Background: Preeclampsia is a major cause of maternal and perinatal morbidity and mortality worldwide. Identifying women with high risk of developing preeclampsia in early pregnancy remains challenging. Extracellular vesicles released from the placenta offer an attractive biomarker but have been elusive to quantify. Methods: Here, we tested ExoCounter, a novel device that immunophenotypes size-selected small extracellular vesicles <160 nm, for its ability to perform qualitative and quantitative placental small extracellular vesicles (psEV) analysis. To investigate disease-specific and gestational age-specific changes, we analyzed psEV counts in maternal plasma samples taken at each of the 3 trimesters from women who had (1) normal pregnancy (n=3); (2) women who developed early-onset preeclampsia (EOPE; n=3); and (3) women who developed late-onset preeclampsia (n=4) using 3 antibody pairs, CD10-placental alkaline phosphatase (PLAP), CD10-CD63, and CD63-PLAP. We further validated the findings in first-trimester serum samples among normal pregnancy (n=9), women who developed EOPE (n=7), and women who developed late-onset preeclampsia (n=8). Results: We confirmed that CD63 was the major tetraspanin molecule coexpressed with PLAP—a known placental extracellular vesicles marker on psEV. Higher psEV counts for all 3 antibody pairs were detected in the plasma of women who developed EOPE than the other 2 groups in the first trimester, which persisted through the second and third trimesters. Significantly higher CD10-PLAP ( P <0.01) and CD63-PLAP ( P <0.01) psEV counts were validated in the serum of the first trimester of women who developed EOPE compared with normal pregnancy. Conclusions: Application of the ExoCounter assay developed here could identify patients at risk of developing EOPE in the first trimester, thereby providing a window of opportunity for early intervention.

Circulating small extracellular vesicles (sEVs) released from the placenta carry bioactive compounds. Placental sEVs (psEVs) have been implicated as drivers of pathology in preeclampsia, a common disorder of pregnancy. This study investigates the hypothesis that psEVs are detectable in cerebrospinal fluid (CSF) during pregnancy, and are present at higher concentrations in preeclamptic and eclamptic pregnancies. Two techniques were used to search for psEVs. Firstly, the ExoCounter assay was performed on neat CSF from normotensive, preeclamptic and eclamptic pregnancies, and non-pregnant controls (n = 11, 12, 10 and 4, respectively). Quantitative PCR was used to search for psEV-associated microRNAs in the CSF of pregnant women. Neither assay found evidence of psEVs in the CSF of pregnant women, regardless of whether they had preeclampsia or eclampsia. This study suggests that psEVs do not reside in CSF during pregnancy and may be more likely to impact central nervous tissues through peripheral changes or interaction with the blood–brain barrier without crossing.

Quantitative knowledge of nanoparticle properties is desirable in a large number of scientific and technological applications, but measurements with a high degree of precision usually prove to be challenging. Among a range of available methodologies, optical techniques with single-particle sensitivity are especially interesting because they can reveal intrinsic heterogeneities in a fast, noninvasive manner. Recently, we presented interferometric nanoparticle tracking analysis (iNTA) as a highly sensitive label-free technique that is capable of determining the size, concentration, and index of refraction of different subpopulations in a suspension mixture. Here, we enhance this method with biochemical specificity through multicolor fluorescence detection at the single-molecule sensitivity limit. We benchmark the performance of the combined technique, which we name iNTA-F, by distinguishing populations of fluorescent and nonfluorescent nanoparticles of different material, size, and fluorescence intensity, with an emphasis on the characterization of lipid vesicles and biological extracellular vesicles (EVs).

Background: Early-onset preeclampsia (EOPE) is frequently complicated by fetal growth restriction (FGR). The sFlt-1/PlGF ratio reflects maternal angiogenic imbalance but provides limited insight into placental pathology-the root cause of preeclampsia. Collagen XVIIA1 (COLXVIIA1), a transmembrane hemidesmosomal collagen expressed in trophoblasts, may serve as a biomarker of placental injury. Methods: Circulating COLXVIIA1 was first measured in gestational age–matched peripheral blood samples from EOPE and normotensive pregnancies (n = 6 per group). Placental origin was assessed using paired uterine and peripheral vein samples (n = 9). Placental COLXVIIA1 expression, localisation, and processing were analysed in normotensive and EOPE placentas. Trophoblast-derived extracellular vesicles (TB-sEVs) were isolated from ex vivo perfused placentas to assess COLXVIIA1 packaging. In a prospective cohort of 167 women with suspected preeclampsia, circulating COLXVIIA1 was analysed alongside PlGF and sFlt-1, and diagnostic performance of the COLXVIIA1/PlGF ratio was compared with sFlt-1/PlGF. Findings: Circulating COLXVIIA1 levels were significantly elevated in EOPE compared with gestational age–matched controls, indicating independence from gestational age. Uterine vein levels exceeded peripheral levels, consistent with placental release. COLXVIIA1 expression was localised to trophoblasts and amniotic epithelium. In EOPE placentas, COLXVIIA1-enriched hotspots co-localised with fibrin nodules, perivillous fibrin deposition, and peri-infarct regions. Increased proteolytic processing (∼120 kDa ectodomain) and preferential packaging of transmembrane COLXVIIA1 into TB-sEVs were observed. The COLXVIIA1/PlGF ratio performed comparably to sFlt-1/PlGF for overall preeclampsia and showed strong predictive accuracy for EOPE with FGR (AUC = 0.858), confirmed by bootstrap analysis. Interpretation: Abnormal COLXVIIA1 expression, processing and release reflects trophoblast response to structural injury and provides a circulating signal complementary to angiogenic markers. The COLXVIIA1/PlGF ratio captures a distinct dimension of placental pathology and may improve risk stratification in EOPE, particularly in pregnancies complicated by FGR.