Polybrominated Diphenyl Ethers in Human Follicular Fluid Dysregulate Mural and Cumulus Granulosa Cell Gene Expression

Abstract

Polybrominated diphenyl ethers (PBDEs), a major class of flame retardants incorporated into numerous consumer products, leach out into dust resulting in widespread exposure. There is evidence from in vitro and in vivo animal studies that PBDEs affect ovarian granulosa cell function and follicular development, yet human studies of their association with female infertility are inconclusive. Here, we tested the hypothesis that exposure to the PBDEs in follicular fluid is associated with dysregulation of gene expression in the mural and cumulus granulosa cells collected from women undergoing in vitro fertilization by intracytoplasmic sperm injection. The median concentration of the ∑ 10PBDEs detected in the follicular fluid samples (n = 37) was 15.04 pg/g wet weight. RNA microarray analyses revealed that many genes were differentially expressed in mural and cumulus granulosa cells. Highest vs lowest quartile exposure to the Σ 10PBDEs or to 2 predominant PBDE congeners, BDE-47 or BDE-153, was associated with significant effects on gene expression in both cell types. Mural granulosa cells were generally more sensitive to PBDE exposure compared to cumulus cells. Overall, gene expression changes associated with BDE-47 exposure were similar to those for ∑ 10PBDEs but distinct from those associated with BDE-153 exposure. Interestingly, exposure to BDE-47 and ∑ 10PBDEs activated the expression of genes in pathways that are important in innate immunity and inflammation. To the best of our knowledge, this is the first demonstration that exposure to these environmental chemicals is associated with the dysregulation of pathways that play an essential role in ovulation.

Keywords: gene expression; granulosa cell; ovary; polybrominated diphenyl ethers.

Figure 1.

Differential gene expression in mural and cumulus granulosa cells. A, Principal component analysis plot of all entities in either mural or cumulus granulosa cells. B, Identification of genes that are expressed in common and differentially between both cell types. A full list of genes is provided in the Supplementary Data 1 Excel file (30).

Figure 2.

Association of polybrominated diphenyl ether exposure with granulosa cell gene expression. Each Venn diagram shows the number of genes that are significantly differentially regulated by 1.5-fold or more, determined by a moderated t test. This analysis was conducted both for mural and cumulus granulosa cell types. The overlap of each Venn diagram represents genes that are differentially regulated in both cell types in the highest- vs the lowest-exposure individuals. A full list of genes is provided in the Supplementary Data 1 Excel file. The tables report the top 5 genes that are differentially regulated (upregulated [blue] or downregulated [red]) for each cell type.

Figure 3.

Polybrominated diphenyl ether (PBDE) congener-dependent differences in gene expression between mural and cumulus granulosa cells. Genes that are differentially regulated by greater than 1.5-fold and deemed significant by moderated t test are represented for a PBDE congener. This was performed both for mural and granulosa cell types. A full list of genes is provided in the Supplementary Data 1 Excel file (30).

Figure 4.

Pathway enrichment in mural and cumulus granulosa cells exposed to polybrominated diphenyl ether (PBDE) congeners. Following the identification of significantly regulated genes, Ingenuity Pathway Analysis software was used to identify gene enrichment in the data set. This provided a list of several pathways. In some cases, pathways were predicted to be activated (green) or inhibited (yellow), depending on relative fold changes provided. The calculated z score is a statistical measure of the match between the expected relationship direction and the observed gene expression. Values greater than 2 or less than –2 are considered significant. When there are not enough data in the data set to make a prediction, the box is undefined (gray), whereas when there is no correlation between the observed data set and the expected relationship, the calculated z score is zero (white).

Figure 5.

Identification of common identities among pathways in mural and cumulus granulosa cells exposed to polybrominated diphenyl ether (PBDE) congeners BDE-47 or BDE-153. Based on pathways that were enriched within the data set, the top 10 pathways were selected and queried for common effector genes. Here the numbers represent the number of genes in common between the relevant pathways. Full details are provided in the Supplementary Data 2 Excel file (30).

Figure 6.

Based on differential gene expression, predictions were made as to the status of upstream regulators of gene expression. Those in green are predicted to be activated, whereas those in yellow are predicted to be inhibited. The calculated z score is a statistical measure of the match between the expected relationship direction and the observed gene expression. Values greater than 2 or less than –2 are considered significant. When there are not enough data in the data set to make a prediction, the box is undefined (gray), whereas when there is no correlation between the observed data set and the expected relationship, the calculated z score is zero (white).