We have shown an association between placental inflammation and a fetal inflammatory response that is consistent with previous studies of term and pre-term births.(11
) Previous work has shown similar associations, most commonly for IL-6 and IL-8,(12
) as well as IL-1β, TNF-α and MMP-9, and other chemokines, regulatory proteins, and growth factors.(14
) The range of biomarkers analyzed in this study is one of the largest simultaneously assessed in published reports and represents all categories of inflammatory mediators.
Placental inflammatory infiltrates include both maternal and fetal contributions. Chorionic plate infiltration is viewed as histologic evidence of a maternal response, (17
) while inflammation of umbilical cord vessels and fetal stem vessels in the chorionic plate, so called fetal vasculitis, are the histologic hallmarks of a fetal inflammatory response.(4
) Neonatal morbidity has tended to be better predicted by fetal vasculitis than by maternal inflammation at the chorionic plate. (11
However, we describe a strong association between histologic markers of both maternal and fetal inflammation and systemic inflammatory response in the newborn. The intensity of placental inflammation is thought to evolve in a sequence developing from chorionic plate inflammation to fetal vasculitis at the chorionic plate to cord inflammation.(18
) The intensity may reflect the duration and extent of infection.(20
) This sequence implies that maternal response precedes fetal response. Consistent with this model, we observe that most cord inflammation occurs in combination with high grade/stage plate inflammation. In addition, we see a dose response relationship between maternal fetal plate inflammation and blood proteins (–).
Despite the model, an accelerated fetal response may sometimes occur. A subset of placentas showed isolated umbilical cord inflammation. Similar findings have been observed in 5–8% of preterm and 17% of term placentas.(21
) Infants with cord-only inflammation tended to have elevated concentrations of blood acute phase reactants. Although the maternal response is generally thought to precede the fetal response, this subset of cases suggests that an accelerated fetal response is possible. We have previously reported that some microorganisms including Actinomyces sp., Group B, Group D, and alpha-hemolytic Streptococci are more likely to promote fetal vasculitis than high-grade chorionic plate inflammation.(4
This study also shows that the inflammatory response is not merely a local neutrophil mediated process in the placenta, but is systemic. The fetus is exposed to intra-amniotic infection at three interfaces; the subamniotic tissue of the placental disc and cord, in the lungs, and in the GI tract through oral intake of amniotic fluid. With placental histologic inflammation, we see increased levels of circulating factors that reflect neutrophil activation (MPO), and endothelial activation allowing chemotaxis and leukocyte migration (e.g.
, MMP-9, E-selectin, VCAM-1, ICAM-1, and ICAM-3). We also see a systemic response in the acute phase reactants produced by the liver (SAA, CRP). In sepsis, circulating blood cells and vascular cells produce TNF-α and IL-1β leading to activation of nuclear factor-kappa B (NF-κB) and subsequent production of IL-6, IL-8, and IFNγ which mediate systemic effects including acute-phase reactants such as CRP and SAA as well as MMP-9. TNF-α and IL-1β can also induce premature labor.(22
Some of the vulnerability of the brain, lung, bowel, and eye in extremely low gestational age newborns has been attributed to their propensity to respond to inflammatory stimuli more vigorously than infants born at term.(12
) Consequently, our finding such strong links between circulating proteins and evidence of a putative stimulus raises the possibility that these circulating proteins might be intermediates between the inflammatory stimulus and organ damage.
Because our study utilized whole blood lysates, our analyses are based on measuring both the soluble and cell-bound forms of membrane receptors (e.g.
E-selectin, VCAM-1, ICAM-1, ICAM-3, TNF-R1 and 2, IL-6R, and VEGF-R1 and 2). Circulating forms of VCAM-1, E-selectin, and ICAM-1 have been detected in plasma and are elevated during systemic inflammatory conditions as well as on endothelial cells. The origins of circulating VCAM-1, E-selectin, and ICAM-1 are unclear, but they may arise from shedding or proteolytic cleavage from endothelial cells.(23
) Similarly, other membranous proteins (e.g.
