Extracellular vesicles during the three trimesters of pregnancy.

OBJECTIVES: Extracellular vesicles (EVs) are cell-derived particles released during different pathophysiological processes and emerging as relevant players in inter-cellular crosstalk. Previous studies have highlighted the role of EVs as potential biomarkers for several pregnancy complications, including miscarriage, pre-eclampsia and gestational diabetes. Despite that, the actual distribution of EVs through gestation has not been reported yet. The aim of this study was to report the concentration of different sub-types of EVs in the first, second and third trimester of pregnancy and to correlate them with different pregnancy and ultrasound characteristics. STUDY DESIGNS: Prospective observational study including uncomplicated pregnancies in the first, second and third trimester of pregnancy. The first aim of the study was to report the concentration of the EVs derived from endothelial, epithelial, platelet and leukocyte cells of maternal peripheral blood samples in the first, second and third trimester pregnancy using polychromatic flow cytometry. The secondary aim was to correlate EVs with neonatal birthweight and fetal Dopplers, including uterine and umbilical arteries. Un and multivariate analyses were used to compute the data. RESULTS: 64 women (20 in the first, 22 in the second and 22 in the third trimester of pregnancies) were included in the analysis. There was no difference in the median concentration of either platelet, leukocyte and endothelial EVs between the first, second and third trimester of pregnancy. The concentration of epithelial derived EVs was higher in the third compared to first and second trimester of pregnancy. When analyzing the percentage of EV vesicles through gestation, there was no difference in the percentage of either leukocyte or endothelial EVs through gestation. Conversely, the median percentage of platelet derived vesicles was higher in the first (48.7 %, IQR 34.1-58.5) compared to second (34.0 %, IQR 22.7-44.9) and third (9.13 %, IQR 5.01-12.1) trimester of pregnancy, while the median percentage of third trimester (6.01, IQR 2.42-7.34) epithelial derived vesicles was higher than that of the second (1.53 %, IQR 0.65-2.98), but not of the first (4.45 %, IQR 1.44-6.07) trimester. Finally, we found no association between the median concentration or percentage of endothelial, epithelial, leukocyte vesicles, neonatal birthweight and fetal or maternal Dopplers. CONCLUSIONS: Distribution of EVs examined does not change during the three trimesters of pregnancy and is not influenced by neonatal birthweight or maternal and fetal Dopplers. The findings from this study allows a more objective interpretation of studies comparing EVs in pregnancies with compared to those without obstetric complication. EVs in future can be used for "liquid biopsy" for the early diagnosis of pathological pregnancies up to the development of possible screening protocols.

Circulating Extracellular Vesicles: Their Role in Patients with Abdominal Aortic Aneurysm (AAA) Undergoing EndoVascular Aortic Repair (EVAR).

Abdominal aortic aneurysm (AAA) is a frequent aortic disease. If the diameter of the aorta is larger than 5 cm, an open surgical repair (OSR) or an endovascular aortic repair (EVAR) are recommended. To prevent possible complications (i.e., endoleaks), EVAR-treated patients need to be monitored for 5 years following the intervention, using computed tomography angiography (CTA). However, this radiological method involves high radiation exposure in terms of CTA/year. In such a context, the study of peripheral-blood-circulating extracellular vesicles (pbcEVs) has great potential to identify biomarkers for EVAR complications. We analyzed several phenotypes of pbcEVs using polychromatic flow cytometry in 22 patients with AAA eligible for EVAR. From each enrolled patient, peripheral blood samples were collected at AAA diagnosis, and after 1, 6, and 12 months following EVAR implantation, i.e. during the diagnostic follow-up protocol. Patients developing an endoleak displayed a significant decrease in activated-platelet-derived EVs between the baseline condition and 6 months after EVAR intervention. Furthermore, we also observed, that 1 month after EVAR implantation, patients developing an endoleak showed higher concentrations of activated-endothelial-derived EVs than patients who did not develop one, suggesting their great potential as a noninvasive and specific biomarker for early identification of EVAR complications.

Extracellular Vesicles in pregnancy: Their potential role as a liquid biopsy.

