SARS-CoV-2 Delta and Omicron Variants alter Trophoblast Cell Fusion and Syncytiotrophoblast Dynamics: New Insights into Placental Vulnerability

SARS-CoV-2 Delta and Omicron Variants alter Trophoblast Cell Fusion and Syncytiotrophoblast Dynamics: New Insights into Placental Vulnerability | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article SARS-CoV-2 Delta and Omicron Variants alter Trophoblast Cell Fusion and Syncytiotrophoblast Dynamics: New Insights into Placental Vulnerability Marie-Benoite Cohen, Manel Essaidi-Laziosi, catia alvarez, Michal Yaron, and 10 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6246871/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 07 Oct, 2025 Read the published version in Cell Death & Disease → Version 1 posted 7 You are reading this latest preprint version Abstract Pregnancy constitutes an at-risk factor for severe COVID-19. Several clinical studies have previously suggested increased risks of adverse obstetrical outcomes and placental pathological changes in pregnant women infected with SARS-CoV-2. In this study, our goal was to assess the susceptibility of trophoblast to SARS-CoV-2 infection at early stage of pregnancy and its impact on trophoblast cell fusion in vitro . We showed that first trimester cytotrophoblast (CTB) and syncytiotrophoblast (STB) are permissive to SARS-CoV-2 in variant- and donor-dependent manner. Delta variant showed a higher efficiency of replication in STB and CTB compared to Omicron BA.1, BA.2 and BA.5. In STB, despite a slight subsequent increase of type III IFN response, no correlation was observed between virus replication and the induction of the overall host response (including expression of entry receptors and immune response) after infection. In CTB, virus replication significantly correlated with the increased level of trophoblast cell fusion leading to syncytia formation. In line with increased STB formation in vitro , we in vivo observed an increase of syncytial knots release in early placenta infected by SARS-CoV-2 compared both to SARS-CoV-2- negative areas from the same placenta, and to age matched references. Altogether, our in vitro and in vivo data suggested that efficient replication of SARS-CoV-2 variants in placenta cells during early stage of pregnancy might alter STB turnover. Health sciences/Pathogenesis/Infection Health sciences/Medical research/Experimental models of disease first trimester pregnancy SARS-CoV-2 variants cell fusion syncytial knots trophoblast Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Association between COVID-19 infections and wide range of clinical adverse outcomes during gestation and post-partum period has been reported, as reviewed in 1 , 2 , affecting (i) the fetus, such as miscarriage, (ii) neonate like stillbirth, and (iii) the mother, with higher incidence of intensive care admission and death compared to age-matched non-pregnant women 3 , as well as higher risk of premature delivery and pre-eclampsia. SARS-CoV-2 placentitis may affect both symptomatic and asymptomatic infected pregnant women, and combines chronic histiocytic intervillositis, massive perivillous fibrin deposition, and trophoblast necrosis 1 , 4 . SARS-CoV-2 considerably evolved genetically since its emergence, giving rise to a range of variants with different disease severity and transmission capacities 5 . Across the waves of variants of concerns, several studies have reported a progressive increase in hospitalization of pregnant patients infected with the Alpha variant compared to the ancestral virus, which further increased for the Delta variant that followed the Alpha variant in 2021 6–8 . In contrast, the risk of preterm birth and admission to maternal critical care has decreased during the Omicron- versus the Delta- dominant period 8 . Intrinsic phenotypic differences were observed between viruses, such as Delta and Omicron BA.2 and BA.5, but not BA.1, showed an enhanced fusogenicity compared to the ancestral virus. At the same time, with the emergence of Omicron, underlying population immunity has increased after the emergence of Omicron. Both changes in viral properties and immunity impact virus transmissibility and pathogenicity. The exact mechanisms responsible for pregnancy complications due to SARS-CoV-2 remain unclear. However, enhanced inflammatory responses are thought to be a major driver of the placenta pathologies observed during SARS-CoV-2 maternal infections. Massive infiltration of immune cells and high induction of pro-inflammatory cytokines could be found at the maternal-fetal interface in SARS-CoV-2 infected pregnant women even in the absence of placenta or fetus infections 9 . Although less common (maximum 8.5% according to a recent systematic review 10 ), vertical transmission has been reported. Viral material could be detected in placental tissue and were localized mainly in syncytiotrophoblast (STB), but also in Hofbauer and stroma cells 4 , 11 . Placental infections have been associated