PE is a pregnancy-associated multi-system disorder appearing at the second half of pregnancy, and is the leading cause of maternal and perinatal mortality and morbidity. The cause of PE is unknown, though it involves inadequate blood supply to the placenta leading to hypoxic environment9,10,11. PE remains unpredictable and is diagnosed only in the second or third trimester, consequently there is an increasing demand for better understanding of PE and development of an early diagnostics test. Although clinical symptoms are late, PE starts with placental dysfunction in the first trimester, hence early detection is feasible as early as weeks 10–1412. Developing a non-invasive efficient screening procedure to identify women at risk of PE would be beneficial for early targeted preventive/prophylactic interventions. Recently reported meta-analysis suggests that the effect of low-dose aspirin for the prevention of PE is optimal only when initiated before or at 16 weeks of gestation, hence women at high risk for PE should be identified in early pregnancy15. Some potential biochemical and mRNA markers for PE early prediction were previously reported16, though none has been proven to effectively predict PE. Small ncRNAs have important roles in many cellular processes such as gene regulation, translation, splicing, and many more. They can be purified from the plasma of first trimester pregnant women in sufficient amounts for accurate identification and quantification, which suggest their potential value as biomarkers for PE non-invasive early diagnosis. Though circulating miRNAs in maternal blood that are associated with preeclamptic risk were identified in later stages of the pregnancy61,62,63,64,65,66, currently no such markers were found for an early diagnosis prior to symptoms appearance.

Using small RNA sequencing analyses, we identified significant changes in the circulating ncRNA abundance in maternal plasma samples of first trimester PE pregnancies, compared with uncomplicated pregnancies. Seven of the up-regulated transcripts (and none of the down-regulated ones) were tRNAs and rRNAS encoded in the mitochondria. A growing body of evidence suggests that mitochondrial dysfunction is manifested by oxidative stress, compromised differentiation, and invasion of trophoblasts, which have been associated with PE pathogenesis71,72,73,74,75,76. Moreover, Qiu et al. found that the odds of PE were positively correlated with the copy number of mitochondrial DNA in maternal blood77. Our results suggest that in addition to mitochondrial DNA, mitochondrial non-coding RNA might also be associated with the development of PE. To the best of our knowledge, this is the first evidence of elevated levels of mitochondrial ncRNAs in the maternal plasma of PE patients. Further prospective research is required to assess these results and to investigate the mechanisms through which altered mitochondrial RNA play a role in the pathogenesis of PE.

Furthermore, 12 out of the 25 differentially expressed transcripts were microRNAs, importantly most were previously related to known mechanisms in PE pathogenesis. For example, miR-10 was down-regulated in PE vs. control samples, in agreement with a previous study that reported down-regulation of miR-10 in preeclamptic placenta compared with normal placentas from uncomplicated pregnancies59. MiR-10 directly targets vascular endothelial growth factor receptor 1 (VEGF-R1, Flt-1) and its soluble splice variant, sFlt-1, both anti-angiogenic factors78. Hence, down-regulation of miR-10 causes increased expression of both sFlt-1 and Flt-1, and significantly impairs the angiogenic behavior of human endothelial cells78. It is well known that angiogenesis is a major mechanism involved in PE pathogenesis. Both sFlt-1 and Flt-1 bind vascular endothelial growth factor (VEGF) and placenta growth factor (PlGF), which play a key role in promoting angiogenesis. A number of studies found elevated sFlt-1 levels and reduced levels of free VEGF and PlGF prior to the onset of the clinical symptoms of PE in blood samples from pregnant women who later developed PE19,79,80, which support our results of reduced miR-10 expression.

Most of the remaining differentially expressed micoRNAs were also previously related to angiogenesis (miR-14345, miR-22147,48, and miR-18246), as well as to other mechanisms in PE pathogenesis such as inflammation (miR-22181), hypoxia (miR-9949 and miR-151a50), regulation of blood pressure (miR-14351) and cell differentiation, apoptosis, and migration/remodeling (miR-14352, miR-19153, miR-18246, miR-2554, and let-7 family55). Moreover, similarly to miR-10, altered expression of miR-14382, -22183, -18246, -2584, -151a85,86,87, and -19184 have been detected in placentas from preeclamptic pregnancies in previous studies. Two of the 12 circulating microRNAs: mir-18288 and miR-22166, were also shown as differentially abundant in PE plasma samples in the third trimester. Additionally, we observed moderate yet significant positive correlations between miR-4433b and 2 maternal clinical features, which may suggest prognostic value for it.

From the remaining differentially expressed transcripts, 4 were long non-coding RNAs (linc). Interestingly, HELLP associated long non-coding RNA (LINC-HELLP) was over-expressed in PE vs. control samples. HELLP syndrome is a pregnancy-associated disease, a severe variant of PE, inducing hemolysis, elevated liver enzymes, and low platelet levels in the mother. LINC-HELLP is a novel lincRNA that was recently identified89. It is localized in first-trimester extravillous trophoblasts and negatively affects the differentiation of the extravillous trophoblasts90. Mutations in LINC-HELLP identified in HELLP families negatively affected trophoblast differentiation89, all of which support our findings.

Due to PE heterogeneity and complexed nature, it is unlikely to be accurately early detected by a single variable. Indeed none of the differentially expressed ncRNAs displayed a perfect separation of PE and control samples, hence we utilized a multivariable model that incorporates several transcripts for PE classification. Based on the differentially expressed ncRNA expression, we built a classification pipeline for PE, and displayed its efficiency. Our pipeline generates a generalizable logistic regression model using a 5-fold cross validation on numerous random partitions into training and test sets. We chose a rather strict machine learning procedure for evaluating the classifier, by using multiple randomly chosen blind test sets, in addition to the cross validation method. This procedure enables us to estimate the accuracy of the classifier given a new unseen data set. Additionally, we trained our model on samples from several ethnicities in order to increase its generalization ability. We acknowledge that additional studies are essential in order to validate the differentially expressed ncRNAs, and to test the classification method. Those validation studies should include larger datasets with samples from several other ethnicities and countries. Our findings suggest the predictive value of circulating small ncRNAs in the first trimester, and demonstrate their application in classification PE and control samples. Further study is required in order to determine whether integrating to the model other clinical features could improve its performance.

Our study has several limitations, which are derived mainly from the relatively limited number of samples in our dataset. Using a small dataset to train and test a prediction model might lead to an overestimation of the performance. In order to reduce this effect, we applied a cross validation procedure in 100 repeats and tested the model on numerous outer random test sets. Nevertheless, we acknowledge our results might still be overestimated, and further validation on an independent dataset is required. Acquiring more samples of various origins and integrating more predictive clinical features, will enable to increase our model generalization, test its performance, and to further confirm the differentially expressed ncRNAs.

In summary, our study suggest the potential of circulating small ncRNA as detectible and accurate biomarkers, which should be further validated in additional studies. Our findings lay the foundation for producing a novel early non-invasive diagnostic tool for PE, which will serve as an effective intervention, and consequently, reduce the life-threatening risk for both the mother and fetus.