A cross-sectional analysis of syncytiotrophoblast membrane extracellular vesicles–derived transcriptomic biomarkers in early-onset preeclampsia

Abstract

BackgroundPreeclampsia (PE) is a pregnancy-specific hypertensive disorder affecting 2%–8% of pregnancies worldwide. Biomarker(s) for the disorder exists, but while these have excellent negative predictive value, their positive predictive value is poor. Extracellular vesicles released by the placenta into the maternal circulation, syncytiotrophoblast membrane extracellular vesicles (STB-EVs), have been identified as being involved in PE with the potential to act as liquid biopsies.ObjectiveThe objective of this study was to identify the difference in the transcriptome of placenta and STB-EVs between preeclampsia and normal pregnancy (NP) and mechanistic pathways.Methods/study designWe performed RNA-sequencing on placental tissue, medium/large and small STB-EVs from PE (n = 6) and NP (n = 6), followed by bioinformatic analysis to identify targets that could be used in the future for EV-based diagnostic tests for preeclampsia. Some of the identified biomarkers were validated with real-time polymerase chain reactions.ResultsOur analysis identified a difference in the transcriptomic STB-EV cargo between PE and NP. We then identified and verified the differential expression of FLNB, COL17A1, SLC45A4, LEP, HTRA4, PAPP-A2, EBI3, HSD17B1, FSTL3, INHBA, SIGLEC6, and CGB3. Our analysis also identified interesting mechanistic processes via an in silico prediction of STB-EV-based mechanistic pathways.ConclusionsIn this study, using comprehensive profiling of differentially expressed/carried genes of three linked sample subtypes in PE, we identified potential biomarkers and mechanistic gene pathways that may be important in the pathophysiology of PE and could be further explored in future studies.

MicroRNA analysis of medium/large placenta extracellular vesicles in normal and preeclampsia pregnancies

“…We also explored mRNA targets associated with differentially expressed microRNAs to illuminate the underlying regulatory mechanisms. Through this analysis, we sought to discern the extent to which previously identified messenger RNA changes could be attributed to alterations in the microRNA profile ( 16 ). Specific interactions, particularly within the m/lSTB-EVs, were corroborated by others using CHIP-Seq validation methods ( Table 4 ).…”

“…We phenotyped the isolated m/lSTB-EV with flow cytometry (BD Biosciences, LSRII), transmission electron microscopy (TEM, for morphology), and western blot (for immunophenotyping). We used antibodies to placental alkaline phosphatase—PLAP- (to confirm syncytiotrophoblast origin), CD41 (to identify co-isolated platelet EVs, CD235a/b (to identify co-isolated red blood cell EVs), HLA class I and II (to identify co-isolated white blood cell EVs) for flow cytometric analysis as previously described in our previous paper ( 16 ). For immunophenotyping, we characterized the m/lSTB-EVs by probing for known EV markers CD63 [(200 mg/ml) at 1:1,000 dilution, Sc-59286, Santa Cruz Biotechnology], placental alkaline phosphatase [PLAP (1.667 mg/ml) at 1:1,000 dilution, in house antibody], and the known negative EV marker Cytochrome C [(200 mg/ml) at 1:1,000 dilution, Sc-13156, Santa Cruz Biotechnology] as recommended by the international society for extracellular vesicles (ISEV) ( 19 ).…”

“…We subsequently focused on predicting and visualizing mRNA targets modulated by the differentially expressed microRNAs detected in our study to elucidate the regulatory dynamics associated with Preeclampsia. A vital part of this study was identifying some microRNAs responsible for the differentially expressed mRNAs previously reported in our transcriptomics paper (16). The raw fastQ files and the processed file of the transcriptomics data can be accessed on NCBI with the following ID: GSE190971.…”

“…We hypothesized that PE m/lSTB-EVs contain a different microRNA profile than NP, which may offer potential diagnostic and mechanistic insights. We also correlated our microRNA analysis results with our previously described messenger RNA (mRNA) analysis from the same population ( 16 ). By correlating the microRNA and mRNA profiles, we aim to enhance our understanding of the molecular mechanisms underlying Preeclampsia and pave the way for developing novel diagnostic and therapeutic strategies.…”

Syncytiotrophoblast extracellular vesicles contain functional kynurenine metabolising enzymes: potential implications for preeclampsia

“…STB-EVs were obtained from placentae using a modified ex-vivo dual lobe placental perfusion and differential ultracentrifugation method, as previously detailed by our group [32][33][34][35] . Placentae were subjected to a 3-hour perfusion process, after which the maternal side perfusate was collected and promptly centrifuged twice using a Beckman Coulter Avanti J-20XP centrifuge and a Beckman Coulter JS-5.3 swing-out rotor.…”