Are there foetal extracellular vesicles in maternal blood? Prospects for diagnostic biomarker discovery

Abstract

Prenatal diagnosis of congenital disease improves clinical outcomes; however, as many as 50% of congenital heart disease cases are missed by current ultrasound screening methods. This indicates a need for improved screening technology. Extracellular vesicles (EVs) have attracted enormous interest in recent years for their potential in diagnostics. EVs mediate endocrine signalling in health and disease and are known to regulate aspects of embryonic development. Here, we critically evaluate recent evidence suggesting that EVs released from the foetus are able to cross the placenta and enter the maternal circulation. Furthermore, EVs from the mother appear to be transported in the reverse direction, whilst the placenta itself acts as a source of EVs. Experimental work utilising rodent models employing either transgenically encoded reporters or application of fluorescent tracking dyes provide convincing evidence of foetal-maternal crosstalk. This is supported by clinical data demonstrating expression of placental-origin EVs in maternal blood, as well as limited evidence for the presence of foetal-origin EVs. Together, this work raises the possibility that foetal EVs present in maternal blood could be used for the diagnosis of congenital disease. We discuss the challenges faced by researchers in translating these basic science findings into a clinical non-invasive prenatal test.

Keywords: Congenital disease; Diagnostics; Exosome; Extracellular vesicle; Microvesicle; Placenta; Trophoblast.

Fig. 1

Anatomy of the placenta. In both humans (A–C) and rodents (D–F), the syncytiotrophoblast forms a barrier separating maternal blood (pink) from foetal blood (purple). In humans, the syncytiotrophoblast surrounds the foetal blood vessels which are located in villi projecting into the maternal blood space. In the rodent placenta, the syncytiotrophoblast surrounds the maternal blood space and separates this from foetal capillaries within a structure known as the labyrinth

Fig. 2

Trafficking and production of extracellular vesicles by the placental syncytiotrophoblast. Three classes of EVs produced by the syncytiotrophoblast are detectable in maternal blood. Exosomes (30–150 nm) originate within the endosomal pathway, whilst microvesicles (100–1000 nm) are produced by blebbing from the plasma membrane. Syncytial nuclear aggregates are a class of large vesicles unique to the trophoblast which contain nuclei. The placenta releases endogenous EVs, but also traffics EVs from the foetal to the maternal circulation. It is hypothesised that foetal EVs are endocytosed and processed through the endosomal pathway. ESE, early sorting endosome; LSE, late sorting endosome; MVB, multivesicular body

Fig. 3

The mT/mG mouse model expresses fluorescently labelled EVs. A mT/mG mice have been engineered to carry a single copy of a transgene inserted randomly into the genome. The transgene (left) consists of two expression cassettes for the lipid-anchored MARCKS protein fused to a fluorescent reporter (mT: tandem dimer tomato; mG: enhanced green fluorescent protein [EGFP]). The ubiquitously expressed pCAG promoter drives expression. LoxP sites flank the mT cassette. In the unrecombined allele, an upstream STOP codon prevents translation of mG, and thus, EVs express mT (right). B The enzyme CRE-recombinase recognises the LoxP sites and mediates recombination of the allele, resulting in excision of the mT cassette. Following recombination, the pCAG promoter drives expression of mG, and thus, EVs express mG (right)

Fig. 4

Tracking dyes commonly used to label EVs. Two classes of fluorescent dyes are commonly used for EV labelling: membrane-permeating and lipophilic dyes. A Membrane-permeating dyes (DDAO-SE and CFDA-SE) are lipophilic molecules that can cross the EV membrane. CFDA-SE is minimally fluorescent in the native state, but is activated once inside the EVs by endogenous esterases which cleave the acetate groups. DDAO-SE, on the other hand, does not require esterases to be activated; instead, it forms covalent attachments to amines both inside and outside of EVs. B Lipophilic dyes (DiR, DiI and PKH26) are a family of fluorescent stains for labelling membranes and other hydrophobic structures. The fluorescence of these dyes is enhanced once incorporated into the EV membrane

Fig. 5

Endogenous markers of placental EVs. EVs express various proteins, glycoproteins and lipid-associated molecules at or near the membrane. These include tetraspanins (CD9, CD63, CD81), antigen-presenting molecules (MHC class I and class II) and ESCRT complex proteins. EVs also carry a cargo including nucleic acids (DNA, miRNA, mRNA). Placental-specific EV markers are shown in red and include HLA-G, syncytin, the lipid-anchored phosphatase PLAP and miRNA expressed from the C19MC and C14MC clusters