Clinical and in Vitro Evidence against Placenta Infection at Term by Severe Acute Respiratory Syndrome Coronavirus 2
2021; Elsevier BV; Volume: 191; Issue: 9 Linguagem: Inglês
10.1016/j.ajpath.2021.05.009
ISSN1525-2191
AutoresArthur Colson, Christophe Depoix, Géraldine Dessilly, Paméla Baldin, Olivier Danhaive, Corinne Hubinont, Pierre Sonveaux, Frédéric Debiève,
Tópico(s)Reproductive System and Pregnancy
ResumoDespite occasional reports of vertical transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) during pregnancy, the question of placental infection and its consequences for the newborn remain unanswered. Herein, we analyzed the placentas of 31 coronavirus disease 2019–positive mothers by reverse transcriptase PCR, immunohistochemistry, and in situ hybridization. Only one case of placental infection was detected, which was associated with intrauterine demise of the fetus. Differentiated primary trophoblasts were then isolated from nonpathologic human placentas at term, differentiated, and exposed to SARS-CoV-2 virions. Unlike for positive control cells Vero E6, the virus inside cytotrophoblasts and syncytiotrophoblasts or in the supernatant 4 days after infection was undetectable. As a mechanism of defense, we hypothesized that trophoblasts at term do not express angiotensin-converting enzyme 2 and transmembrane protease serine 2 (TMPRSS2), the two main host membrane receptors for SARS-CoV-2 entry. The quantification of these proteins in the placenta during pregnancy confirmed the absence of TMPRSS2 at the surface of the syncytium. Surprisingly, a transiently induced experimental expression of TMPRSS2 did not allow the entry or replication of the virus in differentiated trophoblasts. Altogether, these results underline that trophoblasts are not likely to be infected by SARS-CoV-2 at term, but raise concern about preterm infection. Despite occasional reports of vertical transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) during pregnancy, the question of placental infection and its consequences for the newborn remain unanswered. Herein, we analyzed the placentas of 31 coronavirus disease 2019–positive mothers by reverse transcriptase PCR, immunohistochemistry, and in situ hybridization. Only one case of placental infection was detected, which was associated with intrauterine demise of the fetus. Differentiated primary trophoblasts were then isolated from nonpathologic human placentas at term, differentiated, and exposed to SARS-CoV-2 virions. Unlike for positive control cells Vero E6, the virus inside cytotrophoblasts and syncytiotrophoblasts or in the supernatant 4 days after infection was undetectable. As a mechanism of defense, we hypothesized that trophoblasts at term do not express angiotensin-converting enzyme 2 and transmembrane protease serine 2 (TMPRSS2), the two main host membrane receptors for SARS-CoV-2 entry. The quantification of these proteins in the placenta during pregnancy confirmed the absence of TMPRSS2 at the surface of the syncytium. Surprisingly, a transiently induced experimental expression of TMPRSS2 did not allow the entry or replication of the virus in differentiated trophoblasts. Altogether, these results underline that trophoblasts are not likely to be infected by SARS-CoV-2 at term, but raise concern about preterm infection. Within a year, the coronavirus disease 2019 (COVID-19) pandemic has become a worldwide health and social crisis, deeply affecting human lives and questioning the future of humankind. Although our knowledge about COVID-19 has exponentially increased, many questions remain to be answered. Pregnant women are particularly vulnerable to respiratory infectious diseases because of the remodeling of their immune and cardiovascular systems.1Qiao J. What are the risks of COVID-19 infection in pregnant women?.Lancet. 