key: cord-343712-gn7fw891 authors: Taglauer, Elizabeth; Benarroch, Yoel; Rop, Kevin; Barnett, Elizabeth; Sabharwal, Vishakha; Yarrington, Christina; Wachman, Elisha M. title: Consistent localization of SARS-CoV-2 spike glycoprotein and ACE2 over TMPRSS2 predominance in placental villi of 15 COVID-19 positive maternal-fetal dyads date: 2020-08-25 journal: Placenta DOI: 10.1016/j.placenta.2020.08.015 sha: doc_id: 343712 cord_uid: gn7fw891 INTRODUCTION: While the COVID-19 pandemic continues to have a significant global health impact, rates of maternal to infant vertical transmission remain low (<5%). Parenchymal changes of placentas from COVID-19 infected mothers have been reported by several groups, but the localization and relative abundance of SARS-CoV-2 viral proteins and cellular entry machinery has not been fully characterized within larger placental tissue cohorts. METHODS: An extended placental tissue cohort including samples from 15 COVID-19 positive maternal-fetal dyads (with n = 5 cases with evidence of fetal transmission) in comparison with 10 contemporary COVID-19 negative controls. Using comparative immunofluorescence, we examined the localization and relative tissue abundance of SARS-CoV2 spike glycoprotein (CoV2 SP) along with the co-localization of two SARS-CoV2 viral entry proteins angiotensin-converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2). RESULTS/CONCLUSIONS: CoV2 SP was present within the villous placenta in COVID-19 positive pregnancies with and without evidence of fetal transmission. We further identified the predominance of ACE2 expression in comparison with TMPRSS2. Importantly, both CoV2 SP and ACE2 expression consistently localized primarily within the outer syncytiotrophoblast layer placental villi, a key physiologic interface between mother and fetus. Overall this study provides an important basis for the ongoing evaluation of SARS-CoV-2 physiology in pregnancy and highlights the importance of the placenta as a key source of primary human tissue for ongoing diagnostic and therapeutic research efforts to reduce the global burden of COVID-19. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the novel infectious 2 agent responsible for Coronavirus disease 2019 (COVID-19), has caused over ten million 3 confirmed cases and has accounted for over 500,000 deaths worldwide in the six months since 4 the first reported case in December 2019 [1] . Despite its severity in some populations 5 (particularly older adults), studies to date have shown that COVID-19 does not cause increased 6 morbidity or mortality in pregnant women compared to non-pregnant women [2] [3] [4] . There is a 7 low rate of positive SARS-CoV-2 tests in infants born to mothers with COVID-19, with cohort 8 studies reporting a range of 0-4.5% [5, 6] . These rates have remained low even as more 9 obstetrical and neonatal settings are implementing universal testing. Clinical manifestations in 10 neonates related to SARS-CoV-2 exposure are still being fully characterized, but overall the 11 majority of the literature has reported healthy neonates born to mothers with COVID-19 [2, 3, 7- highlight that the placenta undergoes significant parenchymal changes secondary to maternal 23 COVID-19 infection, yet SARS-CoV-2 transmission to the fetus is prevented in the majority of 24 pregnancies, suggesting organ-specific antiviral mechanisms at the maternal-fetal interface. 25 J o u r n a l P r e -p r o o f As the primary anatomical and physiological interface between the mother and fetus 26 during pregnancy, the placenta and its accompanying immune cell repertoire dictate blockade 27 versus transmission of viral pathogens through highly interrelated pathways [22] . A unifying 28 theme among most studies examining viral infections in pregnancy is the importance of the 29 villous placenta and in particular the villous outer syncytiotrophoblast layer (sTB). This syncytial 30 cell layer fed by underlying cytotrophoblast (cTB) cells remains in direct contact with the 31 maternal blood space throughout pregnancy. sTB serve a variety of functions including bi-32 directional maternal-fetal trafficking and importantly, are known to express multiple key viral 33 receptors for pathogens with varying rates of vertical transmission, such as HIV and Hepatitis C 34 CoV-2 by PCR of nasopharyngeal swabs was instituted at our hospital in mid-April 2020 for all 59 women admitted in labor. We collected placental samples from women who tested positive at 60 the time of delivery, and contemporary controls who tested negative. Selected patient 61 demographic data and institutional pathology reports were collected for all cases and controls. 62 Approval was obtained from the Boston University Medical Campus Institutional Review Board, 63 with an informed consent waiver obtained for this study. There were no significant demographic differences between cases with positive maternal 129 SARS-CoV-2 testing (COVID-19 Maternal ) and control mother-infant dyads (Table 1) . Placental 130 pathology diagnoses, listed in Table 2 , were notable for the presence of fibrin deposition and 131 signs of inflammation in COVID-19 Maternal placentas, however this did not meet statistical 132 significance. These diagnoses corresponded with gross pathology assessment of tissue 133 biopsies obtained for the study, with notable intervillous and subchorionic fibrosis in the majority 134 of COVID-19 Maternal samples (Fig. 1) . 135 We next surveyed tissues for the presence and localization of SARS-Co-V-2 spike 136 glycoprotein (CoV2 SP). This viral protein was present in the villous compartment of all COVID-137 19 Maternal placentas and absent from control tissues, as visualized by non-fluorescent and 138 fluorescent immunohistochemical techniques (Fig. 2 A,B) . No expression was observed within 139 chorionic plate or decidual tissues (data not shown). Within placental villi, CoV2 SP was 140 consistently expressed within the sTB layer with intermittent localization to cTB among certain 141 villi (Fig. 2B) . Using quantitative microscopy, we then surveyed the presence of CoV2 SP 142 among all tissues. COVID-19 Maternal cases with evidence of fetal transmission did not have 143 significant alterations of CoV2 SP in comparison to pregnancies with negative infant COVID-19 144 testing (Fig. 2C) . 145 Comparative expression and localization of ACE2 and TMPRSS2 receptors was then 146 evaluated using double immunofluorescence to simultaneously evaluate both proteins within the 147 same tissue section. While ACE2 was consistently found in the sTB layer of all tissues 148 surveyed (COVID-19 Maternal and controls), TMPRSS2 expression was absent in both groups of 149 placentas (Fig. 3 A,B) . 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