Carlos Palma

Oxygen tension regulates the miRNA profile and bioactivity of exosomes released from extravillous trophoblast cells-liquid biopsies for monitoring complications of pregnancy

Our understanding of how cells communicate has undergone a paradigm shift since the recent recognition of the role of exosomes in intercellular signaling. In this study, we investigated whether oxygen tension alters the exosome release and miRNA profile from extravillous trophoblast (EVT) cells, modifying their bioactivity on endothelial cells (EC). Furthermore, we have established the exosomal miRNA profile at early gestation in women who develop pre-eclampsia (PE) and spontaneous preterm birth (SPTB). HTR-8/SVneo cells were used as an EVT model. The effect of oxygen tension (i.e. 8% and 1% oxygen) on exosome release was quantified using nanocrystals (Qdot®) coupled to CD63 by fluorescence NTA. A real-time, live-cell imaging system (Incucyte™) was used to establish the effect of exosomes on EC. Plasma samples were obtained at early gestation (<18 weeks) and classified according to pregnancy outcomes. An Illumina TrueSeq Small RNA kit was used to construct a small RNA library from exosomal RNA obtained from EVT and plasma samples. The number of exosomes was significantly higher in EVT cultured under 1% compared to 8% oxygen. In total, 741 miRNA were identified in exosomes from EVT. Bioinformatic analysis revealed that these miRNA were associated with cell migration and cytokine production. Interestingly, exosomes isolated from EVT cultured at 8% oxygen increased EC migration, whilst exosomes cultured at 1% oxygen decreased EC migration. These changes were inversely proportional to TNF-α released from EC. Finally, we have identified a set of unique miRNAs in exosomes from EVT cultured at 1% oxygen and exosomes isolated from the circulation of mothers at early gestation, who later developed PE and SPTB. We suggest that aberrant exosomal signalling by placental cells is a common aetiological factor in pregnancy complications characterised by incomplete SpA remodeling and is therefore a clinically relevant biomarker of pregnancy complications.

Amniotic fluid exosome proteomic profile exhibits unique pathways of term and preterm labor

Our objective was to determine the amniotic fluid-derived exosomal proteomic profile in patients who had spontaneous preterm birth (PTB) or preterm premature rupture of membranes (pPROM) compared with those who delivered at term. A cross-sectional study of a retrospective cohort was used to quantify and determine the protein content of exosomes present in amniotic fluid, in PTB or pPROM, and normal term labor (TL) or term not in labor (TNIL) pregnancies. Exosomes were isolated by differential centrifugation and quantified using nanocrystals (Qdot) coupled to CD63 and placental alkaline phosphatase (PLAP) by fluorescence nanoparticle tracking analysis. The exosomal proteomic profile was identified by liquid chromatography-tandem mass spectrometry, and a small ion library was constructed to quantify the proteomic data by Sequential Window Acquisition of All Theoretical analysis. Ingenuity Pathway Analysis determined canonical pathways and biofunctions associated with dysregulated proteins. Amniotic fluid exosomes have similar shape and quantity regardless of the conditions; however, the PLAP/CD63 ratios for TL, PTB, and pPROM were significantly higher (∼3.8-, ∼4.4-, and ∼3.5-fold, respectively) compared with TNIL. The PLAP/CD63 ratio was also significantly higher (∼1.3-fold) in PTB compared with pPROM. Biological functions primarily indicated nonspecific inflammatory response regardless of condition, but unique profiles were also identified in cases (PTB and pPROM) compared with term. Amniotic fluid exosomes provide information specific to normal and abnormal parturition. Inflammatory marker enrichment and its uniqueness in term and preterm pregnancies support the value of exosomes in determining underlying physiology associated with term and preterm parturition.

Placental exosomes profile in maternal and fetal circulation in intrauterine growth restriction - Liquid biopsies to monitoring fetal growth

INTRODUCTION Placenta-derived exosomes may represent an additional pathway by which the placenta communicates with the maternal system to induce maternal vascular adaptations to pregnancy and it may be affected during Fetal growth restriction (FGR). The objective of this study was to quantify the concentration of total and placenta-derived exosomes in maternal and fetal circulation in small fetuses classified as FGR or small for gestational age (SGA). METHODS Prospective cohort study in singleton term gestations including 10 normally grown fetuses and 20 small fetuses, sub-classified into SGA and FGR accordingly to birth weight (BW) percentile and fetoplacental Doppler. Exosomes were isolated from maternal and fetal plasma and characterized by morphology, enrichment of exosomal proteins, and size distribution by electron microscopy, western blot, and nanoparticle tracking analysis, respectively. Total and specific placenta-derived exosomes were determined using quantum dots coupled with CD63+ve and placental-type alkaline phosphatase (PLAP)+ve antibodies, respectively. RESULTS Maternal concentrations of CD63+ve and PLAP+ve exosomes were similar between the groups (all p > 0.05). However, there was a significant positive correlation between the ratio of placental-derived to total exosomes (PLAP+ve ratio) and BW percentile, [rho = 0.77 (95% CI: 0.57 to 0.89); p = 0.0001]. The contribution of placental exosomes to the total exosome concentration in maternal and fetal circulation showed a significant decrease among cases, with lower PLAP+ve ratios in FGR compared to controls and SGA cases. DISCUSSION Quantification of placental exosomes in maternal plasma reflects fetal growth and it may be a useful indicator of placental function.