2020; 395: 760-762Abstract Full Text Full Text PDF PubMed Scopus (326) Google Scholar Initially, it was shown that pregnant women infected by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are more likely to require intensive care treatment, and their pregnancies are associated with increased incidence of miscarriage, preterm birth, preeclampsia, cesarean delivery, and perinatal deaths.2Di Mascio D. Khalil A. Saccone G. Rizzo G. Buca D. Liberati M. Vecchiet J. Nappi L. Scambia G. Berghella V. D'Antonio F. Outcome of coronavirus spectrum infections (SARS, MERS, COVID-19) during pregnancy: a systematic review and meta-analysis.Am J Obstet Gynecol MFM. 2020; 2: 100107Abstract Full Text Full Text PDF PubMed Scopus (677) Google Scholar The meta-analyses are less clear, as maternal COVID-19 was not necessarily associated with adverse pregnancy and neonatal complications.3Karimi L. Makvandi S. Vahedian-Azimi A. Sathyapalan T. Sahebkar A. Effect of COVID-19 on mortality of pregnant and postpartum women: a systematic review and meta-analysis.J Pregnancy. 2021; 2021: 8870129Crossref PubMed Scopus (41) Google Scholar, 4Allotey J. Stallings E. Bonet M. Yap M. Chatterjee S. Kew T. Debenham L. Llavall A.C. Dixit A. Zhou D. Balaji R. Lee S.I. Qiu X. Yuan M. Coomar D. van Wely M. van Leeuwen E. Kostova E. Kunst H. Khalil A. Tiberi S. Brizuela V. Broutet N. Kara E. Kim C.R. Thorson A. Oladapo O.T. Mofenson L. Zamora J. Thangaratinam S. on behalf of the PregCOV-19 Living Systematic Review ConsortiumClinical manifestations, risk factors, and maternal and perinatal outcomes of coronavirus disease 2019 in pregnancy: living systematic review and meta-analysis.BMJ. 2020; 370: m3320Crossref PubMed Scopus (1088) Google Scholar, 5Huntley B.J.F. Mulder I.A. Di Mascio D. Vintzileos W.S. Vintzileos A.M. Berghella V. Chauhan S.P. 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Vertical transmission of coronavirus disease 2019: a systematic review and meta-analysis.Am J Obstet Gynecol. 2021; 224: 35-53.e3Abstract Full Text Full Text PDF PubMed Scopus (351) Google Scholar Direct involvement of the placenta is rare, and robust virological evidence, whether by reverse transcriptase PCR, immunohistochemistry (IHC), or in situ hybridization (ISH), is often missing in the reported cases.8Smithgall M.C. Liu-Jarin X. Hamele-Bena D. Cimic A. Mourad M. Debelenko L. Chen X. Third-trimester placentas of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-positive women: histomorphology, including viral immunohistochemistry and in-situ hybridization.Histopathology. 2020; 77: 994-999Crossref PubMed Scopus (96) Google Scholar, 9Levitan D. London V. McLaren R.A. Mann J.D. Cheng K. Silver M. Balhotra K.S. McCalla S. Loukeris K. Histologic and immunohistochemical evaluation of 65 placentas from women with polymerase chain reaction-proven severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection.Arch Pathol Lab Med. 2021; 145: 648-656Crossref PubMed Scopus (31) Google Scholar, 10Tolu L.B. Ezeh A. Feyissa G.T. Vertical transmission of severe acute respiratory syndrome coronavirus 2: a scoping review.PLoS One. 2021; 16: e0250196Crossref PubMed Scopus (29) Google Scholar However, the debate was revived with the documentation of intrauterine transmission and placental infection.11Facchetti F. Bugatti M. Drera E. Tripodo C. Sartori E. Cancila V. Papaccio M. Castellani R. Casola S. Boniotti M.B. Cavadini P. Lavazza A. SARS-CoV2 vertical transmission with adverse effects on the newborn revealed through integrated immunohistochemical, electron microscopy and molecular analyses of placenta.EBioMedicine. 