Eicosanomic profiling reveals dominance of the epoxygenase pathway in human amniotic fluid at term in spontaneous labor

Abstract

Lipid mediators play an important role in reproductive biology, especially, in parturition. Enhanced biosynthesis of eicosanoids, such as prostaglandin E2 (PGE2) and PGF2α, precedes the onset of labor as a result of increased expression of inducible cyclooxygenase 2 (COX-2) in placental tissues. Metabolism of arachidonic acid results in bioactive lipid mediators beyond prostaglandins that could significantly influence myometrial activity. Therefore, an unbiased lipidomic approach was used to profile the arachidonic acid metabolome of amniotic fluid. In this study, liquid chromatography-mass spectrometry was used for the first time to quantitate these metabolites in human amniotic fluid by comparing patients at midtrimester, at term but not in labor, and at term and in spontaneous labor. In addition to exposing novel aspects of COX pathway metabolism, this lipidomic study revealed a dramatic increase in epoxygenase- and lipoxygenase-pathway-derived lipid mediators in spontaneous labor with remarkable product selectivity. Despite their recognition as anti-inflammatory lipid mediators and regulators of ion channels, little is known about the epoxygenase pathway in labor. Epoxygenase pathway metabolites are established regulators of vascular homeostasis in cardiovascular and renal physiology. Their presence as the dominant lipid mediators in spontaneous labor at term portends a yet undiscovered physiological function in parturition.

Keywords: LC-MS; epoxy fatty acids; hydroxy fatty acids; lipidomics; parturition.

Figure 1.

Epoxygenase and ω-hydroxylase pathway products of arachidonic acid detected in amniotic fluid. Each data point represents nanomolar concentration of the analyte in a sample above the detection limit for that analyte (represented by the dashed line). Circles, MT; squares, TNL; triangles, TLB; X, median for each analyte and group.

Figure 2.

COX pathway metabolites detected in amniotic fluid samples. Each data point represents nanomolar concentration of the analyte in a sample above the detection limit for that analyte (represented by the dashed line). Circles, MT; squares, TNL; triangles, TLB; X, median for each analyte and group.

Figure 3.

LOX pathway products of arachidonic acid detected in amniotic fluid samples. Each data point represents nanomolar concentration of the analyte in a sample above the detection limit for that analyte (represented by the dashed line). Circles, MT; squares, TNL; triangles, TLB; X, median for each analyte and group.

Figure 4.

Schematic representation of the amniotic fluid eicosanoid profile. All eicosanoids significantly increased at term; their biosynthetic pathways from arachidonic acid (included in the heat map; see Fig. 6) are shown. Eicosanoids that showed significant differences between TLB and TNL groups are highlighted in bold. Intermediates of the biosynthetic pathways that were not detected (or were not detectable due to their inherent instability) are shown in square brackets. Where known, the primary enzymes of arachidonic acid metabolism are given in italics. Dashed arrows represent the metabolic step involving ≥1 enzyme.

Figure 5.

Scatterplot of calculated ratios of the concentrations of PGE₂ to 19-OH PGE₂ for each sample at term. Each data point represents nanomolar concentration of the analyte in a sample above the detection limit for that analyte. Squares, TNL; triangles, TLB; X, median for each analyte and group. P was calculated using a 2-independent-sample Wilcoxon rank sum test.

Figure 6.

Heat map representation of analyte concentration. In this representation, only analytes that change significantly between groups are included. The original concentration levels were converted into ranks for each analyte separately, with the highest value for each analyte represented by the dark red color and the lowest value by the darkest green color. Values under the detection limit that were offset are shown in light yellow.