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Biological insight into the extracellular vesicles in women with and without gestational diabetes

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Abstract

Purpose

Gestational diabetes mellitus (GDM) is the most common metabolic disorder in pregnancy, with increasing prevalence worldwide and still unclear pathogenic mechanisms. Extracellular vesicles (EVs) are emerging as potential biomarkers of disease-specific pathways in metabolic disorders, but their potential role in GDM is not fully understood. Therefore, the main aim of this study was to evaluate the link between EVs and hyperglycaemia during pregnancy.

Methods

We assessed 50 GDM women and 50 controls at the third trimester of pregnancy in whom we collected demographic characteristics and clinical and anthropometric parameters. In addition, the circulating total EVs (tEVs) and their subpopulations were assessed using flow cytometry.

Results

The levels of tEVs and EVs subtypes, expressed as median and interquartile range, were not significantly different between two groups; however, adipocyte-derived EVs (aEVs) concentration, expressed as percentage, was higher in controls than in GDM women (p = 0.045). In addition, a significant correlation was observed between aEVs (%) and third trimester total cholesterol (p = 0.022) within the GDM group. Furthermore, a significant correlation between endothelial-derived EVs (eEVs) and platelet-derived EVs (pEVs) within both groups was found, as well as a significant relation between aEVs and pEVs.

Conclusions

These data, although preliminary, represent the starting point for further studies to determine the role of circulating EVs in GDM.

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Data availability

The datasets generated and analysed during the current study are available from the corresponding author on reasonable request.

Abbreviations

GDM:

Gestational diabetes mellitus

EVs:

Extracellular vesicles

T2DM:

Type 2 diabetes mellitus

CVD:

Cardiovascular diseases

MetS:

Metabolic syndrome

eEVs:

Extracellular vesicles derived from endothelium

tEVs:

Total extracellular vesicles

TC:

Total cholesterol

HDL-C:

High-density lipoprotein cholesterol

LDL-C:

Low-density lipoprotein cholesterol

TG:

Triglycerides

lEVs:

Extracellular vesicles derived from leukocytes

pEVs:

Extracellular vesicles derived from platelets

aEVs:

Extracellular vesicles derived from adipocytes

PdEs:

Placenta-derived exosomes

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Author notes

  1. P. Lanuti, F. Fraticelli and M. Marchioni have contributed equally to this work.

Authors and Affiliations

  1. Department of Medicine and Aging, School of Medicine and Health Sciences, “G. D’Annunzio” University, Chieti-Pescara, Via dei Vestini, 66100, Chieti, Italy

    M. Franzago, P. Lanuti, F. Fraticelli, M. Liberati, S. Miscia & E. Vitacolonna

  2. Center for Advanced Studies and Technology (CAST), “G. D’Annunzio” University, Chieti-Pescara, Chieti, Italy

    M. Franzago, P. Lanuti, F. Fraticelli, S. Miscia, L. Stuppia & E. Vitacolonna

  3. Laboratory of Biostatistics, Department of Medical, Oral and Biotechnological Sciences, “G. D’Annunzio” University, Chieti-Pescara, Chieti, Italy

    M. Marchioni & M. Di Nicola

  4. Department of Obstetrics and Gynaecology, SS. Annunziata Hospital, “G. D’Annunzio” University, Chieti-Pescara, Chieti, Italy

    D. Buca

  5. Department of Psychological, Health and Territorial Sciences, School of Medicine and Health Sciences, “G. D’Annunzio” University, Chieti-Pescara, Chieti, Italy

    L. Stuppia

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  1. M. Franzago
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  10. E. Vitacolonna
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Contributions

The study was designed by EV and MF. ML and EV contributed to clinical evaluation and support to the recruitment of patients. MF, FF, and EV contributed to data acquisition and interpretation. PL conducted the experiments. MDN and MM were the biostatisticians that performed and supervised the statistical analysis and they also helped the preparation of the figures and tables. The manuscript was drafted by MF and EV. LS contributed to the reviewed of the manuscript. All authors were involved in critical revision and approved the final version of the manuscript before submission. EV and MDN are the guarantors of this work.

Corresponding author

Correspondence to E. Vitacolonna.

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The authors declare no competing interests.

Ethical approval

The procedures used were in accordance with the guidelines of the Helsinki Declaration on human experimentation. The investigational nature of the study was explained to all participants.

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Informed consent was obtained, according to the Ethics Committee of the University “G. d’Annunzio", Chieti-Pescara.

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Supplementary file1 (DOC 35 kb)

40618_2020_1262_MOESM2_ESM.jpg

Supplementary file2 (JPG 187 kb) Work Flow of the flow cytometry protocol for EV analysis. Peripheral blood samples were obtained from a cohort of 50 GDM pregnant women and 50 healthy volunteers. 5 µl of whole blood were added to 195 µl of PBS 1X, then the reagents detailed in Supplementary Table S1 were mixed to the diluted blood. After 45 min of staining (RT, in the dark), 500 µl of PBS 1X were added to each tube and 1 x 106 events/sample were acquired by flow cytometry (FACSVerse, BD). By applying the gating strategy described in Supplementary Figure 2, absolute numbers and percentages of total EVs (tEVs), leukocyte-derived EVs (lEVs), platelet-derived EVs (pEVs), endothelium-derived EVs (aEVs) and adipocyte-derived EVs (aEVs) were obtained

40618_2020_1262_MOESM3_ESM.jpg

Supplementary file3 (JPG 170 kb) Gating strategy for EV identification and subtyping. A. The area with scattered parameters lower than that of platelets, on a FSC-H/SScSSC-H dotcontour-plot was established. B. This area was shown on a Phalloidin-H/LCD-H dot plot and EVs were identified as LCD positive/phalloidin negative events. C. Extracellular vesicles (LCD+/Phalloidin- events) were analysed on a CD45-H/CD41a-H dot-plot and CD45+ events were identified as leukocyte-derived EVs (lEVs). D. A logical gate excluding all the CD45+ events was then obtained and the resulting population was plotted on a CD31-H/CD41a-H dot-plot. Events showing the CD31+/CD41a+ phenotype were identified as platelet-derived EVs (pEVs), while the CD31+/CD41a- compartment represented endothelium-derived EVs (eEVs). E Adipocyte-derived were finally obtained on a CD45-H/CD36-H dot-plot

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Franzago, M., Lanuti, P., Fraticelli, F. et al. Biological insight into the extracellular vesicles in women with and without gestational diabetes. J Endocrinol Invest 44, 49–61 (2021). https://doi.org/10.1007/s40618-020-01262-0

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