2020; 59: 102951Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar, 12Raschetti R. Vivanti A.J. Vauloup-Fellous C. Loi B. Benachi A. De Luca D. Synthesis and systematic review of reported neonatal SARS-CoV-2 infections.Nat Commun. 2020; 11: 5164Crossref PubMed Scopus (196) Google Scholar, 13Vivanti A.J. Vauloup-Fellous C. Prevot S. Zupan V. Suffee C. Do Cao J. Benachi A. De Luca D. Transplacental transmission of SARS-CoV-2 infection.Nat Commun. 2020; 11: 3572Crossref PubMed Scopus (9) Google Scholar As SARS-CoV-2 has been detected in the blood of severely ill patients, circulating viral particles are likely to interact with the maternofetal interface.14Bwire G.M. Majigo M.V. Njiro B.J. Mawazo A. Detection profile of SARS-CoV-2 using RT-PCR in different types of clinical specimens: a systematic review and meta-analysis.J Med Virol. 2021; 93: 719-725Crossref PubMed Scopus (138) Google Scholar Especially, the mononuclear villous cytotrophoblasts (CTBs) fuse and differentiate into syncytiotrophoblasts (STBs), which are in direct contact with maternal blood.15Colson A. Sonveaux P. Debieve F. Sferruzzi-Perri A.N. Adaptations of the human placenta to hypoxia: opportunities for interventions in fetal growth restriction.Hum Reprod Update. 2021; 27: 531-569Crossref PubMed Scopus (28) Google Scholar SARS-CoV-2 is a β-coronavirus composed of spike, envelope, membrane, and nucleocapsid proteins encompassing a single-stranded RNA genome of 29,891 nucleotides.16Kadam S.B. Sukhramani G.S. Bishnoi P. Pable A.A. Barvkar V.T. SARS-CoV-2, the pandemic coronavirus: molecular and structural insights.J Basic Microbiol. 2021; 61: 180-202Crossref PubMed Scopus (74) Google Scholar Its infectivity depends primarily on the expression, localization, and structure of the transmembrane proteins angiotensin-converting enzyme 2 (ACE2) and the transmembrane protease serine 2 (TMPRSS2).17Hoffmann M. Kleine-Weber H. Schroeder S. Kruger N. Herrler T. Erichsen S. Schiergens T.S. Herrler G. Wu N.H. Nitsche A. Muller M.A. Drosten C. Pohlmann S. SARS-CoV-2 cell entry depends on ACE2 and TMPRSS2 and is blocked by a clinically proven protease inhibitor.Cell. 2020; 181: 271-280.e8Abstract Full Text Full Text PDF PubMed Scopus (12190) Google Scholar To infect human cells, SARS-CoV-2 engages ACE2 as the entry receptor and employs TMPRSS2 for spike protein priming, allowing the fusion of viral and cellular membranes. ACE2 and TMPRSS2 expression at the surface of cells is therefore considered as a biological indicator of their susceptibility for SARS-CoV-2 infection. ACE2 and TMPRSS2 were detected in the human placenta throughout pregnancy, but their co-expression by syncytiotrophoblasts remains controversial.18Ashary N. Bhide A. Chakraborty P. Colaco S. Mishra A. Chhabria K. Jolly M.K. Modi D. Single-cell RNA-seq identifies cell subsets in human placenta that highly expresses factors driving pathogenesis of SARS-CoV-2.Front Cell Dev Biol. 2020; 8: 783Crossref PubMed Scopus (78) Google Scholar, 19Taglauer E. Benarroch Y. Rop K. Barnett E. Sabharwal V. Yarrington C. Wachman E.M. Consistent localization of SARS-CoV-2 spike glycoprotein and ACE2 over TMPRSS2 predominance in placental villi of 15 COVID-19 positive maternal-fetal dyads.Placenta. 2020; 100: 69-74Crossref PubMed Scopus (82) Google Scholar, 20Pique-Regi R. Romero R. Tarca A.L. Luca F. Xu Y. Alazizi A. Leng Y. Hsu C.D. Gomez-Lopez N. Does the human placenta express the canonical cell entry mediators for SARS-CoV-2?.Elife. 