, TNF-α receptors) undergo shedding through the actions of MMPs and thus increased levels of these receptors may reflect MMP upregulation in addition to upregulation of the specific receptor genes. (26
The concentrations of a few protein biomarkers (e.g.
, IGFBP-1 and VEGF-R1), decreased with placental inflammation and increased with syncytial knots and infarcts, which are presumably histologic indicators of vascular insufficiency. The concentrations of IGFBP-1, one of the binding proteins that control serum levels of insulin-like growth factor, and VEGF-R1, the soluble fms-like tyrosine kinase-1,(27
) are abnormally high in women who have preeclampsia, a syndrome associated with poor placental vascularization and fetal growth restriction. In-vitro
IGFBP-1 can be induced by chronic hypoxia.(28
) Thus, an increased level might be expected with histologic characteristics attributed to vascular insufficiency. The low levels of IGFBP-1 seen with histologic inflammation may be explained by increased degradation by MMP-9 and other MMPs that were not measured in this study since this phenomenon has been observed in inflamed amniotic fluid. (29
Newborns whose placenta had infarcts and increased knots were unlikely to show increased concentrations of inflammatory proteins. This is consistent with previous observations that vascular and inflammatory characteristics of the placenta tend not to occur together.(5
Vascular endothelial growth factor (VEGF) promotes blood vessel formation and endothelial maintenance when bound to the second of its circulating receptors, VEGF-R2 (KDR/Flk-1). VEGF is upregulated by proinflammatory activation downstream from IL-1β and TNF-α signaling.(31
) In keeping with this, we found that VEGF concentrations were elevated in newborns whose placenta had both maternal and fetal inflammation and lower in newborns whose placenta did not have inflammation, but did have syncytial knots and or infarcts.
The first VEGF receptor, VEGF-R1 (Flt-1), appears to function as a competitive inhibitor, minimizing the physiologic capability of VEGF bound to it. VEGF-R1 in the maternal circulation, likely produced by placental trophoblast, is elevated in preeclampsia. (12
) VEGF-R1 in the newborn has not been extensively reported. We were not surprised to see elevated concentrations of day-1 VEGF-R1 in newborns whose placenta had infarcts and increased syncytial knots since both these histologic lesions are common in preeclampsia. However, decreased VEGF-R1 associated with placental inflammation was a new finding and may be a result of physiologic degradation of VEGF-R1 by matrix metaloproteinases. This is a normal regulatory function of MMP that increases the bioavailability of VEGF for endothelial cells.(32
) It is possible that inflammation in the placenta or systemic activation of MMP may have a similar but non-localized effect.
Our not finding any significant relationship between decidual hemorrhage (i.e. abruption) and any protein in the newborn’s blood might truly indicate no relationship. On the other hand, abruption is seen among preterm deliveries associated with both severe chorioamnionitis(33
) and preeclampsia.(34
) Perhaps this heterogeneity diminished our ability to identify a relationship between inflammation and decidual hemorrhage
The major strengths of our study are the large number of proteins uniformly measured at one time, the large number of infants born before the 28th week of gestation, selection of our sample on the basis of gestational age rather than birth weight, and recording of all histologic findings in a uniform manner after efforts to reduce observer variability. A limitation of our study is the small number of newborns who had isolated umbilical cord inflammation. We were also, as in all observational studies, unable to distinguish between causation and association as explanations for what we found.
In summary, we found a strong inflammatory signal in the blood of newborns delivered before the 28th week of gestation whose placenta had moderately severe inflammation of the chorionic plate alone, severe inflammation of both chorionic plate and umbilical cord inflammation, or severe inflammation of just umbilical cord alone. Our findings suggest a need for placental examination in all ELGAN with emphasis on a detailed description of the pattern of inflammation since the presence of placental inflammation predicted increased odds ratios of newborn inflammatory response within the first three days of life regardless of gestation age. Histologic placental inflammation, especially when of high stage in the chorionic plate or causing fetal vasculitis should be regarded as a fetal inflammatory response. This information may be useful in stratifying ELGAN for studies of intervention.