If you were born before the end of the 37th week of pregnancy, you were born premature – as were around 10.6% of babies worldwide.1 Whilst many premature children may be ‘okay’ (including the author of this article – hi!), many are not so lucky – especially those born in developing countries where the rate of premature birth is high (Figure 1) and there is a lack of access to high quality medical facilities.1 The impact of prematurity is significant; it is:
But why is preterm birth so common? And, could it be predicted using extracellular vesicles (EVs)?
Figure 1. World map showing preterm birth rate (%) in 2014. Created with data from1. Grey indicates data not available
In the search for biomarkers of premature birth, EVs are showing promise. EVs are released by all cells, including those of the placenta, allowing for peripheral sampling of placental biomarkers. EVs also protect their cargo which is delivered to target cells, providing a source of circulating biomarkers which would otherwise be degraded. These advantages of EVs as biomarker sources has driven a surge of research into EV biomarkers for preterm birth.
The causes of preterm birth are not currently fully understood. However, risk factors for spontaneous preterm birth include a short cervix, previous preterm birth, multiparous pregnancy (i.e., carrying more than one baby), preeclampsia and other morbidities (e.g., polycystic ovarian syndrome), smoking and low socioeconomic status.
In around 30-40% of cases, however, preterm birth is initiated by medical professionals through the induction of labour or by performing caesarean sections.4 These cases are known as iatrogenic preterm births, a term which is in contrast to spontaneous preterm birth which occurs, well, spontaneously. Iatrogenic preterm births are most often necessitated by preeclampsia or fetal risk of stillbirth (e.g., fetal growth restriction or reduced fetal movements).4
Despite the multitude of risks for preterm birth, biomarkers are severely lacking. The only existing biomarker is vaginal fetal fibronectin which can identify pregnancies at risk of ending in preterm birth within seven days.5,6 However, such a short predictive timeframe is insufficient for the appropriate clinical management of at-risk pregnancies. This short window also cheats researchers of a time window to study spontaneous preterm birth as it is developing, thereby limiting the development of interventions that could prevent prematurity. Importantly, it also does not allow the family to mentally prepare for the infant’s early arrival. Biomarkers with significantly earlier predictive value would be invaluable for addressing these shortfalls – and EVs carrying early warning signs from the placenta could provide the answers.
Much of the research into preterm labour focuses on the placenta (Figure 2). The placenta is often called the life support of the fetus, and with good reason. Nutrient and gas exchange occurs via the placenta, as does the production of hormones for fetal growth and maintenance of pregnancy. The placenta also forms an imperfect barrier to protect the fetus from toxins. Without a functioning placenta, a fetus cannot survive. When the placenta is functioning insufficiently, the fetus cannot thrive.
The blood of the pregnant woman/person is in direct contact with the syncytiotrophoblast, meaning that EVs from the placenta can be directly released into the blood, giving them immense potential as biomarkers. It is worth noting, however, that studies aiming to measure placental-derived EVs using immunocapture for placental alkaline phosphatase (PLAP) may be hindered by two factors. The first is the non-specificity of PLAP which is also expressed in the lung alveolar cells and the endometrium7, meaning that not all PLAP+ EVs will be placental in origin. Secondly, the placental EVs in venous blood are likely not representative of those released from the placenta, as those readily targeting specific cell types or organs will be depleted in peripheral circulation.8 Whilst this can be avoided by sampling from the uterine artery, this can only be done during caesarean section, which rules out uterine artery blood for predictive liquid biopsy.
Figure 2. Schematic representation of a cross-section of a placental villus. The intervillous space-facing side of the placenta is lined with the syncytiotrophoblast, a giant multinucleated cell. The syncytiotrophoblast is responsible for gas and nutrient exchange, meaning that as the placenta and fetus grows, this layer must expand. The nuclei of the syncytiotrophoblast are transcriptionally inactive and so new proteins must be fed into the syncytiotrophoblast via the fusion of underlying cytotrophoblast cells.
Nevertheless, circulating EVs, be they PLAP+ or total EVs, have diagnostic potential. Changes in proteins, lipids and miRNAs have been identified in circulating EVs at 10-13 weeks in pregnancies that subsequently ended in spontaneous preterm birth.9-14 In primiparous pregnancies (i.e., in cases where this is the woman/person’s first pregnancy ending in birth), preterm birth could be predicted using a panel of proteins including TRFE, IC1, ITIH4 and LCAT.10 Having a positive result on this test gave patients a 20% increased risk of preterm delivery as compared to a 2% risk if the test was negative, suggesting that it had some potential for development into a diagnostic test. However, one study investigating the spontaneous premature rupture of membranes – which leads to preterm birth – did not find any altered EV cargo in early pregnancy.15 This might mean that the events leading to premature rupture of membranes have not occurred at the time of sampling, or that a larger cohort is needed to identify their resulting biomarkers in EVs.
Overall, these studies offer hope that circulating EVs could be used to predict spontaneous preterm labour early in pregnancy. Leading the way in this space is NX Prenatal, a company developing diagnostic tests to predict preterm birth and other pregnancy complications based on EVs.
As explained by Brian Brohman, CEO of NX Prenatal, EVs look set to have a place in the development of future tests for premature birth and beyond:
"Advances in personalised prenatal care have been limited by the inability to access relevant information in the maternal-fetal microenvironment. Our EV-based human clinical data leads us to the conviction that, going forward, any biomarker assessment for adverse pregnancy conditions is incomplete without evaluating biomarkers derived from the bioactive EVs in maternal circulation."
Given their utility as predictors of preterm birth, it is perhaps unsurprising that EVs are implicated in the pathogenesis of preterm birth. For instance, inflammatory cargo in circulating EVs increases throughout gestation in mice,16 and the administration of late pregnancy mouse EVs to mice in mid-pregnancy (via intraperitoneal or intraamniotic injection) results in preterm birth – possibly via alarmin HMGB1.16,17 As parturition is an inflammatory process, it makes sense that an inflammatory protein may contribute to preterm birth. Preeclampsia is also associated with inflammation, and does itself carry an increased risk of spontaneous preterm birth, supporting a role for inflammation as a driver of preterm birth.18
Further work is needed to determine whether spontaneous preterm birth is associated with increased inflammatory cargo in EVs in the lead up to parturition, as has been suggested by previous studies.9 If a mechanistic driver of preterm birth could be identified, this could enable the development of a therapeutic which could extend at risk pregnancies towards term. To identify patients for such research, the continued development of early biomarkers for preterm birth is essential. Furthermore, given its contribution to spontaneous and iatrogenic preterm birth, gaining a better understanding of preeclampsia and developing diagnostic and therapeutic strategies could have a significant impact on addressing prematurity.
In the next article in this series, we discuss preeclampsia, diving into the role of EVs in the biogenesis, diagnostics and treatment of this disease.