2020; 9: e58716Crossref PubMed Scopus (3) Google Scholar This study aimed to explore the probability of vertical transmission through placental infection by SARS-CoV-2. In a cohort of 31 pregnant women with maternal COVID-19, the viral expression and clinical/immune responses of the mother-infant dyad to SARS-CoV-2 infection were characterized. Then, using a combination of histologic observations and culture of human primary trophoblasts, placental suscptibility to infection was tested and potential mechanisms of resistance to the virus entry were investigated. This study was conducted with the approval of the Ethical Committee of the Catholic University of Louvain (approval number 2020/18AVR/228) in Saint-Luc University Hospital (Brussels, Belgium). Between April 1, 2020, and December 1, 2020, 31 pregnant women who were tested positive for SARS-CoV-2 by reverse transcriptase PCR during their pregnancy were included after informed consent. After delivery, one piece of the placenta was quickly stored in RNAlater (Invitrogen, Thermo Fisher Scientific, Waltham, MA) at −80°C. Placental samples were fixed in formalin and paraffin embedded for histologic analyses. For a subset of patients, additional samples were collected during their hospitalization for delivery: maternal plasma, vaginal swab, rectal swab, maternal urine, maternal milk, plasma from cord blood, nasopharyngeal neonatal swab, and fetal urine. Detection of SARS-CoV-2 RNA was performed using reverse transcriptase PCR, according to the protocol of the Pasteur Institute (Paris, France).21Corman V.M. Landt O. Kaiser M. Molenkamp R. Meijer A. Chu D.K. Bleicker T. Brunink S. Schneider J. Schmidt M.L. Mulders D.G. Haagmans B.L. van der Veer B. van den Brink S. Wijsman L. Goderski G. Romette J.L. Ellis J. Zambon M. Peiris M. Goossens H. Reusken C. Koopmans M.P. Drosten C. Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR.Euro Surveill. 2020; 25: 2000045Crossref PubMed Scopus (4619) Google Scholar Briefly, viral RNA was extracted from liquid samples using QIAamp MinElute Virus Spin Kit (Qiagen, Hilden, Germany) and from tissue samples with PureLink RNA Mini Kit (Invitrogen, Thermo Fisher Scientific). Extracted RNA was eluted in 30 μL of elution buffer. The PCR amplification regions (positions according to SARS-CoV, NC_004718; https://www.ncbi.nlm.nih.gov/nuccore/nc_004718.3, last accessed August 4, 2021) were nCoV_IP2/12621-12727 and nCoV_IP4/14010-14116. A 25-μL reaction containing 5 μL of extracted RNA and 0.4 μmol/L of specific primers [RdRp_nCoV_IP2, 5′-ATGAGCTTAGTCCTGTTG-3′ (forward) and 5′-CTCCCTTTGTTGTGTTGT-3′ (reverse), AGATGTCTTGTGCTGCCGGTA [5']Hex [3']BHQ-1; RdRp_nCoV_IP4, 5′-GGTAACTGGTATGATTTCG-3′ (forward) and 5′-CTGGTCAAGGTTAATATAGG-3′ (reverse), TCATACAAACCACGCCAGG [5']Fam [3']BHQ-1] was performed with Takyon One-Step ROX Probe 5× MasterMix dTTP blue (Eurogentec, Seraing, Belgium) and amplified on a StepOne Real-Time PCR System (Applied Biosystems, Thermo Fisher Scientific). In addition to unknown samples, each assay includes one negative control (water) and five positive controls consisting of in vitro synthesized RNA transcripts (107, 106, 105, 104, and 103 copies genome equivalent). The presence of antibodies in maternal and fetal plasma samples was investigated by using the Elecsys anti–SARS-CoV-2 immunoassay (Roche, Mannheim, Germany), according to manufacturer's protocol. If the test was positive, the Ig class was determined by the MAGLUMI 2019-nCoV IgM/IgG assay (Snibe, Shenzhen, China). Results higher than one arbitrary unit per milliliter were considered to be significant. Three antibodies adapted for IHC were initially tested: two rabbit polyclonal antibodies directed against the spike protein and one mouse monoclonal