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 ^[1], ^[2]. This has led to an increased prevalence of surviving preterm children with handicaps. In spite of its importance, the etiology of preterm labor is not well understood. Intrauterine infection is strongly linked to birth prior to 30 weeks ^[3], ^[4]. However, diverse factors including inflammation, race, environmental stress, and genetics are also associated with preterm labor.

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 ^[5], ^[6], ^[7], ^[8]. Maternal, non-obstetrical infections such as pyelonephritis, pneumonia, and periodontal disease are also associated with preterm birth, implying that systemic inflammation can lead to preterm parturition. Studies demonstrating that antibiotics prolong gestation following preterm, premature rupture of membranes, with an increased latency period and improved neonatal outcome within antibiotic-treated women, support a link between infection, inflammation and preterm birth ^[9], ^[10].

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 ^[11]. Acute inflammation is initially mediated by the innate immune system, producing vasodilation, complement activation, and the release of inflammatory mediators including cytokines and chemokines. Subsequently, as inflammation progresses, the adaptive immune system becomes involved via recruitment by soluble factors, innate immune cells, Toll-like receptor ligands, and antigen presenting cells ^[6], ^[12]. Although several investigators have assessed the role of innate immune activation in preterm parturition ^[11], ^[13], ^[14], much less is known about the role of T cell activation in preterm delivery.

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 ^[15]. To define the role of fetal lymphocyte activation in preterm birth we assessed neonatal CD4⁺ T lymphocyte activation and memory phenotype ex vivo utilizing fluorescently labeled antibody staining followed by flow cytometric analysis. This permits simultaneous identification of the cell type and the activation state in addition to other phenotypic markers; activation markers assessed include: CD69, CD25, HLA-DR and CD45RO. This methodology was previously used, whereby we found that spontaneous labor at term is not associated with neonatal T cell activation ^[16]. In this study we examined the association between preterm birth neonatal T lymphocyte activation, as well as levels of memory CD45RO⁺ T cells to determine whether there is a link between the fetal adaptive immune response and preterm birth.

Preterm parturition is associated with fetal and neonatal inflammation. Several researchers have described increased neonatal serum cytokine levels following preterm delivery; ^[11], ^[12], ^[17], ^[18], ^[19], ^[20], ^[21] however, it is not understood whether the inflammation originates from the mother, fetus, or both. There is also evidence that innate immune cells are activated at the time of preterm birth; whereby investigators have described a fetal inflammatory response syndrome associated with preterm parturition ^[11], ^[13], ^[14]. Although acute inflammation is typically mediated by the innate immune system, established inflammation potentially involves both the innate and adaptive immune system. Most studies to date assessing neonatal immune activation have focused on the innate immune system. To better understand if preterm parturition is associated with adaptive immune activation, we assessed neonatal T cell activation following idiopathic preterm birth and uncomplicated term birth.

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 ^[3], ^[4] To confi.rm that our observations are related to inflammation and not secondary to gestational age, we need to assess T cell activation in gestational age matched infants delivered for indications which do not involve inflammation, studies are ongoing to address this issue.

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 ^[22], ^[23]. We determined the frequency of CD4⁺ memory T cells in neonates born preterm, in the absence of clinical chorioamnionitis, and found that they had an increased percentage of memory T cells compared to infants born at term without complications. This correlated with a previously published study that found preterm neonates had fewer naïve T cells ^[24].

The present study extends the work of other investigators, who have described innate immune activation following preterm birth ^[13], ^[14]. Our observation that preterm delivery is associated with T cell activation, suggests that the inflammation associated with preterm parturition is well established, as acute inflammation is more typically restricted to the innate immune system. Expansion of inflammation to involve the adaptive immune system, might occur via soluble factors, professional antigen presenting cells, ligation of Toll-like receptors or a combination of factors ^[25]. It is not clear if this phenomenon initiates a cascade of events that predisposes to preterm parturition or if this immune activation is the result of preterm labor. Genetic predisposition and environment interactions also potentially modulate immune activation, these possibilities are currently under investigation.

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.

Competing Interests: The authors have declared that no competing interests exist.

Funding: This study was supported by the American Cancer Society Grant #RSG-07-070-01-LIB. The American Cancer Society had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

We thank Dr. Method Duchon for critical reading of this manuscript.