Elsevier

Placenta

Volume 61, January 2018, Pages 61-71
Placenta

Identification of potential early biomarkers of preeclampsia

https://doi.org/10.1016/j.placenta.2017.11.011Get rights and content

Highlights

  • The expression of identified miRNAs depends on the gestational age.

  • Early and late-onset PE differ in their miRNA expression levels.

  • Placental miR-423-5p, -629-5p, and -519a-3p and let-7c-5p levels are increased in the blood plasma in early-onset PE.

  • miR-423-5p was proposed as a potential candidate for the early diagnosis of PE.

Abstract

Introduction

It is thought that poor placental perfusion caused by inadequate remodelling of the maternal spiral arteries leads to preeclampsia (PE). To identify novel signalling pathways that contribute to PE pathogenesis and to create prerequisites for the non-invasive diagnosis of PE before clinical manifestations of the disease, this study aimed to evaluate miRNA expression levels in the placenta and blood plasma of pregnant women.

Methods

miRNA deep sequencing followed by real-time quantitative RT-PCR was applied to compare miRNA expression profiles in the placenta and blood plasma from women with early- and late-onset PE relative to the control group.

Results

A more than two-fold decrease in miR-532-5p, -423-5p, -127-3p, -539-5p, -519a-3p, and -629-5p and let-7c-5p expression levels was observed in the placenta, while a more than two-fold increase in miR-423-5p, 519a-3p, and -629-5p and let-7c-5p was observed in the blood plasma of pregnant women with PE. The above-listed miRNAs are associated with PE for the first time in this study, except for miR-519a-3p, whose role in PE has already been postulated. Using a logistic regression, plasma samples were classified into the early-onset PE group (probability p = 0.01, 80% specificity, 87.5% sensitivity and 87.5% precision) and showed increased miR-423-5p expression levels that were confirmed by the 9.8-fold up-regulation (р = 0.0002498) of miR-423-5p expression observed in the blood plasma at 11–13 GW by RT-PCR in a group of pregnant women manifesting severe PE clinical signs at 28–33 GW.

Conclusions

miR-423-5p may be considered a potential candidate for the early diagnosis of PE during the targeted management of high-risk pregnancies.

Introduction

Preeclampsia (PE), a multisystem pathological condition that occurs in 3–5% of pregnant women, is one of the leading causes of maternal and perinatal mortality [1]. Typically, the disease manifests in the second half of pregnancy with a classic triad of symptoms including hypertension, proteinuria and peripheral oedema [2]. Although the aetiology and pathogenesis of PE have not been fully elucidated, the development of PE is considered to occur following a two-step process. The first stage involves a placentation defect, probably as a result of a maladjustment of the maternal local immune response against foetal tissues [3] coupled with an abnormal differentiation of cytotrophoblast cells during their invasion of the spiral uterine arteries [4], [5], [6], [7]. This symptom results in a decreased placental size and restricted utero-placental blood flow, which does not meet the needs of the growing foetus [8]. As a consequence, ischaemia/hypoxia of the placenta is followed by an increase in syncytiotrophoblast apoptosis and necrosis and by the release of microvesicles and cellular debris by the trophoblasts [9]. There is also a release of damaging factors of placental origin, such as soluble fms-like tyrosine kinase 1 (sFlt1), proinflammatory cytokines, antibodies to the angiotensin 1 receptor, soluble endoglin, tumour necrosis factor alpha, interleukin 1, fibronectin, and blood coagulation factor VIII into the mother's bloodstream [10]. Overall, these changes lead to the onset of the second stage of PE that occurs after 20 weeks of gestation (GW) and is characterized by systemic endothelial dysfunction in many organs and systems including the kidneys, cardiovascular system, liver, brain, and others [11], [12]. The application of different molecular biology approaches has enabled the identification of the entire gamut of differentially expressed genes in the placenta of pregnant women with PE compared to that of women with physiological pregnancies [13], [14]. Nevertheless, the genetic and epigenetic mechanisms regulating these differences in gene expression are still poorly understood. miRNAs form a class of small non-coding RNAs with a length of 21–25 nucleotides that epigenetically control of the expression target genes, mainly at the post-transcriptional level by destabilizing mRNAs and inhibiting protein translation [15]. The role of miRNAs in the regulation of placental development and function has been analyzed in several recent studies. In one of these studies, changes in the levels of 33 miRNA clusters including miR-17-92, C14MC, miR-371-3, the C19MC clusters, the miR-29 cluster, the let-7 family, miR-195 and miR-181c were observed in the placenta depending on 35 gestational age [16]. Large-scale studies of miRNA gene expression in the placenta of pregnant women with physiological pregnancies and pregnant women with PE [17], [18], [19], [20] and premature births [21] allowed for the identification of more than a dozen differentially expressed miRNAs in each study. Overall, the lists of identified miRNAs differed between the studies with minimal overlap. The causes for these discrepancies may be associated with the use of different test systems for the analysis of specific sets of miRNAs or to differences in the topology of the studied fragment of placental tissue. The aim of the present study was to perform a miRNA screen on placental tissue and blood plasma for pregnant women with early- and late-onset PE using miRNA deep sequencing. This method allows us to simultaneously analyse all the miRNAs in a sample with high accuracy and specificity, rather than a defined number of molecules, such as the ones present in available microarrays. Additionally, microarrays may not have high specificity due to the possible cross-hybridization of homologous miRNAs. Some of the miRNA that we identified appear usable in early terms of pregnancy and are therefore interesting potential biomarkers. The functional analysis of the identified differentially expressed miRNAs while considering their potential gene-targets will provide additional information on deregulated signalling pathways which have not been previously considered in terms of PE pathogenesis.

Section snippets

1st patient cohort

In total, 54 pregnant women aged between 27 and 40 years with Caesarean section indications were enrolled in the study and comprised the following four groups (Table 1): 1) women with full-term physiological pregnancy (n = 16, 37–40 GW); 2) pregnant women with an indication for an emergency Caesarean section due to the lack of prolonging the pregnancy because of cervical insufficiency, placental abruption or premature rupture of the foetal membrane (n = 10, 24–34 weeks) without clinical

miRNA sequencing

To identify miRNAs associated with PE, placenta and blood plasma samples from 12 women in the 1st cohort of patients (three women from each of the four groups in Table 1) were analyzed by miRNA deep sequencing. The read counts for all the miRNAs normalized by the DESeq2 package were filtered, only including miRNAs in the differential expression analysis that had at least 20 counts in at least one sample. Comparison of the severe early-onset PE (PE < 34 GW) and moderate late-onset PE

Discussion

PE remains an unresolved problem in modern obstetrics. High maternal and perinatal mortality in PE have hindered the development of a non-invasive early preclinical PE diagnostic to prevent PE and its complications. With the aim of identifying placenta-specific miRNAs circulating in blood plasma as potential biomarkers of PE, we performed high-throughput screening study based on miRNA NGS. We found that the miRNA expression pattern in the placenta in early- and late-onset PE were markedly

Conflict of interest

There are no conflicts of interest between any of the authors of the manuscript.

Acknowledgements

Sample collection, total RNA isolation, NGS and RT-PCR were performed as part of the State assignment (State Registration No. 116082210002). Bioinformatics processing of the NGS and RT-PCR data was supported by the Russian Science Foundation project (No. 16-14-00029).

We thank Anastasia Snezhkina and Anna Kudryavtseva from the EIMB RAS “Genome” centre (http://www.eimb.ru/rus/ckp/ccu_genome_c.php) for their assistance in using the next-generation sequencing equipment.

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