Pre-eclampsia and intrauterine growth restriction (IUGR) are major causes of maternal and perinatal morbidity and mortality.1,2 Pre-eclampsia affects approximately 2–5% of pregnancies, and usually develops after 20 weeks of gestation.2 It is characterized by chronic or gestational hypertension combined with proteinuria caused by defective placentation resulting from inadequate uteroplacental blood perfusion and ischaemia.3,4 Recent findings suggest that pre-eclampsia should be subdivided according to gestational age at the time of required delivery into early pre-eclampsia (before 34 weeks of gestation), intermediate pre-eclampsia (at 34–37 weeks of gestation) and late pre-eclampsia (after 37 weeks of gestation). This enables us to distinguish more clearly between women at high, low and no risk of pre-eclampsia as early as possible, optimally around 11–13 weeks of gestation,5,6 when prophylactic treatment may be considered and eventually started. A meta-analysis found that low-dose aspirin therapy (50–150 mg of acetylsalicylic acid daily) initiated in early pregnancy (before 16 weeks of gestation) was associated with a significant reduction in the incidence and severity of pre-eclampsia, IUGR and preterm birth (before 34 weeks of gestation) in women identified to be at moderate or high risk of pre-eclampsia using various inclusion criteria, mostly anamnestic maternal risk factors and subsequently abnormal uterine artery Doppler velocimetry.7 It is believed that low-dose aspirin could inhibit thromboxane-mediated vasoconstriction and thereby prevent endothelial dysfunction and activation of the platelet and clotting system.8,9
The underlying causes of pre-eclampsia and IUGR have not yet been discovered. Nevertheless, it has been clearly demonstrated that placenta is continuously remodelled during normal placental development, and establishment of an optimal balance between trophoblast proliferation and apoptosis is crucial.10 As a result, extracellular nucleic acids of both fetal and placental origin,11,12 packed in trophoblast-derived apoptotic bodies,13–15 may be detected in the maternal circulation during the course of normal gestation. Increasing levels of circulating fetal DNA with advancing gestation reflect the growth of the placenta.12 In addition, the placenta sheds syncytiotrophoblast microparticles, which are also released into the maternal circulation.16
Placental insufficiency-related pregnancy complications are associated with excessive levels of extracellular fetal DNA. A hypoxic environment is likely to be responsible for inducing trophoblast cell death and increased shedding of placental debris into the maternal circulation.15,16
In this article, recent advances in this field demonstrated by our group are reviewed, with a focus on the diagnostic potential of particular markers and their implementation in algorithms to predict and diagnose placental insufficiency-related complications by quantification of extracellular nucleic acids in the maternal circulation.
Quantification of extracellular DNA in maternal circulation – discrimination between normal pregnancies and placental insufficiency-related complications
Our previous extracellular DNA quantification studies17–19 proved to have high accuracy for differentiation between normal pregnancies and those associated with the onset of pre-eclampsia with or without IUGR (RASSF1A, 93.1%; SRY, 93.6%; and GLO, 92.1%) and/or IUGR itself (RASSF1A, 91.6%; SRY, 92.5% and GLO, 89.5%).19
We also reported that the DYS-14 sequence is not an optimal marker for extracellular fetal DNA quantification because of considerable variation in DYS-14 copy numbers in males and discrepancies in DYS-14 copy numbers between extracellular fetal DNA and the original fetal genome.17
Quantitative aberrations of extracellular DNA in maternal circulation as risk markers for predicting placental insufficiency-related complications
Our next pilot study demonstrated elevation of extracellular DNA in three out of five (SRY), one out of eight (hypermethylated RASSF1A) and four out of eight (GLO) patients at various stages of gestation ranging from 26 weeks to 2 weeks before the onset of clinical symptoms of pre-eclampsia (n = 4), IUGR (n = 3) and/or chronic placentopathy causing hypoxia. SRY and GLO markers provided superior results to the hypermethylated RASSF1A sequence.19
Identification of placenta-specific micro-RNAs in maternal circulation
The goal of our recent study20,21 was to identify extracellular placenta-specific micro-RNAs in the maternal circulation. We focused mainly on testing those micro-RNAs which had been shown to be placenta specific according to the miRNAMap database22 and a study presented by Liang et al.23 Finally, we introduced a panel of seven extracellular placenta-specific micro-RNAs (miR-516-5p, miR-517*, miR-518b, miR-520a*, miR-520h, miR-525, miR-526a) with the greatest potential to enable non-invasive monitoring throughout gestation of the growth and condition of the placenta in all pregnancies without interference from expression of micro-RNAs arising from maternal peripheral blood and tissues. Absolute and relative quantification approaches revealed significant increases in extracellular placenta-specific micro-RNAs level over time in normally progressing pregnancies.20,21
Extracellular micro-RNAs may differentiate between normal pregnancies and placental insufficiency in early gestation
The data derived from our pilot study demonstrated that extracellular placenta-specific micro-RNA levels and their expression profile (miR-516–5p, miR-517*, miR-518b, miR-520a*, miR-520h, miR-525 and miR-526a) did not differ significantly between normal and complicated pregnancies at the time of pre-eclampsia and/or IUGR onset.24,25 Nevertheless, significant elevation of extracellular micro-RNAs was observed during early gestation (12–16 weeks) in all pregnancies in which pre-eclampsia and/or IUGR later developed.
We have developed a panel of extracellular placenta-specific micro-RNA markers that might be reliably detected in the maternal circulation during the course of gestation. We have demonstrated that the level of micro-RNA markers increases continuously in the maternal circulation with advancing gestation, reflecting the growth of the placenta. Despite the fact that placental insufficiency-related complications have been shown to be associated with excessive levels of extracellular fetal and total DNA in the maternal circulation, neither absolute nor relative quantification of placenta-specific extracellular micro-RNAs was able to differentiate between normal and complicated pregnancies at the time of pre-eclampsia and/or IUGR onset. Interestingly, the experiments pioneered by our group, evaluating the diagnostic potential of the newly identified micro-RNA markers to predict the onset of placenta insufficiency-related complications as early as possible, provided promising results. Significant elevation of extracellular placenta-specific micro-RNAs was observed at 12–16 weeks of gestation in all pregnancies with later onset of pre-eclampsia and/or IUGR, whereas elevation of extracellular fetal (hypermethylated RASSF1A and SRY sequences) and total DNA (GLO sequence) was observed in only some of these patients. Based on the results of this pilot study, a large-scale analysis was initiated. The panel of selected extracellular micro-RNAs is being validated for incorporation into first-trimester screening to identify high-risk pregnancies. These data strongly support the need for a more detailed exploration of extracellular micro-RNAs in the maternal circulation with a view to their routine assessment in everyday practice and recognition as potential biomarkers for placental insufficiency-related complications.