antibody raised against the nucleocapsid of the virus (Table 1). Positive control slides consisted of tissues that were tested positive by reverse transcriptase PCR: a lung autopsy from a COVID-19–deceased patient and the preterm infected placenta. Negative controls were retrieved from 2017 samples: a normal lung biopsy (for diagnosis), a lung biopsy from acute respiratory distress syndrome (from nonvirological origin), and a term placenta from uncomplicated pregnancy (Supplemental Figures S1–S3).Table 1Antibodies Used for WB Analysis, IHC, and IFTargetSourceReferenceUse and technical notesSARS-CoV-2 spikeSino Biological, Beijing, China40150-V08B1IHC: 1:2000, citrate buffer (pH 5.7)SARS-CoV-2 spikeGenetex, Irvine, CAGTX632604IHC: 1:200, citrate buffer (pH 5.7)SARS-CoV-2 nucleocapsidInvitrogen, Thermo Fisher Scientific, Waltham, MAMA17404IHC: 1:100, TE buffer (pH 9)IF: 1:100, methanolDesmoplakin I + IIAbcam, Cambridge, UKAb16434IF: 1:200, methanolCytokeratin 7Cell Signaling Technology, Danvers, MA4465IF: 1:100, methanolAlexa Fluor 488 anti-mouseCell Signaling Technology4408SIF: secondary antibody, 1:500Alexa Fluor 555 anti-rabbitCell Signaling Technology4413IF: secondary antibody, 1:500Human ACE2Sigma-Aldrich, St. Louis, MOAMAb91268IHC: 1:200, citrate buffer (pH 5.7)Human ACE2Novus Biologicals, Centennial, COAF933WB analysis: 1:400, blocking with milk 5%Human TMPRSS2Sigma-AldrichHPA035787IHC: 1:300, citrate buffer (pH 5.7)IF: 1:200, PFAWB analysis: 1:1250, blocking with milk 5%ACE2, angiotensin-converting enzyme 2; IF, immunofluorescence; IHC, immunohistochemistry; PFA, paraformaldehyde; TMPRSS2, transmembrane protease serine 2; WB, Western blot. Open table in a new tab ACE2, angiotensin-converting enzyme 2; IF, immunofluorescence; IHC, immunohistochemistry; PFA, paraformaldehyde; TMPRSS2, transmembrane protease serine 2; WB, Western blot. COVID-19 immunostaining was confirmed with ISH by using the RNAscope 2.5 HD Assay-RED kit (Advanced Cell Diagnostics, Bio-Techne, Minneapolis, MN). A specific probe directed against nCoV2019-S-sense (catalog number 848561, NC_045512.2, https://www.ncbi.nlm.nih.gov/nuccore/nc_045512.2, last accessed August 4, 2021) was used to detect viral RNA.22Liu J. Babka A.M. Kearney B.J. Radoshitzky S.R. Kuhn J.H. Zeng X. Molecular detection of SARS-CoV-2 in formalin-fixed, paraffin-embedded specimens.JCI Insight. 2020; 5: e139042Crossref PubMed Scopus (63) Google Scholar The positive probe was directed against UBC (catalog number 310041, NM_021009, https://www.ncbi.nlm.nih.gov/nuccore/1519312341, last accessed August 4, 2021), the gene encoding ubiquitin C, to assess sample quality, whereas the negative probe was directed against bacterial gene DapB (catalog number 310043, EF191515, https://www.ncbi.nlm.nih.gov/nuccore/ef191515, last accessed August 4, 2021) (Supplemental Figure S4). As a supplementary precaution, only diethylpyrocarbonate-treated water was used to avoid RNA degradation during the experiment. Placentas were collected from uncomplicated pregnancies at term to isolate and differentiate primary CTBs into STBs, following a previously validated protocol.23Colson A. Depoix C.L. Baldin P. Hubinont C. Sonveaux P. Debieve F. Hypoxia-inducible factor 2 alpha impairs human cytotrophoblast syncytialization: new insights into placental dysfunction and fetal growth restriction.FASEB J. 2020; 34: 15222-15235Crossref PubMed Scopus (14) Google Scholar Pieces of fresh placenta retrieved from uncomplicated pregnancy at term were digested with Dispase II and DNase I, grade II (Roche). The digested tissue was then sequentially filtered, and the cells were separated by density Percoll gradient