Prenatal inflammation as a link between placental expression signature of tryptophan metabolism and preterm birth

Abstract

Spontaneous preterm birth is a serious medical condition responsible for substantial perinatal morbidity and mortality. Its phenotypic characteristics, preterm labor with intact membranes (PTL) and preterm premature rupture of the membranes (PPROM), are associated with significantly increased risks of neurological and behavioral alterations in childhood and later life. Recognizing the inflammatory milieu associated with PTL and PPROM, here, we examined expression signatures of placental tryptophan metabolism, an important pathway in prenatal brain development and immunotolerance. The study was performed in a well-characterized clinical cohort of healthy term pregnancies (n = 39) and 167 preterm deliveries (PTL, n = 38 and PPROM, n = 129). Within the preterm group, we then investigated potential mechanistic links between differential placental tryptophan pathway expression, preterm birth and both intra-amniotic markers (such as amniotic fluid interleukin-6) and maternal inflammatory markers (such as maternal serum C-reactive protein and white blood cell count). We show that preterm birth is associated with significant changes in placental tryptophan metabolism. Multifactorial analysis revealed similarities in expression patterns associated with multiple phenotypes of preterm delivery. Subsequent correlation computations and mediation analyses identified links between intra-amniotic and maternal inflammatory markers and placental serotonin and kynurenine pathways of tryptophan catabolism. Collectively, the findings suggest that a hostile inflammatory environment associated with preterm delivery underlies the mechanisms affecting placental endocrine/transport functions and may contribute to disruption of developmental programming of the fetal brain.

Figure 1

Differentially expressed genes involved in placental tryptophan homeostasis in preterm versus term deliveries. Relative expression of several key enzymes of the kynurenine (A) and serotonin pathways (B), as well as genes encoding tryptophan and serotonin transporters (C), was significantly altered in preterm samples. Data presented in A–C are relative levels of transcripts of indicated genes, normalized with respect to the geometric mean expression of β2-microglobulin (B2M) and TATA-binding protein (TBP) after log2 transformation, and median values with IQR. Asterisks indicate significance according to type III ANOVA with an FDR correction: ^**(q ≤ 0.01), ^***(q ≤ 0.001), ^****(q ≤ 0.0001). (D) Volcano plot summarizing the intensity of differential expression between preterm and term placentas. The horizontal axis denotes log2 fold-change, and the vertical axis −log10 transformed corrected P-values (q-values). Red and blue dots represent significantly differentially expressed genes with absolute log2 fold-change higher than 1.0 and q-value of <0.05 (red upregulation and blue downregulation). Gray dots indicate genes with an absolute log2 fold-change less than 1.0 but q-value of <0.05, and black dots indicate genes with unaltered expression in preterm births.

Figure 2

Results of comparative analysis of placental tryptophan pathway gene expression. Volcano plots used to establish the differentially expressed genes between (A) PPROM versus term and (B) PTL versus term placentas. The horizontal axis denotes the log2 fold-change and the vertical axis represents -log10 transformed corrected P-values (q-values). Red and blue dots indicate significantly differentially expressed genes with absolute log2 fold-change >1.0 and q < 0.05 (red upregulation and blue downregulation). Gray dots indicate genes with absolute log2 fold-change <1.0 but q < 0.05, and black dots indicate genes with unaltered expression in preterm births. (C) Heatmap generated from the log2-transformed expression data for differentially regulated genes, with preterm samples subgrouped into PTL and PPROM samples. The color intensity indicates expression levels (red upregulated and blue downregulated. Between the hierarchical clustering and heatmap, individuals are colored by category. Group separation includes preterm (red) and term (gray) placentas, while diagnosis highlights term (gray), PPROM (orange) and PTL (yellow) placentas.

Figure 3

Principal component analysis (PCA) biplots visualizing placental tryptophan pathway gene expression in preterm delivery placentas. (A) Positions of individual and subgroups of placentas in the first, second and third dimensions (accounting for 37.2, 20.5 and 10.9% of explained variance, respectively) in biplots [MIAC (colonization), MIAC and IAI (intra-amniotic infection), IAI (sterile IAI), and none (neither MIAC nor IAI)] and vectors of expression of indicated genes (black arrows). (B) Positions of placentas associated with indicated HCA grades [fetal (fetal inflammatory response), maternal (maternal inflammatory response) or none (absence of HCA)] in the first, second and third dimensions (accounting for 40.7, 17.2 and 11.1% of explained variance, respectively) in biplots and vectors of expression of indicated genes variables (black arrows). A 95% confidence ellipse was drawn around the barycenter for each group. The gene expression data were log2 transformed prior to analysis.

