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Transcriptomic perturbations in placental gene expression following developmental exposure to perfluorooctanoic acid (PFOA) or hexafluoropropylene oxide-dimer acid (HFPO-DA or GenX) in CD-1 mice are consistent with placental insufficiency

Bevin E. Blake, Vesna A. Chappell, Colette N. Miller, Helen Nguyen, Trina P. Phan, Dhiral P. Phadke, Michele R. Balik-Meisner, Ignacio J. Tripodi, Deepak Mav, Ruchir R. Shah, Suzanne E. Fenton.

Publication:
Environment and Health DOI: https://doi.org/10.1021/envhealth.5c00350
NCBI GEO Accession number GSE262609: https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE262609

DOI: https://doi.org/10.22427/NTP-DATA-109-003-003-000-6

Publication

Abstract

Per- and polyfluoroalkyl substances (PFAS) comprise a group of synthetic chemicals that are ubiquitous global contaminants. Exposure to PFAS has been associated with adverse pregnancy outcomes in humans and animal models, including preeclampsia and low birth weight. The placenta is critical for a healthy pregnancy and evidence suggests adverse pregnancy outcomes associated with PFAS exposure may arise from aberrant placental development and/or function. To investigate effects of PFAS exposure on the placenta, samples collected from a prior study of pregnant CD-1 mice exposed to perfluorooctanoic acid (PFOA; 1 or 5 mg/kg) or hexafluoropropylene oxide-dimer acid (HFPO-DA, or GenX; 2 or 10 mg/kg) via oral gavage from embryonic day (E) 1.5 to E 11.5 or E 17.5 were used. Placenta were evaluated for morphometric, transcriptomic, and pathway-level effects. Although relatively few genes were significantly differentially expressed, multiple significantly enriched pathways were identified and were involved in biological processes related to hemostasis (e.g., coagulation, angiogenesis), the innate immune response (e.g., acute phase response signaling), and metabolism, synthesis, and transport of cholesterol, lipids, and bile acids (e.g., FXR/RXR activation, fatty acid metabolism). These pathway-level observations provide a mechanistic basis for observed morphometric changes, fetal:placental weight changes and previously reported histopathological changes. Overall, these findings confirm proposed Adverse Outcome Pathways consistent with placental insufficiency and fetal nutrient restriction, and PFAS-induced maternal vascular malperfusion of the placenta.

Tables

Table 1

Number of significant differentially expressed genes (DEGs) in placenta at embryonic day (E) 11.5 or 17.5 following exposure to PFOA or GenX.

NCBI GEO Accession number GSE262609: https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE262609

Figures

Figure 1

Heatmap indicating significant DEGs identified in placenta after exposure to PFOA or GenX at E11.5 and E17.5. Color shading indicates fold-change for the 96 genes with significant expression levels in at least one experimental group, with an absolute fold change ≥ 1.5 and p ≤ 0.05. White spaces represent comparisons where the gene did not meet the significance threshold.

NCBI GEO Accession number GSE262609: https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE262609

Figure 2

Volcano plots illustrating the perturbations in the distribution of the prolactin gene family in the placenta at (A) E11.5, (B) female placentas at E17.5 and (C) male placentas at E17.5 caused by PFOA and GenX. Transcriptomic data plotted based on –log10(Pvalue) as a function of log(fold change), labeling the statistically significant genes (p>0.05) denoted by grey dotted line. 

Figure 3

Normalized enrichment scores (NES) for significantly enriched MSigDB Hallmark Pathways in E 11.5 placenta (top panel), E 17.5 female placenta (middle panel), and E 17.5 male placenta (bottom panel). Dot color corresponds to the range of p values associated with the pathway NES (yellow: p ≤ 0.05, green: 0.05 ≤ p ≤ 0.1, and grey: p > 0.1). Dot size corresponds to log10(p value) with larger dots indicating more significant/smaller p values and smaller dots indicating less significant/large p values. Of the 50 MSigDB Hallmark Pathways, only 23 are shown in this figure and represent pathways with a significant NES for at least one tissue/treatment group.

Figure 4

Ingenuity Pathway Analysis Comparison of Shared Canonical Pathways and Upstream Regulators. (A) Heatmap indicating significantly enriched pathways identified in placenta after exposure to PFOA or GenX at E 11.5 and E 17.5. Color shading indicates –log(Benjamini-Hochberg adjusted p value), * p ≤ 0.05. White spaces represent comparisons where the pathway did not meet the significance threshold. (B) Heatmap indicating significantly enriched upstream genes identified in placenta after exposure to PFOA or GenX at E 11.5 and E 17.5. Color shading indicates –log(Benjamini-Hochberg adjusted p value), * p ≤ 0.05. White spaces represent comparisons where the pathway did not meet the significance threshold.

