Risk of COVID-19 maternofetal transmission at term: new evidence against placental infection by SARS-CoV-2

Abstract

s / Placenta 112 (2021) e1ee89 e79 P2.60. COMPARATIVE ANALYSIS OF MIRNA EXPRESSION PROFILES IN SMALL EXTRACELLULAR VESICLES AND THEIR ORIGINATING PRIMARY HUMAN TROPHOBLAST CELLS FROM NORMAL AND GESTATIONAL DIABETES MELLITUS PLACENTAE Soumyalekshmi Nair , Nanthini Jayabalan , Katherin Scholz Romero , Dominic Guanzon , Andrew Lai , Harold David McIntyre , Martha Lappas , Carlos Salomon . 1 Exosome Biology Laboratory, Centre for Clinical Diagnostics, University of Queensland Centre for Clinical Research, Brisbane, Australia; Mater Research InstituteUniversity of Queensland, Brisbane, Australia; 3 Translational Research Institute, Woolloongabba, Australia; Department of Obstetrics and Gynaecology, University of Melbourne, Melbourne, Australia; Mercy Perinatal Research Centre, Mercy Hospital for Women, Heidelberg, Australia; Department of Clinical Biochemistry and Immunology, Faculty of Pharmacy, University of Concepcion, Concepcion, Chile Objectives: The aim of this study was to analyse the miRNA profile of primary human trophoblast (PHT) cells and their secreted small extracellular vesicles (sEVs) obtained from women with normal glucose tolerance (NGT) and gestational diabetes mellitus (GDM). Methods: PHT were isolated from placentae obtained from NGT and GDM. sEVs were isolated from PHT-conditioned media from NGT and GDM by size exclusion chromatography. A small RNA library of PHT and sEVs was constructed using Illumina NextSeq 500 platform. Bioinformatic analysis to identify the potential targets of the miRNAs was performed using the Ingenuity Pathway Analysis (IPA) software and Gene ontology analysis by Cytoscape. Results: After correction for multiple testing using the False Discovery Rate method, a total of 59 miRNAs were significantly different (p<0.05) in PHT from GDM compared to NGT, and 26miRNAs were significantly different in sEVs in GDM compared to NGT. Interestingly, a positive correlation in the fold changes of the miRNAs in PHT compared with sEVs was identified in GDM compared to NGT. However, certain miRNAs were significantly different in sEVs compared to their cells of origin, indicating specific packaging of miRNAs into sEVs in NGT and GDM. We identified a specific set of miRNAs that were highly enriched in sEVs compared to the PHT of origin in NGT (hsa-miR-4700-5p, hsa-miR-150-5p, hsa-miR-1323, hsamiR-7975), in GDM (hsa-miR-1468-5p, hsa-miR-4507, hsa-miR-1285-5p, hsa-miR-6807-5p) and common to NGT and GDM (hsa-miR-486-5p, hsamiR-486-2-5p, hsa-miR-1273g-3p, hsa-miR-7704). On Cytoscape analysis and IPA, the differentially expressed miRNAs were targeting the pathways associated with insulin signalling and glucose metabolism. Conclusion: The content of sEVs released from the placenta may reflect the cell of origin, but differences in GDMmay be indicative of independent functions that sEVs serve in promoting metabolic changes during GDM pregnancies through the modulation of insulin signalling in the target organs. P2.61. PLATELET-DERIVED FACTORS INDUCE PLACENTAL PLASMINOGEN ACTIVATOR INHIBITOR-1 EXPRESSION VIA TGFBR3 D esir ee Forstner , Jacqueline Guettler , Olivia Nonn , Beatrice Brugger , Gerhard Cvirn , Shrey Kohli , Berend Isermann , Berthold Huppertz , Martin Gauster . Division of Cell Biology, Histology and Embryology, Gottfried Schatz Research Center, Medical University of Graz, Graz, Austria; Division of Physiological Chemistry, Otto Loewi Research Center, Medical University of Graz, Graz, Austria; 3 Institute of Laboratory Medicine, Clinical Chemistry and Molecular Diagnostics, University of Leipzig, Leipzig, Germany Objectives: Proand anticoagulant mechanisms in the placenta play an important role in very early stages of gestation to guarantee a successful implantation, but also when it comes to term to avoid haemorrhage. Previous studies revealed that adherence of maternal platelets to the placental villous surface is a common process even in very early stages of gestation. We have recently shown that platelet-derived factors significantly downregulate