivered following spontaneous, idiopathic preterm labor (gestational age less than 35 weeks, n = 15), and preterm and term infants delivered from mothers with documented, clinical chorioamnionitis, defined as maternal fever (Temperature above 100.4°F), tachycardia, and uterine tenderness requiring antibiotic treatment (n = 8). Exclusion criteria included: pre-eclampsia, premature rupture of membranes, congenital anomalies, significant maternal illness, maternal or fetal indications for delivery, and prostaglandin induction of labor. Demographic and clinical details from patients delivering at University Hospitals were obtained from the medical record (
Demographics | Term (n = 17) | Preterm (n = 12) | p value |
Mean maternal age (SD) |
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Mean gestational age in weeks (SD) |
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Mean birth weight in grams (SD) |
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Median APGAR 1 min (IQR) |
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Median APGAR 5 min (IQR) |
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Prenatal steroids (%) |
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Prenatal antibiotics (%) |
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Prenatal magnesium sulfate (%) |
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Cord blood from term and preterm pregnancies was collected according to the National Cord Blood Program collection protocol. Activation was assessed in whole blood within 4 hours of delivery; this rapid analysis permits accurate assessment of the in vivo activation state. Minimal sample processing and rapid
For each stain, 100 µL of blood was placed in a fluorescence-activated cell sorter tube, washed once, and incubated for 15 minutes with 20 µL of human AB serum for blocking. Conjugated antibodies were then added to each tube and incubated for 15 minutes at room temperature as described
The Shapiro-Wilk normality test was used to assess the distribution of data using an alpha value of 0.05. Based on normality, group means or medians were compared using either a Student's t-test or Wilcoxon Rank Sum test; a p-value of 0.05 was considered nominally significant. Data was analyzed using Stata11.0 (StataCorp LP, College Station, TX) and Prism 5.0 Graphpad software (La Jolla, CA).
We thank Dr. Method Duchon for critical reading of this manuscript.