extracellular vesicles (ev) size in shallow … · measured using image-pro premier 9.3 program....

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EXTRACELLULAR VESICLES (EV) SIZE IN SHALLOW TROPHOBLAST INVASION IN OLD WORLD NON-HUMAN PRIMATES

Objective

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 Women's 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

We evaluated plasma from three non-pregnant,four pregnant, and one fetal baboon at 130-165days of gestation. The total numbers of EV werequantified by nanoparticle tracking analysis(NTA). The different population of vesicles wasdetermined based in their size and classified as<50, 50-150, 150-200 and >200nm. Additionally,we performed the analyses of microphotographs,obtained from baboon placenta at the end ofgestation, which were originally analyzed inPlacenta 2009 Sep;30(9):752-60. Inapproximately 80 microphotographs theintervillous space and fetal capillaries wereidentified and examined for the presence of theextracellular vesicles, which in turn weremeasured using Image-Pro Premier 9.3program. The largest diameters are reported.

Results and Discussion

Acknowledgements

Extracellular vesicles are playing a critical rolein pathophysiological responses. Pregnancy-related changes in extracellular vesicles (EV)compositions, and the presence of trophoblastdebris in maternal tissues, are well described inhuman normal and pathological pregnancies.Extracellular vesicles, including exosomes areplaying a critical role in cell-to-cell and organcommunication However, the concentration ofcirculating vesicles in baboons, which is amodel to study pregnancy-related physiology,has not been established yet.

The total concentration of EVs was ~8-fold higher in the peripheral circulation in maternal compared with fetalplasma. EVs <50 nm were not present in the peripheral circulation of the non-pregnant animals.Concentrations of EV of 50-150nm and >200 nm were approximately two times higher in pregnant comparedto the non-pregnant animals, with no difference in the concentration 150-200mn.

We previously suggested the absence of thetrophoblast debris in the maternal circulation inbaboon (Papio spp.) (SRI, March 2012, SanDiego, CA, USA), based on the evaluation of theavailable lung samples from the pregnantanimals. It was proposed that shallow trophoblastinvasion in Papio spp. (as compared to humanplacentation) does not result the trophoblastshedding. We further analyzed placental samplesfrom the stillbirths in the baboons and in humansand found, that syncytiotrophoblast thickness isdecreased in the placenta from the stillbornbaboons, similar to the finding in humans.

Figure 3. Exosome profiling across thepregnancy. Number of exosomes acrossthe pregnancy. Data are presented asaligned dot plot and values are mean ±SEM.

Extracted from: C. Salomon, et al. Agestational profile of placental exosomes inmaternal plasma and their effects onendothelial cell migration PLoS One, 9(2014), p. e98667.

This investigation was supported by Southwest National PrimateResearch Center grant P51 RR013986 from the National Centerfor Research Resources and the National Institutes of Health,which are currently supported by the Office of ResearchInfrastructure Programs through P51 OD011133.

Figure 1. Serum extracellular vesicles distribution in baboons (Papio spp.) : non-pregnant female (n=3), pregnant females (n=4) and fetus at term.

C

Figure 2. Extracellular vesicles size distribution in fetal placental capillaries (A), intervillous space (B), and maternal placental capillaries (C), determined by evaluation of electron microscopy images (n=80).

The comparison of the numbers of exosomes in theperipheral circulation revealed some differences,despite similar in both species increase in pregnantcompared to the non-pregnant femalesconcentrations of EV. However, the number of theexosomes in baboons per ml of serum was 1000times lower, compared to humans. While thedifferences of concentrations of exosomes betweenpregnant and non-pregnant women were in themagnitude of 50 times while in the baboons in ourstudy was 2. Interestingly, the number of exosomesdramatically increased in pre-eclampsia, thepathology, which has not been documented in Papiospp. The mechanisms, leading to placentalshedding in Papio spp. remain to be elucidated.

Fetal Circulation Maternal Circulation

Discussion

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This investigation was conducted in facilitiesconstructed with support from the Office ofResearch Infrastructure Programs (ORIP) ofthe National Institutes of Health through grantnumbers C06 RR015456 and C06RR014578.This work was supported bystart-up funds to NSL.

B

Presenting Author –Marcel Chuecos

Figure 4. Comparablehistology of human andbaboon placentas.

Extracted from: TheSyncytiotrophoblast (ST)Volume Is Critical for Human,but Not Non- Human Primates(NHP) Stillbirths (SB) at Term.SRI meeting, 2012).

natalia.schlabritz-lutsevich@ttuhsc.edu