Unique transcriptomic landscapes identified in idiopathic spontaneous and infection related preterm births compared to normal term births

Abstract

Preterm birth (PTB) is leading contributor to infant death in the United States and globally, yet the underlying mechanistic causes are not well understood. Histopathological studies of preterm birth suggest advanced villous maturity may have a role in idiopathic spontaneous preterm birth (isPTB). To better understand pathological and molecular basis of isPTB, we compared placental villous transcriptomes from carefully phenotyped cohorts of PTB due to infection or isPTB between 28-36 weeks gestation and healthy term placentas. Transcriptomic analyses revealed a unique expression signature for isPTB distinct from the age-matched controls that were delivered prematurely due to infection. This signature included the upregulation of three IGF binding proteins (IGFBP1, IGFBP2, and IGFBP6), supporting a role for aberrant IGF signaling in isPTB. However, within the isPTB expression signature, we detected secondary signature of inflammatory markers including TNC, C3, CFH, and C1R, which have been associated with placental maturity. In contrast, the expression signature of the gestational age-matched infected samples included upregulation of proliferative genes along with cell cycling and mitosis pathways. Together, these data suggest an isPTB molecular signature of placental hypermaturity, likely contributing to the premature activation of inflammatory pathways associated with birth and providing a molecular basis for idiopathic spontaneous birth.

Fig 1. A comparative approach to identifying molecular signatures of isPTB and AHC.

A. Differentially expressed genes were identified using pairwise comparisons edgeR. Red dots represent significant genes that have an absolute log2 fold-change of 1.0 and Benjamini Hochberg adjusted P-value of <0.05. Green dots represent genes with an absolute log2 fold-change of 1.0 and Benjamini Hochberg adjusted P-value of <0.1. Blue lines represent log2 fold-change of 1.0. B. The Venn diagram represents the intersection of significant genes from Panel A which was utilized to further refine the classification into molecular signatures for isPTB and AHC.

Fig 2. Identification of isPTB specific transcriptomic signature.

A. Pathological isPTB candidate genes were identified by assessing the differential expression across all three pairwise comparisons. isPTB candidate genes had significant upregulated expression in isPTB samples compared to TB and AHC, with the either no change in expression pattern between AHC and TB or upregulation in AHC, although to a lesser extent than observed in the isPTB samples. This slight upregulation in AHC samples indicates a baseline expression is higher in samples of earlier gestational age and decreases over term. B. isPTB hypermaturity genes were also identified in the same manner as those in Panel A with the reversal of expression assessment between AHC and TB. In this analysis, we observed that the expression of these genes increases over gestation with expression being increased in TB samples compared to AHC. More importantly, when comparing isPTB to AHC which overlap in gestational age, there is a significant upregulation in isPTB samples which is suggestive of hypermaturity. Genes are arranged in order of Log2 fold change in the isPTB vs AHC comparison. Values = Log2 fold change.

Fig 3. isPTB placental tissue samples demonstrated increased isPTB signature gene expression.

A. DKK1, IGFBP1, IGFBP2, and IGFBP6 localization the syncytiotrophoblast in the control term births with increased expression in isPTB samples. Images are taken at 40x magnification and scale bar = 50um. B. QPCR validation of the upregulation of DKK1, IGFBP1, IGFBP2, and IGFBP6 in isPTB vs TB samples. C. QPCR validation of hypermaturity signature genes TNC and C3 in isPTB vs TB samples. Student’s two-tailed T-test was utilized for statistical analyses and error bars represent standard deviation.

Fig 4. Identification of an AHC transcriptomic signature.

AHC candidate genes were identified by assessing the expression pattern across all three pairwise comparisons. In this instance, we observed greater differential expression, both upregulated and downregulated, in the AHC samples compared to isPTB or TB with either no difference or non-significant differences in isPTB vs TB comparisons. Genes are arranged in order of Log2 fold change in the AHC vs TB comparison. Values = Log2 fold change.