DISCUSSION
Early prediction of PE remains a challenge in current clinical practice. Known traditional risk factors inadequately identify women who will develop PE early in gestation . To aim the discovery of novel serological markers with better predictive power for PE at early gestations, we applied a multi-omics approach, integrating differentially expressed gene (DEGs) from placental mRNA expression multiplex analysis and lipidomic database mining of existing literature, to identify novel PE biomarkers, which are Lep and Cer. By characterizing the maternal serological profiles of Lep and Cer using commercial ELISA and reported liquid chromatography-tandem mass spectrometry (LC/MS/MS) assays, we validated the up-regulated Lep and down-regulated Cer(d18:1/25:0) in a case-control testing cohort with maternal sera collected at confirmative diagnosis of PE. With a cohort of longitudinally collected maternal sera from both PE and non-PE women, we further assessed the PE-predictive power of the Lep/Cer (18:1/25:0) ratio early in gestation. Our results validated this ratio as a better serological predictor of impending PE than the established sFlt-1/PlGF ratio. Our findings demonstrated that the use of the Lep/Cer (d18:1/25:0) ratio can identify women at high risk of developing PE at a substantially earlier time window during pregnancy (at 5 to 25 wks) than the sFlt-1/PlGF ratio (after 25 wks). Moreover, our results showed that, compared with the sFlt-1/PlGF ratio, the Lep/Cer (d18:1/25:0) ratio has better sensitivity (40% vs 85%), specificity (45% vs 90%), PPV (42% vs 89%), and NPV (43% vs 86%). Therefore, early in gestation, the Lep/Cer (d18:1/25:0) ratio outperforms the established sFlt-1/PlGF ratio and is a predictor of impending PE. The fact that the Lep/Cer (d18:1/25:0) ratio increases early in gestation in pregnant women who later develop PE offers an opportunity for predicting PE prior to the onset of clinical signs and symptoms. Integration of the ratio into a high-risk screening tool might allow patient identification at a pre-symptomatic stage. In addition, the concept of integrating a transcriptomic approach in placenta tissue with a lipidomic approach in serum is novel, as it combines the merits of studies in tissue whose focuses are more towards the pathogenesis and pathophysiology with those study in serum whose focuses are more towards the clinical translation. Taking the candidates obtained from the discovery phase to the validation phase makes the findings of this study translatable into clinical practice.
Previous studies have suggested that placental trophoblast cells are a leading source of circulating Lep in pregnancy , where Lep increases progressively in the first and second trimesters, peaks in the third, and returns to pre-pregnancy levels prior to parturition . In early gestation, Lep may play a critical role in modulating essential biological activities such as proliferation, protein synthesis, invasion, and apoptosis of trophoblast cells . Failure of trophoblastic invasion might result in incomplete remodeling of the maternal spiral arteries and inadequate placental perfusion to the embryo , leading to various disorders of reproduction and gestation such as intrauterine growth restriction , PE , gestational diabetes mellitus , and recurrent miscarriage . Other recent studies documented significant elevations of Lep expression in preeclamptic placentas . In the current study, we found a significant upregulation of Lep in maternal sera of PE women, which is consistent with previous reports .
The gestational dysregulation of Cer metabolism is believed to induce the aberrant de novo synthesis and lysosomal breakdown of Cer, which leads to trophoblast cell autophagy, dysfunctional development of placenta, and eventually pregnancy complications like PE . Cer (d18:1/25:0) is an unusual odd-chain species of the Cer family that is generated by de novo synthesis based on 25:0 fatty acid. Such odd-chain fatty acids are mainly from dairy products and meat from ruminant animals . Cer (d18:1/25:0) was previously described as a potential urinary marker of inflammation-induced alcoholic liver disease . Recently, an elevation of the Cer (d18:1/25:0) was also identified as a serum prognostic marker to predict various acute diseases, including cardiovascular death, myocardial infarction, and stroke in patients with acute myocardial infarction within an ensuing 12-month period . Our results suggested the pathological implications of Cer (d18:1/25:0) in the development of pregnancy complicated by PE, which might provide additional insights into the mechanistic roles of Cer (d18:1/25:0) and other odd-chain Cer species in PE pathophysiology.
An association between Lep and Cer has been reported in several studies. Lep was shown to exert its anorexigenic action by promoting mitochondrial lipid oxidation in both adipose and non-adipose tissues to alleviate ectopic accumulation of lipotoxic Cer via the activation of AMP-activated protein kinase (AMPKK/AMPK) . The de novo synthesis of Cer was found to play prominent roles in modulating downstream signaling of central Lep’s activity via mediation of malonyl-CoA, carnitine palmitoyl transferase-1c, and serine palmitoyl transferase . Persistent elevation of circulating Lep also appears to induce resistance at the level of the Lep receptor, which accounts for attenuated potency of Lep to alleviate the accumulated cytotoxic Cer . Our data suggest a crosstalk between Lep’s activity and de novo Cer synthesis. Lep functions well as a predictor of PE early in gestation, while Cer (d18:1/25:0) performs better at mid-gestation. The Lep/Cer (d18:1/25:0) ratio has a better predictive performance than Lep or Cer (d18:1/25:0) levels alone. Our findings revealed a correlation between the biological patterns of the two markers during PE progression, which might add value to existing knowledge about the Lep-Cer relationships.
Our study has several limitations. First, the sample sizes of the cohorts were small and lacked racial heterogeneity–thus, the generalizability of the results awaits larger and more racially diverse study populations. Second, longitudinal collections of blood samples were not evenly distributed over gestation. Finally, we did not investigate the exact tissue of origin, where Lep is overexpressed in PE women. By using a conditional knock-in placental Lep transgenic mouse model, it may be possible to elucidate the mechanistic role of placental Lep in the pathogenesis of PE early in pregnancy.