Figure 4

Unsupervised clustering of placentas from preterm births based on expression of genes involved in tryptophan metabolism. (A) Analysis of 167 placentas identified two main clusters: Cluster 1 represented in red (n = 125) and Cluster 2 in blue (n = 42). (B) Cluster 1 was positively linked with high relative expression of IDO1 and SLC6A4 gene expression, while Cluster 2 was positively associated with high relative expression of KYAT1, HAAO, IDO2, KYNU, MAO-A, MAO-B, SLC7A5, SLC22A3, SPR, TDO2 and TPH1. (C) Cluster 2 was associated with higher gestational age at delivery, but lower concentrations of inflammatory markers, specifically amniotic fluid IL-6 (Roche dataset, n = 111), and maternal serum CRP (D). The significance of differences between the clusters was assessed using the chi-square test (B) or type III ANOVA (C and D) with fetal sex and maternal BMI at admission treated as covariates. Gene expression data were log2 transformed, while amniotic fluid IL-6 and maternal serum CRP concentrations were log transformed before analysis. ^*(P ≤ 0.05), ^**(P ≤ 0.01).

Figure 5

Comparison of characteristics of samples in Clusters 1 and 2. Distributions of fetal sex (A), diagnosis (B), presence of HCA (C), parity (D), delivery mode (E), maternal BMI (prepregnancy and at admission) and pregnancy weight gain (F). ^*(P ≤ 0.05).

Figure 6

Relationships between placental gene expression, gestational age at delivery and inflammatory markers in preterm samples. Significant correlations (P < 0.05) between relative gene expression and gestational age at delivery (A), amniotic fluid IL-6 concentration (Roche dataset, n = 111) (B), and serum CRP concentration (whole dataset, n = 167) (C) are shown. (D) Correlation plot (Pearson correlation coefficient, r > 0.19) indicating the dependence between gene expression, gestational age at delivery (GA) and concentration of inflammatory markers. The intensity of correlations varies between red (negative) and blue (positive). The closer the variables, the stronger the correlation. (E) Results of mediation analysis: numbers displayed are beta-coefficients from the linear model, and those marked with asterisks significantly differ from zero. a is the direct effect between relative gene expression and inflammatory marker concentration. b is the direct effect between inflammatory marker concentration and gestational age at delivery. c′ is the direct effect between relative gene expression and gestational age at delivery after adjusting for inflammatory marker concentration. c is the total effect, i.e. direct effect of relative gene expression on gestational age at delivery. ab is the mediation effect, i.e. effect of gene expression on gestational age at delivery through the concentration of inflammatory markers. If c′ remains significant, the mediation is partial, but if insignificant, the mediation is complete. Indicated significance was obtained by the Pearson test (a, b and c) or from a linear model (e). For mediation analysis, delivery mode and pregnancy weight gain were treated as covariates. Gene expression data were log2 transformed, and amniotic fluid IL-6 and maternal serum CRP concentrations were log transformed. ^*(P ≤ 0.05), ^**(P ≤ 0.01), ^***(P ≤ 0.001), ^****(P ≤ 0.0001).

Figure 7

Relationship between placental IL6 gene expression and relative expression of genes involved in placental tryptophan homeostasis. (A) Significant correlations (r > 0.15, P < 0.05; according to the Pearson test) were detected for several genes involved in metabolism/transport of tryptophan. (B) Correlation plot depicting the intensity of relationships varying between red (negative) and blue (positive); the closer the variables, the stronger the correlation. ^*(P ≤ 0.05), ^**(P ≤ 0.01), ^****(P ≤ 0.0001).

Figure 8

Graphical representation of the dominant molecular placental subtype associated with preterm delivery, identified by unsupervised clustering analysis. This cluster is characterized by a highly inflammatory environment with upregulated amniotic fluid IL-6 and maternal serum CRP levels. Compared with the low inflammation subtype, this high inflammation profile is associated with differentially expressed placental expression of tryptophan metabolism pathways (red upregulation, blue downregulation).