Figure 5

Placental morphometry in male and female placentas following gestational exposure to PFOA or GenX. (A) Representative images of whole placenta cross-sections stained with hematoxylin and eosin (H&E) used for morphometric analyses (3.5x), (B) Stacked bar chart showing the area of placental regions relative to total placental area, (C) Representative images of spiral arteries used for vessel and luminal area and length measurements stained with H&E, (D) Average vessel to luminal area, (E) Total placental area, (F) Labyrinth area, (G) Labyrinth to decidua ratio. Other morphometry results are reported in Supplemental Table 9-10. Boxplots show median (bold center line), 25th and 75th quantiles (lower and upper hinges, respectively), smallest observation ≥ lower hinge - 1.5 * IQR (lower whisker), and largest observation ≤ upper hinge - 1.5 * IQR. IQR = inter-quartile range, *p<0.05 by ANOVA.

Figure 6

DEGs significantly correlated with placenta endpoints in E17.5 male (A-D) and female (E-G) placenta following gestational exposure to PFOA or GenX. Gene expression of (A) Apoa2, (B) Serpina1b, and (C) Gm8893 was positively correlated with the ratio of decidua to junctional zone area, with higher expression levels and ratios in E17.5 male controls. Gene expression of (D) Fam189a2 was negatively correlated with decidua necrosis, with higher expression levels and lower decidua necrosis in E17.5 male controls. Gene expression of (E) 2310036O22Rik, (F) Gm13316, and (G) Met were positively correlated with maternal sinus dilation, with lower expression levels and lower maternal sinus dilation in E17.5 female controls.

Figure 7

Mapping of transcriptomic data to the AOP network proposed by Rogers et al. (2023). Molecular initiating events and Key Events are marked with symbols to indicate significant enrichment of the AOP component by placental transcriptomic results. Pink symbols = 1 mg/kg PFOA, red symbols = 5 mg/kg PFOA, light blue symbols = 2 mg/kg GenX, dark blue symbols = 10 mg/kg GenX. Circles = pooled E 11.5 placenta, triangles = E 17.5 male placenta, stars = E 17.5 female placenta. Figure adapted from Rogers et al. (2023).

Supplemental Tables

Table S1

IDT PrimeTime Primer Assay IDs. PrimeTime primer assays ordered from IDT.DNA.COM as primers only, for use with SYBR reagents. GOI = gene of interest. Mm = Mus musculus. HKG = housekeeping gene. Timepoint (E) = embryonic day.

Table S2

Number of significant differentially expressed genes (DEGs) in placenta at embryonic day (E) 11.5 or 17.5 following exposure to PFOA or GenX.

NCBI GEO Accession number GSE262609: https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE262609

Table S3

Gene ontology biological process analysis for up-regulated DEGs identified in placentas exposed to PFOA or GenX

NCBI GEO Accession number GSE262609: https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE262609

Table S4

Gene ontology biological process analysis for down-regulated DEGs identified in placentas exposed to PFOA or GenX

NCBI GEO Accession number GSE262609: https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE262609

Table S5

Gene expression fold change and pvalues from qPCR gene expression analysis

Table S6

Hallmark pathway normalized enrichment scores (p value) in E 11.5 placentas after exposure to PFOA or GenX

Table S7

Hallmark pathway normalized enrichment scores (p value) in E 17.5 female placentas after exposure to PFOA or GenX

Table S8

Hallmark pathway normalized enrichment scores (p value) in E 17.5 male placentas after exposure to PFOA or GenX

Table S9

E17.5 female placenta morphometry (mean ± SD).

Table S10

E17.5 male placenta morphometry (mean ± SD)

Table S11

Number of genes significantly correlated with placental and fetal endpoints.

Sciome_placenta_phenotype_expression_correlation_results_12202024.xlsx

Supplemental Figures

Figure S1

Principal components analysis of transcriptomic profiles for placenta showing separation of samples by timepoint (A) E 11.5 vs E 17.5, whereas samples did not exhibit a clear separation by treatment group or dose at E 11.5 (B) or E 17.5 for female placenta (C) or male placenta (D). Low Dose = 1 mg/kg-d for PFOA or 2 mg/kg-d for GenX, High Dose = 5 mg/kg-d PFOA or 10 mg/kg-d GenX.

NCBI GEO Accession number GSE262609: https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE262609

Figure S2

Hierarchical clustering of placenta transcriptomic profiles after outlier removal at E 11.5 (A) and E 17.5 (B). No obvious separation between treatment groups or doses were observed.

NCBI GEO Accession number GSE262609: https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE262609

Figure S3

Heatmap of select genes from E 11.5 and E 17.5 placenta transcriptomic microarray analysis and corresponding qPCR gene expression. Scale bars indicating the magnitude and direction of gene expression fold change are illustrated in the heatmap. Data is presented as mean fold change over control. Gray cells correspond to non-significant genes or genes with non-detectable levels of expression. Statistical cutoffs were applied to each gene: *p-value<0.05, ǂp-value<0.1 and an absolute fold-change ≥1.5.