synthesis of placental human chorionic gonadotropin, while plasminogen activator inhibitor-1 (PAI-1, encoded by SERPINE1) was upregulated. Platelet-derived factors include several factors that regulate growth and differentiation, such as the transforming growth factor (TGF)-b. The TGF-b type III receptor (TGFBR3), also known as betaglycan, is a co-receptor for the TGF-b superfamily and an important regulator of reproduction and fetal development. Here, we investigate the involvement of TGFBR3 in platelet-mediated upregulation of placental PAI-1. Methods: Placental first trimester explants and the human trophoblast cell line BeWowere incubated in presence or absence of human pooled platelet lysate (pHPL). Additionally, BeWo cells were treated with forskolin and different concentrations of the peptide P144, which encompasses amino acids 730e743 from the membrane-proximal ligand-binding domain of TGFBR3. Respectively, total RNA was subjected to quantitative real-time PCR. Furthermore, trophoblast cells were treated with defined concentrations of differentially-induced platelet releasate from healthy and pregnant donors. Results: Gene expression analysis of TGF-b receptor type I, II and III revealed a predominant expression of TGFBR3 in untreated BeWo cells. SERPINE1 expression was substantially upregulated in response to differentially-induced platelet releasate as well as to pHPL, whereas peptide P144 blocked the pHPL-induced effect to control levels in a concentration dependent manner in both undifferentiated and differentiated BeWo cells. The expression of the TGFBR3 per se was not affected by pHPL. Conclusion: In conclusion, our results suggest that platelet-derived factors induce placental PAI-1 expression via TGFBR3. P2.62. RISK OF COVID-19 MATERNOFETAL TRANSMISSION AT TERM: NEW EVIDENCE AGAINST PLACENTAL INFECTION BY SARS-COV-2 Arthur Colson , Christophe Depoix , G eraldine Dessilly , Pamela Baldin , Olivier Danhaive , Corinne Hubinont , Pierre Sonveaux , Fr ed eric Debi eve . Catholic University of Louvain (IREC), Brussels, Belgium; 2 Saint-Luc University Hospital, Brussels, Belgium Objectives: The vertical transmission of SARS-CoV-2 has not been proven yet, but several cases of positive newborns have been reported. Combining clinical samples and cultures of primary human trophoblasts, this project aims to determine if SARS-CoV-2 can infect the fetus by replicating inside the syncytium. Methods: Pregnant women tested positive by RT-PCR at their admission for delivery in the Cliniques Universitaires Saint-Luc (Brussels) were included. Plasmas, swabs, urines, placenta, and maternal milk were collected to detect the virus. Primary trophoblasts were exposed to SARSCov-2 and replication was assessed. The two main proteins mediating the entry of the virus (ACE2 and TMPRSS2) were studied in trophoblasts and normal placentas throughout gestation. Results: Between April 1 and November 1, 20 patients were included (10 asymptomatic, 9 mildly symptomatic, 1 ICU hospitalization). One patient gave birth prematurely and one fetus died in utero. All the babies were healthy and were tested negative for SARS-CoV-2. The virus was detected in the plasma of the hospitalized patient, confirming the rare viremia occurring in severely ill patients. Besides, SARS-CoV-2 was only evidenced in the placenta of the deceased fetus by IHC and ISH. Several mothers had SARS-CoV-2 IgM and/or IgG but their newborns only had IgG, which suggests that none of the fetuses were infected by SARS-CoV-2. In vitro, exposure of undifferentiated and differentiated trophoblasts to increasing MOI did not result in infection and viral replication, contrary to the positive control cells Vero E6. The mechanistic study revealed that the absence of infection is likely due to the very low co-expression of ACE2 and TMPRSS2 by the placenta. Conclusion: Altogether, these results underline that trophoblasts are not likely to be infected by SARS-CoV-2 at term. However, variable expression of ACE2 TMPRSS2 by the placenta throughout gestation raises concern about infection before term, which could lead to dramatic pregnancy complications.