Placental biomarkers of phthalate effects on mRNA transcription: application in epidemiologic research

Abstract

Background: CYP19 and PPARgamma are two genes expressed in the placental trophoblast that are important to placental function and are disrupted by phthalate exposure in other cell types. Measurement of the mRNA of these two genes in human placental tissue by quantitative real-time polymerase chain reaction (qPCR) offers a source of potential biomarkers for use in epidemiologic research. We report on methodologic challenges to be considered in study design.

Methods: We anonymously collected 10 full-term placentas and, for each, sampled placental villi at 12 sites in the chorionic plate representing the inner (closer to the cord insertion site) and outer regions. Each sample was analyzed for the expression of two candidate genes, aromatase (CYP19) and peroxisome proliferator activated receptor protein gamma (PPARgamma) and three potential internal controls: cyclophilin (CYC), 18S rRNA (18S), and total RNA. Between and within placenta variability was estimated using variance component analysis. Associations of expression levels with sampling characteristics were estimated using mixed effects models.

Results: We identified large within-placenta variability in both transcripts (>90% of total variance) that was minimized to <20% of total variance by using 18S as an internal control and by modelling the means by inner and outer regions. 18S rRNA was the most appropriate internal control based on within and between placenta variability estimates and low correlations of 18S mRNA with target gene mRNA. Gene expression did not differ significantly by delivery method. We observed decreases in the expression of both transcripts over the 25 minute period after delivery (CYP19 p-value for trend = 0.009 and PPARgamma (p-value for trend = 0.002). Using histologic methods, we confirmed that our samples were comprised predominantly of villous tissue of the fetal placenta with minimal contamination of maternally derived cell types.

Conclusion: qPCR-derived biomarkers of placental CYP19 and PPARgamma gene expression show high within-placental variability. Sampling scheme, selection of an appropriate internal control and the timing of sample collection relative to delivery can be optimized to minimize within-placenta and other sources of underlying, non-etiologic variability.

Figure 1

Placental sampling scheme and device. a) Samples S1 – S4 represent the "inner" region and samples Q1 – Q8 represent the "outer" region and b) Placenta is pictured looking down at the fetal side and the chorionic membrane. Sampling was carried out just below this membrane.

Figure 2

Histologic confirmation of cell type composition done with H&E stained cross-sections of RNA-Later treated placental biopsies. a) inner region of a vaginally-delivered placenta and b) outer region of a vaginally-delivered placenta.

Figure 3

Raw data shows high within-placenta variation in gene expression that is highest when cyclophilin is used as an internal control compared to 18S. a) CYP19 mRNA adjusted for CYC mRNA b). CYP19 mRNA adjusted for 18S mRNA. The vertical lines and black dots represent values for individual biopsies within a placenta. The gray lines connect the median values per placenta.

Figure 4

The means by inner and outer region show less within-placenta variation. a) mean CYP19 mRNA adjusted for CYC mRNA b) mean CYP19 mRNA adjusted for 18S mRNA. Black points represent the outer region and gray represents the inner region.