centrifugation (GE Healthcare Bio-Sciences AB, Uppsala, Sweden). CTBs were cultured in Iscove's modified Dulbecco's medium (Gibco, Thermo Fisher Scientific) complemented with 50 μg/mL of gentamicin (Carl Roth GmbH & Co, Karlsruhe, Germany) and 10% of fetal bovine serum (Gibco, Thermo Fisher Scientific). Cultures were maintained up to 7 days in an atmosphere-controlled humidified incubator under 21% O2 and 5% CO2 at 37°C. The SARS-Cov-2 strains were isolated from patient samples (clade 20E/EU1). At 24 or 96 hours after isolation, the cells were exposed to three concentrations of viral particles (109, 108, and 107 copies/mL). Two hours after, the cells were washed three times with phosphate-buffered saline, and fresh medium was added. A negative control (uninfected cells) was included in each experiment, while Vero E6 cells (CRL-1586; ATCC, Manassas, VA) served as a positive control of the infection. Four days later, the supernatant was collected for rapid antigen assessment with CORIS COVID-19 Ag Respi-Strip (International Medical Products, Brussels, Belgium) and viral RNA detection by reverse transcriptase PCR. The cells were also fixed with methanol to detect the viral nucleocapsid by immunofluorescence. Thirty-six hours after isolation (day 2), primary CTBs were transfected with a vector expressing the human isoform 1 of TMPRSS2 by using the Lipofectamine 3000 Transfection Reagent (Invitrogen, Thermo Fisher Scientific). DNA (1 μg) was used in a 12-well format, according to manufacturer's protocol. The plasmid was a gift from Roger Reeves (plasmid number 53887; Addgene, Watertown, MA).24Edie S. Zaghloul N.A. Leitch C.C. Klinedinst D.K. Lebron J. Thole J.F. McCallion A.S. Katsanis N. Reeves R.H. Survey of human chromosome 21 gene expression effects on early development in Danio rerio.G3 (Bethesda). 2018; 8: 2215-2223Crossref PubMed Scopus (22) Google Scholar Total RNA was extracted from cells using PureLink RNA Mini Kit (Invitrogen, Thermo Fisher Scientific), followed by reverse transcription using qScript cDNA SuperMix (Quanta Biosciences, Gaithersburg, MA). Quantitative real-time PCR was performed with 5 ng of cDNA and 0.2 μmol/L of specific primers (Table 2) in Takyon ROX SYBR 1× MasterMix dTTP blue (Eurogentec) on a StepOne Real-Time PCR System (Applied Biosystems, Thermo Fisher Scientific). The relative expression ratio of a target gene was calculated following the Pfaffl method, using succinate dehydrogenase A (SDHA) and TATA-box binding protein (TBP) as reference genes.25Pfaffl M.W. A new mathematical model for relative quantification in real-time RT-PCR.Nucleic Acids Res. 2001; 29: e45Crossref PubMed Scopus (26152) Google ScholarTable 2Primers Used for Quantitative Real-Time PCRGeneForward primerReverse primerCGB5′-GCTACTGCCCCACCATGACC-3′5′-ATGGACTCGAAGCGCACATC-3′ACE25′-CGAGTGGCTAATTTGAAACCAAGAA-3′5′-ATTGATACGGCTCCGGGACA-3′TMPRSS25′-CCTGTGTGCCAAGACGACTG-3′5′-TTATAGCCCATGTCCCTGCAG-3′SDHA5′-TGGGAACAAGAGGGCATCTG-3′5′-CCACCACTGCATCAAATTCATG-3′TBP5′-GAACATCATGGATCAGAACAACA-3′5′-ATAGGGATTCCGGGAGTCAT-3′ Open table in a new tab Whole-cell lysates were prepared using radioimmunoprecipitation assay buffer (Merck, Burlington, MA) supplemented with Halt Protease Inhibitor Cocktail 1× (Thermo Scientific, Thermo Fisher Scientific). For Western blot analysis, 20 μg of proteins was reduced and proceeded according to a previously published protocol.26Depoix C.L. Colson A. Hubinont C. Debieve F. Impaired vascular endothelial growth factor expression and secretion during in vitro differentiation of human primary term cytotrophoblasts.Angiogenesis. 