OBJECTIVE: Extracellular Vesicles (EVs) are cell-derived particles released during different pathophysiological processes, circulating in many body fluids and mediating the inter-cellular crosstalk. We have analyzed, for the first time, different EV phenotypes and concentrations in the peripheral blood of uncomplicated pregnant women. STUDY DESIGN: In this prospective case-control study, uncomplicated singleton pregnant women at term (N = 59) and aged matched non-pregnant women (N = 21) were enrolled. Freshly drowned peripheral blood samples were stained for flow cytometry analyses of EVs. RESULTS: EVs derived from platelets, leukocytes, endothelial and epithelial cells were identified and counted. Platelet-derived EVs were higher in pregnant compared to non-pregnant women, both in terms of absolute counts (2064.4 ± 1156.3 vs 701.1 ± 378.8; p < 0.0001) and percentages (27.6 ± 17.2 vs 10.7 ± 5.9; p < 0.0001). The opposite pattern was observed both for concentrations of endothelial-EV counts (525.8 ± 499.6 vs 844.7 ± 652.9; p = 0.007) and percentages (6.1 ± 5.5 vs 11.8 ± 8.0; p < 0.0001) and leukocyte-derived EV percentages (10.2 ± 7.4 vs 17.9 ± 11.2; p = 0.002) EVs. CONCLUSIONS: Uncomplicated pregnancies are characterized by a specific EV signature. These cell-derived particles may therefore represent promising biomarkers of different pathological conditions complicating pregnancies, such as preeclampsia or preterm birth.

SARS-CoV-2 and Immunity: Natural Infection Compared with Vaccination.

Recently, the protective and/or pathological role of virus-specific T cells in SARS-CoV-2 infection has been the focus of many studies. We investigated the anti-spike IgG levels and SARS-CoV-2-specific T cells in 125 donors (90 vaccinated with four different vaccine platforms, 16 individuals with a previous natural infection, and 19 not vaccinated donors who did not report previous SARS-CoV-2 infections). Our data show that anti-spike IgG titers were similar between naturally infected subjects and those vaccinated with adenoviral vector vaccines. Of note, all immunized donors produced memory CD4+ and/or CD8+ T cells. A sustained polyfunctionality of SARS-CoV-2-specific T cells in all immunized donors was also demonstrated. Altogether, our data suggest that the natural infection produces an overall response like that induced by vaccination. Therefore, this detailed immunological evaluation may be relevant for other vaccine efforts especially for the monitoring of novel vaccines effective against emerging virus variants.

Blood Circulating CD133+ Extracellular Vesicles Predict Clinical Outcomes in Patients with Metastatic Colorectal Cancer.

Colorectal cancer (CRC) is one of the most incident and lethal malignancies worldwide. Recent treatment advances prolonged survival in patients with metastatic colorectal cancer (mCRC). However, there are still few biomarkers to guide clinical management and treatment selection in mCRC. In this study, we applied an optimized flow cytometry protocol for EV identification, enumeration, and subtyping in blood samples of 54 patients with mCRC and 48 age and sex-matched healthy controls (HCs). The overall survival (OS) and overall response rate (ORR) were evaluated in mCRC patients enrolled and treated with a first line fluoropyrimidine-based regimen. Our findings show that patients with mCRC presented considerably higher blood concentrations of total EVs, as well as CD133+ and EPCAM+ EVs compared to HCs. Overall survival analysis revealed that increased blood concentrations of total EVs and CD133+ EVs before treatment were significantly associated with shorter OS in mCRC patients (p = 0.001; and p = 0.0001, respectively). In addition, we observed a correlation between high blood levels of CD133+ EVs at baseline and reduced ORR to first-line systemic therapy (p = 0.045). These findings may open exciting perspectives into the application of novel blood-based EV biomarkers for improved risk stratification and optimized treatment strategies in mCRC.

Diagnostic Impact of Radiological Findings and Extracellular Vesicles: Are We Close to Radiovesicolomics?

