First trimester plasma-derived exosomal proteins: putative biomarker for early detection of pathological pregnancies

Abstract

Background: Of the 130 million babies born each year, 8 million die before their first birthday. A contributing factor in many of these deaths is poor pregnancy outcome as a result of complications of pregnancy. Preeclampsia (PE) and Gestational Diabetes Mellitus (GDM) are the common pregnancy complications that have no effective antenatal treatment other than steroid administration and timely delivery. Each occurs with an incidence of 5-10% and is responsible for the majority of obstetric and paediatric morbidity and mortality. These can permanently impact on lifelong health. Early detection of disease risk and onset is the first step in implementing efficacious treatment and improving patient outcome. In the context of antenatal screening, the objective is to identify biomarkers (e.g. proteins) that are informative of the risk of asymptomatic pregnant women subsequently developing complications of pregnancy. Recent studies highlight the putative utility of tissue-specific nanovesicles (i.e. exosomes) in the diagnosis of disease onset and treatment monitoring. It was hypothesized that presymptomatic women who subsequently develop pregnancy complications display altered exosome profile (i.e. concentration and/or protein content) at first trimester of pregnancy (i.e. 6-12 weeks). The general aim of this thesis was to identify blood-borne biomarkers (i.e. exosomes) that may be used at the first antenatal visit to identify presymptomatic women who are at risk of developing complications of pregnancy. Methods: A time-series experimental design was used to establish pregnancy-associated changes in maternal plasma exosome concentrations during first trimester. Serial samples of plasma were collected from normal healthy pregnant women (n=10) at 6, 7, 8, 9, 10, 11 and 12 weeks of gestation. Plasma samples from pregnant women at 11-14 weeks of gestation who developed preeclampsia (n=15) and gestational diabetes mellitus (n=7) were also collected. Exosomes were isolated by differential and buoyant density centrifugation and characterised by size distribution and morphology using nanoparticles tracking analysis (NTA; NanoSightt) and transmission electron microscopy (TEM) respectively. The total number of exosome vesicles and placenta-derived exosome vesicles were determined by quantifying immunoreactive exosomal CD63 and a placenta-specific marker (PLAP) by ELISA. Finally, the differentially expressed exosomal proteins and peptides were identified by Liquid Chromatography and Mass Spectrometry based approaches (LC-MS/MS). Statistical analysis was performed using the Graph Pad Prism software. Results: EM and NTA identified the presence of 50 - 120 nm spherical vesicles in maternal plasma as early as 6 weeks of pregnancy. The number of exosomes in maternal circulation increased significantly (ANOVA, p=0.002) with the progression of pregnancy (from 6 to 12 weeks). The concentration of placenta-derived exosomes in maternal plasma (i.e. PLAP+) increased progressively with gestational age, from 6 weeks 70.6 p 5.7 pg/ml to 12 weeks 117.5 p 13.4 pg/ml. Regression analysis shows that week is a factor that explains g70% of the observed variation in plasma exosomal PLAP concentration, total exosome number only explains 20%. The total number of exosome was 1.31e12 p 1.60e12 (mean p SD) during the first trimester in women who developed Gestational Diabetes Mellitus (GDM) in second trimester and 1.09e11 p 3.17e10 in women who developed Preeclampsia (PE). The statistical analysis showed that the total number of released exosomes in the first trimester of pregnancy complicated with GDM was three- fold higher than normal pregnancy and the concentration of the exosomes released from the placental cells was twofold compared to PLAP concentration in normal pregnancy. In addition, a significant (pl0.001) increase in the cumulative number of exosomes was observed during the first trimester of preeclamptic condition when compared with the first trimester normal pregnancy condition. Conclusion: This study presents longitudinal data on placental-derived exosomes in the first trimester of pregnancy, starting from as early as 6 weeks. Early detection of women at risk of pregnancy complications would provide opportunity to evaluate appropriate intervention strategies to limit acute adverse sequelae. The rationale for developing early pregnancy screening tests is not only for the management of the contemporaneous pregnancy but also to optimise lifelong and intergenerational health. If this can be achieved, it will provide an opportunity for early assessment of risk and the implementation of an alternative clinical management strategy to improve outcome for both the mother and the baby.