2020; 23: 221-230Crossref PubMed Scopus (20) Google Scholar Proteins were loaded onto a 10% Acrylamide/Bisacrylamide Bolt Bis-Tris mini gel (Invitrogen, Thermo Fisher Scientific) and transferred onto an Invitrolon polyvinylidene difluoride membrane (Invitrogen, Thermo Fisher Scientific). Then, the membrane was blocked before being incubated overnight at 4°C with the primary antibodies (Table 1). The day after, the membrane was washed and incubated for 1 hour with horseradish peroxidase–conjugated secondary antibodies at room temperature (Table 1). Bound antibodies were detected using the SuperSignal West Pico PLUS chemiluminescent substrate (Thermo Fisher Scientific) and Amersham Hyperfilm ECL films (GE Healthcare Limited, Chicago, IL). The supernatant of the last 24 hours was conserved to assess biochemical differentiation of the trophoblasts. Total secreted free β-subunit human chorionic gonadotrophin was quantified with a B.R.A.H.M.S. Kryptor Compact Plus immune analyzer (Thermo Fisher Scientific) and normalized to whole-cell lysates extracted from the same well. Cells were cultured in CELLview (Greiner Bio-One International GmbH, Kremsmünster, Austria). They were fixed with methanol for the detection of intracellular proteins or Image-iT Fixative Solution (methanol-free 4% formaldehyde; Invitrogen, Thermo Fisher Scientific) for the detection of membrane proteins. Specific primary antibodies were added to the wells and incubated overnight at 4°C (Table 1). The day after, the wells were washed and incubated with the appropriate secondary antibodies. Nuclei were counterstained with NucBlue when mounting slides with ProLong Glass Antifade Mountant (Invitrogen, Thermo Fisher Scientific). Fluorescence was examined on an AxioImager microscope combined with ApoTome (Zeiss, Oberkochen, Germany) or on an LSM 800 confocal microscope (Zeiss). Acquisition parameters and color histograms were kept similar between conditions. Expression of ACE2 and TMPRSS2 during pregnancy was quantified by IHC in retrospectively collected formalin-fixed, paraffin-embedded placentas from 2017. This study was approved by the Ethical Committee of the Catholic University of Louvain (approval number 2018/23OCT/397) and with the consent of the patients. Samples were retrieved from spontaneous abortion (before 22 weeks), uncomplicated preterm pregnancies (before 37 weeks of gestation), and term pregnancies (between 37 and 41 weeks of gestation). According to the institutional procedure, all of the collected cases were analyzed by a pathologist and diagnosed as nonpathologic. Histologic slides from recruited placentas were incubated with primary antibodies overnight at 4°C (Table 1). Immunohistochemical reactions were visualized with the EnVision+ System (Dako, Agilent Technologies, Santa Clara, CA), which is a horseradish peroxidase–labeled polymer conjugated with secondary antibodies. Sites of binding were revealed by treatment with diaminobenzidine (Dako, Agilent Technologies). Kidney (ACE2-positive)27Mizuiri S. Hemmi H. Arita M. Ohashi Y. Tanaka Y. Miyagi M. Sakai K. Ishikawa Y. Shibuya K. Hase H. Aikawa A. Expression of ACE and ACE2 in individuals with diabetic kidney disease and healthy controls.Am J Kidney Dis. 2008; 51: 613-623Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar or prostate adenocarcinoma (TMPRSS2-positive)28Ko C.J. Huang C.C. Lin H.Y. Juan C.P. Lan S.W. Shyu H.Y. Wu S.R. Hsiao P.W. Huang H.P. Shun C.T. Lee M.S. Androgen-induced TMPRSS2 activates matriptase and promotes extracellular matrix degradation, prostate cancer cell invasion, tumor growth, and metastasis.Cancer Res. 2015; 75: 2949-2960Crossref PubMed Scopus (93) Google Scholar tissue sections were used as positive controls, whereas negative controls