Currently, several pathologies have corresponding and specific diagnostic and therapeutic branches of interest focused on early and correct detection, as well as the best therapeutic approach. Radiology never ceases to develop newer technologies in order to give patients a clear, safe, early, and precise diagnosis; furthermore, in the last few years diagnostic imaging panoramas have been extended to the field of artificial intelligence (AI) and machine learning. On the other hand, clinical and laboratory tests, like flow cytometry and the techniques found in the "omics" sciences, aim to detect microscopic elements, like extracellular vesicles, with the highest specificity and sensibility for disease detection. If these scientific branches started to cooperate, playing a conjugated role in pathology diagnosis, what could be the results? Our review seeks to give a quick overview of recent state of the art research which investigates correlations between extracellular vesicles and the known radiological features useful for diagnosis.

Role of Extracellular Vesicles in Epithelial Ovarian Cancer: A Systematic Review.

Extracellular vesicles (EVs) are a heterogeneous group of cell-derived submicron vesicles released under physiological or pathological conditions. EVs mediate the cellular crosstalk, thus contributing to defining the tumor microenvironment, including in epithelial ovarian cancer (EOC). The available literature investigating the role of EVs in EOC has been reviewed following PRISMA guidelines, focusing on the role of EVs in early disease diagnosis, metastatic spread, and the development of chemoresistance in EOC. Data were identified from searches of Medline, Current Contents, PubMed, and from references in relevant articles from 2010 to 1 April 2020. The research yielded 194 results. Of these, a total of 36 papers, 9 reviews, and 27 original types of research were retained and analyzed. The literature findings demonstrate that a panel of EV-derived circulating miRNAs may be useful for early diagnosis of EOC. Furthermore, it appears clear that EVs are involved in mediating two crucial processes for metastatic and chemoresistance development: the epithelial-mesenchymal transition, and tumor escape from the immune system response. Further studies, more focused on in vivo evidence, are urgently needed to clarify the role of EV assessment in the clinical management of EOC patients.

Extracellular Vesicles in Feto-Maternal Crosstalk and Pregnancy Disorders.

Extracellular vesicles (EVs) actively participate in inter-cellular crosstalk and have progressively emerged as key players of organized communities of cells within multicellular organisms in health and disease. For these reasons, EVs are attracting the attention of many investigators across different biomedical fields. In this scenario, the possibility to study specific placental-derived EVs in the maternal peripheral blood may open novel perspectives in the development of new early biomarkers for major obstetric pathological conditions. Here we reviewed the involvement of EVs in feto-maternal crosstalk mechanisms, both in physiological and pathological conditions (preeclampsia, fetal growth restriction, preterm labor, gestational diabetes mellitus), also underlining the usefulness of EV characterization in maternal-fetal medicine.

Analytic and Diagnostic Performances of Human Papillomavirus E6/E7 mRNA Test on up-to 11-Year-Old Liquid-Based Cervical Samples. A Biobank-Based Longitudinal Study.

Human Papillomavirus (HPV) E6/E7 mRNA test demonstrated high specificity in detecting HPV infections, but studies assessing its efficacy in terms of cancer risk stratification are lacking. Follow-up studies are arduous and expensive. Biobank would be the answer to the problem, although data investigating the effects of long-term storage on RNA preservation are still needed. We addressed these issues by retrieving 202 residual liquid-based cervical specimens, collected from 149 women attending cervical cancer screening during the years 2001-2012. Samples were stored in Adriatic Biobank at room temperature and without any handing. After calculation of RNA yield and purity, E6/E7 mRNA test was retrospectively performed on each samples, to assess analytic and diagnostic performances. Using automated extraction procedures, RNA of good quantity and quality was obtained. The mean value of RNA concentration was 27.5 ng/µL. The mean A260/A280 ratio was 2.1. An invalid mRNA test result was found in 11.9% of the specimens. Neither RNA integrity, nor analytic performances of mRNA test were influenced by the year of sample collection. In total, 62.4% of the specimens tested as mRNA positive; among these, 89.2% were CIN2+. E6/E7 mRNA was detected in all Squamous Cervical Cancer (SCC) cases. Percentage of positive samples increased with the severity of histological diagnosis. mRNA testing, showing specificity and predictive values of 75.6% and 84.4%, respectively, significantly improved the corresponding values for DNA testing. Thus, the reflex mRNA test was demonstrated to be suitable to triage women with persistent cervical lesions. A "one sample for all" approach is possible, with practical benefits for Biobank-based long-term longitudinal studies, diseases prevention, prediction, diagnosis and treatment.