Circulating cell-free miR-494 and miR-21 are disease response biomarkers associated with interim-positron emission tomography response in patients with diffuse large b-cell lymphoma

MicroRNA (miRNA)s are dysregulated in Diffuse large B-cell lymphoma (DLBCL), where they reflect the malignant B-cells and the immune infiltrate within the tumor microenvironment. There remains a paucity of data in DLBCL regarding cell-free (c-f) miRNA as disease response biomarkers. Immunosuppressive monocyte/macrophages, which are enriched in DLBCL, are disease response markers in DLBCL, with miRNA key regulators of their immunosuppressive function. Our aim was to determine whether plasma miRNA that reflect the activity of the malignant B-cell and/or immunosuppressive monocytes/macrophages, have value as minimally-invasive disease response biomarkers in DLBCL. Quantification of 99 DLBCL tissues, to select miRNA implicated in immunosuppressive monocytes/macrophage biology, found miR-494 differentially elevated. In a discovery cohort (22 patients), pre-therapy c-f miR-494 and miR-21 but not miR-155 were raised relative to healthy plasma. Both miR-494 and miR-21 levels 3-6 months reduced post immuno-chemotherapy. The validation cohort (56 patients) was from a prospective clinical trial. Interestingly, in sequential samples both miRNAs decreased in patients becoming Positron Emission Tomography/Computerized Tomography (PET/CT)-ve, but not in those remaining interim-PET/CT+. Patient monocytes were phenotypically and functionally immunosuppressive with ex-vivo monocyte depletion enhancing T-cell proliferation in patient but not healthy samples. Pre-therapy monocytes showed an immunosuppressive transcriptome and raised levels of miR-494. MiR-494 was present in all c-f nanoparticle fractions but was most readily detectable in unfractionated plasma. Circulating c-f miR-494 and miR-21 are disease response biomarkers with differential response stratified by interim-PET/CT in patients with DLBCL. Further studies are required to explore their manipulation as potential therapeutic targets.

OP01.02: Placenta‐derived exosomes in pregnancies complicated with fetal growth restriction at term

Objectives: Evaluation of the fetus at risk for uteroplacental insufficiency and growth restriction applies spectral Doppler measurements of the fetal circulation. Our objective is to determine if fetuses with abdominal circumference (AC) below the 5th or weight (EFW) below the 10th percentile will have lower values for the cerebroplacental ratio or the cerebrorenal ratio. Methods: We evaluated 2900 unselected women with multiple associated fetal and maternal co morbidities in whom we measured both the CPR and the CRR using previously standardised methodology. Results: No discernible differences were found between fetuses with low AC <5 or 10 percentile and fetuses with EFW <10% and the CRR or CPR of the appropriately grown fetuses. We plotted the values of each on previously created reference curves (figure). Conclusions: The measurement of the CPR or the CRR among fetuses with AC <10th percentile was not better than EFW <10th percentile to identify fetuses that would ultimately have a lower value consistent with centralisation of fetal blood flow.

Pregnancy-Associated Exosomes Changes in Pregnancies Complicated by Small-for-Gestational-Age (SGA) Neonates and Intrauterine Growth Restriction (IUGR)

Figures will be available only online Underline represents presenting author; Asterisk represents senior author; Dagger represents an in-training author.

Exosomal Content in the Plasma of Patients with Ovarian Cancer Reflect Tumor State and Induce the Epithelial to Mesenchymal Transition in Target Cells

Figures will be available only online Underline represents presenting author; Asterisk represents senior author; Dagger represents an in-training author.

Extracellular vesicles (ev) size in shallow trophoblast invasion in old world non-human primates

Materials and Methods Introduction Marcel Chuecos, BS1, Edward Dick2, Gene Hubbard3, Cathy Perez2, Soumyalekshmi Nair4, Carlos Palma4,Vyjayanthi Kinhal4, Carlos Salomon4 and Natalia Shlabritz-Loutsevitch, MD, PhD1. 1OB/GYN Research, TTUHSC-Permian Basin, Odessa, Texas, United States. 2Southwest National Primate Center, Texas Biomedical Research Institute, San Antonio, Texas, USA . 3University of Texas Health Sciences Center at San Antonio, San Antonio, Texas, USA. 4Exosome Biology Laboratory, Centre for Clinical Diagnostics, University of Queensland Centre for Clinical Research, Royal Brisbane and Womens Hospital, The University of Queensland, Brisbane, QLD, Australia. 5Maternal-Fetal Medicine, Department of Obstetrics and Gynecology, Ochsner Clinic Foundation, New Orleans, Louisiana USA; Department of Clinical Biochemistry and Immunology, Faculty of Pharmacy, University of Concepción, Concepción, Chile