Towards Deep Cellular Phenotyping in Placental Histology

The placenta is a complex organ, playing multiple roles during fetal development. Very little is known about the association between placental morphological abnormalities and fetal physiology. In this work, we present an open sourced, computationally tractable deep learning pipeline to analyse placenta histology at the level of the cell. By utilising two deep Convolutional Neural Network architectures and transfer learning, we can robustly localise and classify placental cells within five classes with an accuracy of 89%. Furthermore, we learn deep embeddings encoding phenotypic knowledge that is capable of both stratifying five distinct cell populations and learn intraclass phenotypic variance. We envisage that the automation of this pipeline to population scale studies of placenta histology has the potential to improve our understanding of basic cellular placental biology and its variations, particularly its role in predicting adverse birth outcomes.Comment: Updated MRC funding material. Corrected typo that suggested ensembling and Inception accuracy were the same (updated to reflect the fact the ensemble model is 1% better than previously reported

Placental extracellular vesicles express active dipeptidyl peptidase IV; levels are increased in gestational diabetes mellitus

Gestational diabetes mellitus (GDM) is the most common metabolic disorder in pregnancy and is characterized by insulin resistance and decreased circulating glucagon-like peptide-1 (GLP-1). GDM resolves rapidly after delivery implicating the placenta in the disease. This study examines the biological functions that cause this pathology. The placenta releases syncytiotrophoblast-derived extracellular vesicles (STB-EVs) into the maternal circulation, which is enhanced in GDM. Dipeptidyl peptidase IV (DPPIV) is known to play a role in type 2 diabetes by breaking down GLP-1, which in turn regulates glucose-dependent insulin secretion. STB-EVs from control and GDM women were analysed. We show that normal human placenta releases DPPIV-positive STB-EVs and that they are higher in uterine than paired peripheral blood, confirming placental origin. DPPIV-bound STB-EVs from normal perfused placentae are dose dependently inhibited with vildagliptin. DPPIV-bound STB-EVs from perfused placentae are able to breakdown GLP-1 . STB-EVs from GDM perfused placentae show greater DPPIV activity. Importantly, DPPIV-bound STB-EVs increase eightfold in the circulation of women with GDM. This is the first report of STB-EVs carrying a biologically active molecule that has the potential to regulate maternal insulin secretion

Studies to understand the pathological mechanisms in preeclampsia via extracellular vesicles

This thesis was submitted for the award of Doctor of Philosophy and was awarded by Brunel University LondonPreeclampsia (PE) is a pregnancy-specific disorder, affecting 3-5% of pregnancies worldwide. There is no current cure for PE apart from the removal of the placenta and the fetus, irrespective of gestation. Thus, PE does not only severely affect the mother but also causes a significant impact on the fetus development. The exact mechanisms by which PE occurs remains unclear. Extracellular vesicles (EV) are membrane-encapsulated particles carrying cargo originating from their parental cells; they have been shown to have an essential role in mediating intercellular communication. Here, syncytiotrophoblast derived EV (STBEV) were isolated from PE and normal pregnancies (NP) by dual-lobe ex vivo placental perfusion and differential ultracentrifugation. We show that STBEV carry functional endothelial nitric oxide synthase (eNOS), and its expression and activity were reduced in STBEV isolated from PE placentae. eNOS is an enzyme essential for the synthesis of nitric oxide (NO), which is a potent vasodilator. The reduced NO generated by STBEV-bound eNOS in PE may contribute to an overall decreased NO bioavailability associated with the endothelial dysfunction previously reported in PE. PE has been described as early- (≤34 weeks gestation) and late- (≥34 weeks gestation) onset. We also demonstrated that late-onset PE-derived STBEV do not exacerbate a pro-inflammatory profile on a macrophage/monocytic cell line, THP-1. Instead, a suppressed inflammatory response is being reported, which is distinct from maternal systemic inflammation associated with early-onset PE. Thus, early- and late-onset PE are different pathophysiological entities. We also report, for the first time, the characterisation, protein cargo and possible functions of placental capillary pericytes (PLVP) derived exosomes (PLVPex) cultured under normal (20% O2) and hypoxic (8% and 1% O2) conditions. We show that PLVPex can induce a pro-angiogenic effect on primary human umbilical vein endothelial cells (HUVEC), irrespective of oxygen stimulus, carrying bound molecules needed for angiogenesis. Hypoxia does not alter the function of PLVPex on HUVEC, but stimulates the increased production of PLVPex and growth factors, indicating a potential survival mechanism by PLVP in response to oxygen deprivation, which is common in PE placentae

Placental syncytiotrophoblast-derived extracellular vesicles carry active NEP (neprilysin) and are increased in preeclampsia

NEP (neprilysin) is a widely expressed membrane-bound metalloprotease, which binds and cleaves a variety of peptides including vasodilators, natriuretics, and diuretics. Higher levels of NEP result in hypertension-a cardinal feature of the placental disease preeclampsia. Syncytiotrophoblast-derived extracellular vesicles (EVs), comprising microvesicles and exosomes, are released into the peripheral circulation in pregnancy and are postulated as a key mechanism coupling placental dysfunction and maternal phenotype in preeclampsia. We aimed to determine whether higher levels of active NEP are found in syncytiotrophoblast-derived EVs in preeclampsia compared with normal pregnancy. Using immunostaining and Western blotting, we first demonstrated that NEP levels are greater not only in preeclampsia placental tissue but also in syncytiotrophoblast-derived microvesicles and exosomes isolated from preeclampsia placentas ( P<0.05, n=5). We confirmed placental origin using antibody-coated magnetic beads to isolate NEP-bound vesicles, finding that they stain for placental alkaline phosphatase. NEP on syncytiotrophoblast-derived EVs is active and inhibited by thiorphan ( P<0.01, n=3; specific inhibitor). Syncytiotrophoblast-derived microvesicles, isolated from peripheral plasma, demonstrated higher NEP expression in preeclampsia using flow cytometry ( P<0.05, n=8). We isolated plasma exosomes using size-exclusion chromatography and showed greater NEP activity in preeclampsia ( P<0.05, n=8). These findings show that the placenta releases active NEP into the maternal circulation on syncytiotrophoblast-derived EVs, at significantly greater levels in preeclampsia. NEP has pathological roles in hypertension, heart failure, and amyloid deposition, all of which are features of preeclampsia. Circulating syncytiotrophoblast-derived EV-bound NEP thus may contribute to the pathogenesis of this disease

Proceedings of the 4th World Conference on Research Integrity

CITATION: O’Brien, S. P., et al. 2016. Proceedings of the 4th World Conference on Research Integrity. Research Integrity and Peer Review, 1:9, doi:10.1186/s41073-016-0012-9.The original publication is available at https://researchintegrityjournal.biomedcentral.comThese Proceedings contain the abstracts of the presentations given at the 4th World Conference in concurrent sessions, partner symposia, and poster sessions. Also included are summaries of the discussions in three focus tracks, which allowed delegates to consider and work on questions about the roles of funders, institutions, and countries in improving research systems and strengthening research integrity. Videos of the plenary presentations are available at the conference website (www.wcri2015.org).https://researchintegrityjournal.biomedcentral.com/articles/10.1186/s41073-016-0012-