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Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

For several years, a growing number of studies have highlighted the pivotal role of placental extracellular vesicles (EVs) throughout pregnancy. These membrane nanovesicles, heterogeneous in nature, composition and origin, are secreted by several trophoblastic cell types and are found in both the maternal and fetal compartments. They can be uptaken by recipient cells and drive a wide variety of physiological and pathological processes. In this review, we provide an overview of the different described roles of placental EVs in various aspects of normal pregnancy, from placenta establishment to maternal immune tolerance towards the fetus and protection against viral infections. In the second part, we present selected examples of pathological pregnancies in which placental EVs are involved, such as gestational diabetes mellitus, pre-eclampsia, and congenital infections. Since the abundance and/or composition of placental EVs is deregulated in maternal serum during pathological pregnancies, this makes them interesting candidates as non-invasive biomarkers for gestational diseases and opens a wide field of translational perspectives., (© 2022 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Although placental small extracellular vesicles (sEVs) are extensively studied in the context of pregnancy, little is known about their role during viral congenital infection, especially at the beginning of pregnancy. In this study, we examined the consequences of human cytomegalovirus (hCMV) infection on sEVs production, composition, and function using an immortalized human cytotrophoblast cell line derived from first trimester placenta. By combining complementary approaches of biochemistry, electron microscopy, and quantitative proteomic analysis, we showed that hCMV infection increases the yield of sEVs produced by cytotrophoblasts and modifies their protein content towards a potential proviral phenotype. We further demonstrate that sEVs secreted by hCMV-infected cytotrophoblasts potentiate infection in naive recipient cells of fetal origin, including human neural stem cells. Importantly, these functional consequences are also observed with sEVs prepared from an ex vivo model of infected histocultures from early placenta. Based on these findings, we propose that placental sEVs could be important actors favoring viral dissemination to the fetal brain during hCMV congenital infection.

Usutu virus (USUV) is a neurotropic mosquito-borne flavivirus that has dispersed quickly in Europe these past years. This arbovirus mainly follows an enzootic cycle involving mosquitoes and birds, but can also infect other mammals, causing notably sporadic cases in humans. Although it is mainly asymptomatic or responsible for mild clinical symptoms, USUV has been associated with neurological disorders, such as encephalitis and meningoencephalitis, highlighting the potential health threat of this virus. Among the different transmission routes described for other flaviviruses, the capacity for some of them to be transmitted vertically has been demonstrated, notably for Zika virus or West Nile virus, which are closely related to USUV. To evaluate the ability of USUV to replicate in the placenta and gain access to the fetus, we combined the use of several trophoblast model cell lines, ex vivo human placental explant cultures from first and third trimester of pregnancy, and in vivo USUV-infected pregnant mice. Our data demonstrate that human placental cells and tissues are permissive to USUV replication, and suggest that viral transmission can occur in mice during gestation. Hence, our observations suggest that USUV could be efficiently transmitted by the vertical route.

Extracellular vesicles (EVs) have increasingly been recognized as key players in a wide variety of physiological and pathological contexts, including during pregnancy. Notably, EVs appear both as possible biomarkers and as mediators involved in the communication of the placenta with the maternal and fetal sides. A better understanding of the physiological and pathological roles of EVs strongly depends on the development of adequate and reliable study models, specifically at the beginning of pregnancy where many adverse pregnancy outcomes have their origin. In this study, we describe the isolation of small EVs from a histoculture model of first trimester placental explants in normal conditions as well as upon infection by human cytomegalovirus. Using bead-based multiplex cytometry and electron microscopy combined with biochemical approaches, we characterized these small EVs and defined their associated markers and ultrastructure. We observed that infection led to changes in the expression level of several surface markers, without affecting the secretion and integrity of small EVs. Our findings lay the foundation for studying the functional role of EVs during early pregnancy, along with the identification of new predictive biomarkers for the severity and outcome of this congenital infection, which are still sorely lacking., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Bergamelli, Martin, Bénard, Ausseil, Mansuy, Hurbain, Mouysset, Groussolles, Cartron, Tanguy le Gac, Moinard, Suberbielle, Izopet, Tscherning, Raposo, Gonzalez-Dunia, D’Angelo and Malnou.)

Congenital infection of the central nervous system by human cytomegalovirus (HCMV) is a leading cause of permanent sequelae, including mental retardation or neurodevelopmental abnormalities. The most severe complications include smooth brain or polymicrogyria, which are both indicative of abnormal migration of neural cells, although the underlying mechanisms remain to be determined. To gain better insight on the pathogenesis of such sequelae, we assessed the expression levels of a set of neurogenesis-related genes, using HCMV-infected human neural stem cells derived from embryonic stem cells (NSCs). Among the 84 genes tested, we found dramatically increased expression of the gene PAFAH1B1, encoding LIS1 (lissencephaly-1), in HCMV-infected versus uninfected NSCs. Consistent with these findings, western blotting and immunofluorescence analyses confirmed the increased levels of LIS1 in HCMV-infected NSCs at the protein level. We next assessed the migratory abilities of HCMV-infected NSCs and observed that infection strongly impaired the migration of NSCs, without detectable effect on their proliferation. Moreover, we observed increased immunostaining for LIS1 in brains of congenitally infected fetuses, but not in control samples, highlighting the clinical relevance of our findings. Of note, PAFAH1B1 mutations (resulting in either haploinsufficiency or gain of function) are primary causes of hereditary neurodevelopmental diseases. Notably, mutations resulting in PAFAH1B1 haploinsufficiency cause classic lissencephaly. Taken together, our findings suggest that PAFAH1B1 is a critical target of HCMV infection. They also shine a new light on the pathophysiological basis of the neurological outcomes of congenital HCMV infection, by suggesting that defective neural cell migration might contribute to the pathogenesis of the neurodevelopmental sequelae of infection. © 2021 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd., (© 2021 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.)