Human spontaneous labor without histologic chorioamnionitis is characterized by an acute inflammation gene expression signature.

Human parturition involves “a common pathway” manifested clinically by uterine contractions, cervical ripening, and chorioamniotic membrane/decidual activation, culminating in membrane rupture. The chorioamniotic membranes undergo complex anatomical and biochemical events that lead to membrane rupture. Morphologic, biochemical, and biophysical studies suggest that rupture of membranes results from the application of acute or chronic stress on localized areas of the membranes that are weaker.

Cervical ripening has been likened to an inflammatory response. Indeed, analyses of inflammatory mediators in gestational tissues have demonstrated that the expression of these mediators is increased during normal term labor. Although these studies of individual cytokines have hinted at parturition as an inflammatory process, the full extent of the involvement of inflammation has not yet been established.

Global expression analyses using genomic approaches including oligonucleotide and cDNA microarrays are capable of exploring the full extent of gene expression changes associated with parturition. Multiple studies have used genomic approaches to study gene expression changes in chorioamniotic membranes associated with parturition. The gene expression studies performed with chorioamniotic membranes at term, however, have not been unbiased.

Specifically, gene expression analyses that use arrays with small numbers of genes selected for their involvement in the immune response are biased because of the number of genes and their selection. In addition, other genomic analyses of fetal membranes at term have focused on gene expression profiles after first culturing tissues in organ culture. It is not clear, however, to what degree the culture conditions influence gene expression profiles.

Comment

Examination of a global transcription profile that reflects an acute inflammatory response extends previous gene-by-gene observations of inflammatory proteins. Although other studies have shown that inflammatory genes increase in expression in fetal membranes during labor, no study has yet presented sufficient data to clearly demonstrate an orchestrated gene expression signature. The analysis presented here demonstrates that multiple transcripts controlling each of the defined steps of acute inflammation increase during labor. Specifically, we observed increases in multiple cytokines and chemokines that are known to orchestrate acute inflammation. It is noteworthy that the “acute inflammation gene expression signature” appeared to be coordinately expressed and was not associated with either the interval after rupture or the duration of labor.

Consistent with previous analyses of chorioamniotic membranes measuring 1 or several genes at a time, IL8, IL6, PBEF, TLR2, and SOD2 were overexpressed in TIL samples compared with TNL samples. Indeed, the expression levels of these mRNAs, or their corresponding proteins, have previously been found to be elevated in samples such as chorioamniotic membranes, myometrium, cervix, or amniotic fluid from TIL patients.

Although numerous stimuli can initiate an acute inflammatory response, one possible sequence may begin with the activation of pattern recognition receptors including TLR2 and TLR4, which are found on the surface of epithelial cells and resident macrophages. TLR2 activation can occur by binding to products from yeast, mycoplasmas, and Gram-positive bacteria. TLR4 recognizes bacterial lipopolysaccharide and can also bind host-derived products that are degraded during acute inflammation. Activated TLR2 and TLR4 cause a release of chemokines that result in neutrophil recruitment and activation.

Neutrophils recruited by this mechanism bind to ICAM1 receptors and migrate to the site of chemokine release. It is, therefore, striking that the mRNA levels for both TLR2 and ICAM1 increased 1.6-fold and 2.1-fold, respectively, in the TIL chorioamniotic membrane samples relative to their TNL counterparts. The subsequent phase of the acute inflammatory response involves the recruitment of monocytes that will go on to differentiate into macrophages and dendritic cells at the site of inflammation. This phase of the inflammatory response is preceded by the production of chemokines responsible for monocyte-specific recruitment and differentiation. It is therefore noteworthy that the mRNA levels of monocyte-specific chemokines increased in the TIL samples (discussed below). The transition to the monocyte-specific phase appears to be controlled by IL6 and SOCS3, both of which were increased in the TIL samples relative to TNL samples.

Multiple lines of evidence are consistent with the “acute inflammation gene expression signature” eliciting the recruitment and activation of neutrophils in the context of an acute inflammatory response. First, genes specifically implicated in neutrophil recruitment and activation had statistically the most significant changes in their expression. It is remarkable that IL8, CXCL1, CXCL2, and CXCL3 were among the 10 most discriminant probe sets and are coordinately expressed. Furthermore, the mRNA levels of all of these genes increased in the TIL samples in a similar manner from 4-fold to 6.5-fold relative to the TNL samples. Finally, hierarchical clustering indicated that the same 4 genes exhibited similar expression patterns across multiple samples. Interestingly, CXCL5, another neutrophil-specific chemokine, was also in the same subcluster, even though its P value was not as significant. Taken together, the “acute inflammation gene expression signature” is consistent with neutrophil recruitment and activation seen during a classic inflammatory response.

Changes in the “acute inflammation gene expression signature” were also consistent with monocyte recruitment and differentiation. Specifically, a set of genes that was central to all GO Biological Process categories (with the exception of “viral genome replication”) were 4 genes whose expression increased during spontaneous labor and which are known to be involved in the chemotaxis and differentiation of monocytes. These genes are FPR1, CCL3 (MIP1[alpha]), CCL4 (MIP1[beta]), and CCL20 (MIP3[alpha]). Both CCL3 and CCL4 signal through the CCR1 receptor, which had a decreased expression in TIL samples. Decreased expression of CCR1 occurs when monocytes differentiate into activated and mature dendritic cells.

