BackgroundHuman placenta releases specific nanovesicles (i.e. exosomes) into the maternal circulation during pregnancy, however, the presence of placenta-derived exosomes in maternal blood during early pregnancy remains to be established. The aim of this study was to characterise gestational age related changes in the concentration of placenta-derived exosomes during the first trimester of pregnancy (i.e. from 6 to 12 weeks) in plasma from women with normal pregnancies.MethodsA time-series experimental design was used to establish pregnancy-associated changes in maternal plasma exosome concentrations during the first trimester. A series of plasma were collected from normal healthy women (10 patients) at 6, 7, 8, 9, 10, 11 and 12 weeks of gestation (n = 70). We measured the stability of these vesicles by quantifying and observing their protein and miRNA contents after the freeze/thawing processes. Exosomes were isolated by differential and buoyant density centrifugation using a sucrose continuous gradient and characterised by their size distribution and morphology using the nanoparticles tracking analysis (NTA; Nanosight™) and electron microscopy (EM), respectively. The total number of exosomes and placenta-derived exosomes were determined by quantifying the immunoreactive exosomal marker, CD63 and a placenta-specific marker (Placental Alkaline Phosphatase PLAP).ResultsThese nanoparticles are extraordinarily stable. There is no significant decline in their yield with the freeze/thawing processes or change in their EM morphology. NTA identified the presence of 50–150 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 ± 5.7 pg/ml to 12 weeks 117.5 ± 13.4 pg/ml. Regression analysis showed that weeks is a factor that explains for >70% of the observed variation in plasma exosomal PLAP concentration while the total exosome number only explains 20%.ConclusionsDuring normal healthy pregnancy, the number of exosomes present in the maternal plasma increased significantly with gestational age across the first trimester of pregnancy. This study is a baseline that provides an ideal starting point for developing early detection method for women who subsequently develop pregnancy complications, clinically detected during the second trimester. Early detection of women at risk of pregnancy complications would provide an opportunity to develop and evaluate appropriate intervention strategies to limit acute adverse sequel.
Although there is significant interest in elucidating the role of placenta-derived exosomes (PdEs) during pregnancy, the exosomal profile in pregnancies complicated by gestational diabetes mellitus (GDM) remains to be established. The aim of this study was to compare the gestational-age profile of PdEs in maternal plasma of GDM with normal pregnancies and to determine the effect of exosomes on cytokine release from human umbilical vein endothelial cells. A prospective cohort of patients was sampled at three time points during pregnancy for each patient (i.e., 11–14, 22–24, and 32–36 weeks' gestation). A retrospective stratified study design was used to quantify exosomes present in maternal plasma of normal (n = 13) and GDM (n = 7) pregnancies. Gestational age and pregnancy status were identified as significant factors contributing to variation in plasma exosome concentration (ANOVA, P < 0.05). Post hoc analyses established that PdE concentration increased during gestation in both normal and GDM pregnancies; however, the increase was significantly greater in GDM (∼2.2-fold, ∼1.5-fold, and ∼1.8-fold greater at each gestational age compared with normal pregnancies). Exosomes isolated from GDM pregnancies significantly increased the release of proinflammatory cytokines from endothelial cells. Although the role of exosomes during GDM remains to be fully elucidated, exosome profiles may be of diagnostic utility for screening asymptomatic populations.
Inherent interindividual and intraindividual variation in the length of the menstrual cycle limits the accuracy of predicting days of peak fertility. To improve detection of days of peak fertility, a more detailed understanding of longitudinal changes in cervicovaginal fluid (CVF) biomarkers during the normal menstrual cycle is needed. The aim of this study, therefore, was to characterize longitudinal changes in CVF proteins during the menstrual cycle using a quantitative, data-independent acquisition mass spectrometry approach. Six serial samples were collected from women (n = 10) during the menstrual cycle. Samples were obtained at two time points for each phase of the cycle: early and late preovulatory, ovulatory, and postovulatory. Information-dependent acquisition (IDA) of mass spectra from all individual CVF samples was initially performed and identified 278 total proteins. Samples were then pooled by time of collection (n = 6 pools) and analyzed using IDA and information-independent acquisition (Sequential Windowed Acquisition of All Theoretical Mass Spectra [SWATH]). The IDA library generated contained 176 statistically significant protein identifications (P < 0.000158). The variation in the relative abundance of CVF proteins across the menstrual cycle was established by comparison with the SWATH profile against the IDA library. Using time-series, pooled samples obtained from 10 women, quantitative data were obtained by SWATH analysis for 43 CVF proteins. Of these proteins, 28 displayed significant variation in relative abundance during the menstrual cycle (assessed by ANOVA). Statistical significant changes in the relative expression of CVF proteins during preovulatory, ovulatory, and postovulatory phases of menstrual cycle were identified. The data obtained may be of utility not only in elucidating underlying physiological mechanisms but also as clinically useful biomarkers of fertility status.
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; NanoSight™) 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 placentaspecific 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.ii 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 ± 5.7 pg/ml t...
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