consisted of slides incubated with secondary antibodies alone. Stained slides were digitalized using an SCN400 slide scanner (Leica Biosystems, Wetzlar, Germany) at ×20 magnification. Scanned slides were analyzed by the image analysis tool author version 2017.2 (Visiopharm, Hoersholm, Denmark), following the workflow described in Supplemental Figure S5. All graphs show means ± SEMs, and the tables show means ± SDs. Statistical analyses were performed on GraphPad Prism 8 (GraphPad Software, La Jolla, CA), using t-test or one-way analysis of variance, followed by the Dunnett post-hoc test. P < 0.05 was considered to be statistically significant. Each in vitro experiment was repeated three times (N = 3 human placentas) in technical duplicates (n = 6). Quantification of ACE2 and TMPRSS2 by IHC was performed on three random fields from 35 placentas (N = 35 placentas) with technical triplicates (n = 105). Thirty-one pregnant women who delivered after 22 weeks and who were tested positive for SARS-CoV-2 during their pregnancy were included in the study. Maternal and fetal characteristics are described in Table 3. Gestational age was estimated from the first day of the last menstrual period and confirmed by first-trimester ultrasound examination. Twenty-seven patients tested positive during their third trimester, mainly the week before childbirth. Of these patients, 18 were asymptomatic, five had mild symptoms (fatigue, anosmia, or ageusia), and four had moderate symptoms, including fever and dyspnea. In most cases, the pregnancy ended without complications, and the mothers gave birth to healthy neonates. Four patients tested positive before 30 weeks of gestation. The first one had fever associated to dyspnea. She was diagnosed at 26 weeks 3 days of gestation and gave birth prematurely by cesarean section because of severe preeclampsia. The baby was growth-restricted and tested positive for SARS-CoV-2 by reverse transcriptase PCR, but 7 days after birth.29Piersigilli F. Carkeek K. Hocq C. van Grambezen B. Hubinont C. Chatzis O. Van der Linden D. Danhaive O. COVID-19 in a 26-week preterm neonate.Lancet Child Adolesc Health. 2020; 4: 476-478Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar The second patient was tested at 28 weeks 6 days of gestation. She was hospitalized in the intensive care unit for 14 days for severe respiratory distress, but was able to give birth at term without complications. The third patient tested positive at 23 weeks 3 days of gestation because of dyspnea. She delivered a viable polymalformed newborn (esophageal atresia, anal imperforation, Fallot tetralogy, and multicystic kidney) at 33 weeks 1 day. The CHARGE syndrome (coloboma, heart defects, choanal atresia, retardation of growth and/or development, genitourinary malformation, and ear abnormalities and/or deafness) was excluded by genomic analysis, but the malformations remained compatible with a VACTERL association (vertebral defects, anal atresia, cardiac defects, tracheo-esophageal fistula, renal anomalies, and limb abnormalities). The last patient was referred to obstetrical emergency at 25 weeks 2 days for a fever peak at 39°C, decreased fetal movements, and ultrasonographic signs of fetal distress. A diagnosis of intrauterine fetal demise was made on admission. A positive nasopharyngeal swab of the mother confirmed the medical history, which was compatible with a COVID-19 infection contracted a few days ago.Table 3Maternal and Fetal CharacteristicsCharacteristicValueMother Age, years31 (4.28) BMI, kg/m225.18 (5.53)>30 kg/m2, %12.90 Ethnic group, %White48.39Black22.58Asian0Other2
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