In contrast to the gene expression patterns in TIL chorioamniotic membranes at the end of labor, blood samples of TIL patients in active labor did not exhibit an increase in the “acute inflammation gene expression signature.” Taken together, our results indicate that differentially increased expression of the inflammatory response signature is independent of the duration of labor, as well as the interval after rupture, and does not manifest systemically during the early stages of the active phase of labor.

The fact that the “acute inflammation gene expression signature” is not associated with either the interval after rupture of membranes or the duration of labor suggests that this gene expression signature is not a simple consequence of these 2 events. The results presented here can be interpreted in the context of 2 nonexclusive models. The first has been previously proposed as the hypo- or hyperimmune responder model. An alternative model proposes that genes associated with acute inflammation may function in tissue homeostasis.

The principle line of evidence consistent with the hypo-or hyperimmune responder model derives from hierarchical clustering of the 42 probe sets belonging to enriched GO categories. This hierarchical clustering suggests the existence of TIL and TNL subclasses. While the distribution of gene expression appeared to be unimodal, it was simple to discern TIL samples that were either “high,” “average,” or “low expressors.” We suggest that the TIL “low expressor” and “high expressor” samples may correspond to genetically predetermined hypo- and hyperimmune responders, respectively. Previous studies have measured the expression of the cytokines IL1B, IL6, and IL8 in the cervical fluid and discovered that patients with low concentrations of 2 of 3 of these cytokines were more likely to subsequently develop clinical chorioamnionitis.

We consider that the 5 TIL samples showing high expression were more likely to correspond to hyperimmune responders. Conversely, the 3 TIL low expressors were more likely to correspond to hypoimmune responders. Because 3 TNL samples clustered with the 3 TIL low expressors, it is also possible that these 3 TNL samples correspond to immune hyperresponders that have not yet been subjected to the stimulus of labor. Our results demonstrate clearly that labor increases expression of genes involved in acute inflammation. Given that the “acute inflammation gene expression signature” is overexpressed in the absence of detectable inflammation, the fetal membranes may be primed for a massive inflammation reaction, in the case of a triggering event. Although a previous study analyzed cervical fluid and its effects on chorioamnionitis, our study focused exclusively on normal chorioamniotic membranes that have undergone labor or not. Nevertheless, our results demonstrated that subclasses of TIL samples were evident and could be differentiated on the level of expression of genes associated with acute inflammation.

The second model is predicated on the expression levels of inflammatory genes in TNL samples. Although TIL patient samples demonstrated overexpression of the “acute inflammation gene expression signature,” the TNL samples showed high levels of a considerable fraction of these inflammatory genes. Thus, about half of the genes associated with each of the 3 principle phases of acute inflammation exhibited mean expression levels in the TNL samples that were at least at the 50th percentile or higher in the discriminant probe sets. Specifically, 3 of 6 neutrophil-specific chemokines, 1 of 2 transition genes, and 2 of 4 monocyte-specific chemokines were expressed at high levels in TNL samples. Given that all of the chorioamniotic membrane samples were selected based on their lack of inflammation (as defined by histologic examination of the extraplacental membranes) this result implies that genes involved in acute inflammation may serve functions not traditionally associated with acute inflammation. In this context, TLR2 signaling was shown to be necessary for normal epithelial homeostasis in the mouse intestine. It is therefore conceivable that TLR2 and other genes associated with acute inflammation may be involved in maintaining tissue homeostasis of the chorioamniotic membranes.

This study investigated global transcriptional changes during labor in chorioamniotic membranes. Nevertheless, multiple lines of evidence are consistent with the translation of transcripts from the inflammatory response signature into protein products. First, we observed gene expression changes that have been reported previously to be temporally linked, including the downregulation of CCR1 mRNA expression in response to the binding of CCR1 receptor protein by its ligands CCL3 and CCL4. Second, studies measuring protein in chorioamniotic membranes are consistent with the results presented here and have demonstrated overexpression of IL8, IL6, TLR2, and SOD2 protein in TIL samples compared with their TNL counterparts. Finally, virtually all previous studies measuring amniotic fluid protein levels of genes in the inflammatory response signature are consistent with our results, including the protein levels of IL1A, IL1B, IL6, IL8, and CCL3 (MIP1[alpha]). Because protein in amniotic fluid is most likely derived from the fetus and the fetal membranes, the level probably reflects synthesis and secretion by the membranes and fetal neutrophils.

The previous protein studies were performed for 1 or at most a few gene products at a time. Although they hinted at the role of inflammation during parturition, the transcriptional profile established here provides, for the first time, the full extent of gene expression associated with acute inflammation during parturition in the chorioamniotic membrane. Thus, while transcriptional activity does not necessarily reflect translational activity, for many of the genes we have investigated in the “acute inflammation gene expression signature,” we found that increased transcription was consistent with multiple studies demonstrating increased protein levels.

In conclusion, the results presented here suggest that labor induces gene expression changes in chorioamniotic membranes consistent with a localized acute inflammatory response, despite the absence of histologic evidence of inflammation. Further research is required to determine which of the 2 models we proposed explains the clinical and biochemical findings.