with higher risk of adverse obstetrical outcomes 12 . Although not surprising considering the critical role of this maternal-fetal interface organ in protecting, oxygenizing, and nourishing the developing fetus, a better understanding of the cause of these adverse clinical outcomes is still needed. While the presence of SARS-CoV-2 was demonstrated in placenta of infected women, it is still unclear if it could efficiently replicate in this tissue. During the early steps of SARS-CoV-2 infection, the interplay between virus and placenta response might be determinant for viral replication and overall outcome of the pregnancy. This includes innate immune response mediated by the activation of interferon (IFN) pathways and host factors involved in virus entry such as Angiotensin-converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2) 13 . Tallarek et al. failed to demonstrate replication of SARS-CoV-2 in placental explants 14 . However, Fahmi et al. reported that SARS-CoV-2 could infect and propagate in term placental section explants in association with the expression of ACE2, but not with TMPRSS2 15 . As Maternal-fetal virus transmission and pathogenesis might be dependent on gestational age, similar studies from earlier stage of gestation are relevant but limited by the restricted accessibility of placenta before term. Alternatively, trophoblast stem cells were recently used to generate an in vitro SARS-CoV-2 infection model of placenta 16 . Placental formation and development are complex multistep processes, which start with blastocyst implantation in the endometrium. At the feto-maternal interface, trophoblast cells differentiate according the villous or the extravillous pathway 17 . In this pathway, extravillous cytotrophoblast (CTB) proliferates and differentiates into an invasive phenotype. These cells invade decidual stromal compartments as well as spiral arteries of the decidua and the proximal third of the myometrium 18 . In the villous pathway, villous CTB (vCTB) remains in the fetal compartment, fuses and differentiates to form the syncytiotrophoblast (STB) 19 . Then, vCTB fuses with overlying STB to bring fresh cellular components to the STB. To maintain homeostasis, apoptotic material of STB is packed into syncytial knots (SK) and then released into maternal circulation. Controlled CTB-STB fusion and STB turnover are thus crucial to maintain the integrity of the placental barrier. An increased number of SK has been suggested to induce an inflammatory response by the mother and may lead to adverse obstetrical outcomes such as preeclampsia, a hypertensive disorder of pregnancy and a major cause of maternal mortality and morbidity 20 – 23 . Examination of placenta obtained from patients with and without SARS-CoV-2 infection in pregnancy showed an association between SARS-CoV-2 infection and features of maternal vascular malperfusion and accelerated villous maturation 24 , 25 . The strongest associations were found with infection in the eras of the Delta and Omicron variants 25 , and early in gestation 24 . In line with an accelerated villous maturation, several studies also reported an increase of SK formation in placenta in late stage of pregnancy analysed from SARS-CoV-2 infected pregnant women 26 – 28 . These findings suggest that SARS-CoV-2 infection could alter STB turnover and are supported by a recent in vitro study demonstrating that SARS-CoV-2 can induce trophoblast fusion and apoptosis 29 . However, studies using human trophoblast stem cells (hTSC) to determine the effect of SARS-CoV-2 infection on STB differentiation observed a decrease in cell fusion with SARS-CoV-2 infection 30 . These discrepancies could be due to the different trophoblast models used for infection and cell fusion studies. Indeed, immortalized trophoblast cell lines, especially the BeWo cell line, are often considered as a model for syncytialisation 31 . However, these models require biochemical reagents to induce cell fusion, which may differ from the spontaneous fusion of trophoblast observed in vivo or in vitro . hTSCs is another interesting model where the cells differentiate into different trophoblast cells, including STB 31 . Nevertheless, they have not undergone rigorous comparison with in vivo or in vitro trophoblast cells. CTBs isolated from trophoblast where cells at different gestational stage spontaneously fuse and differentiate into STB, with a fusion index of 90% within 72h are considered to be a relevant model for studying trophoblast fusion 31 . This model is also considered as a valuable model in placental infection studies. However, the usage of first trimester CTB is limited by the difficulty to obtain patient samples. In this work, we evaluate replicative capacity of SARS-CoV-2 Delta variant and Omicron BA.1, BA.2 and BA.5 in primary first trimester CTB and STB. We also investigated host response induction in the context of infection and the effect of SARS-CoV-2 infection on trophoblast cell fusion. Methods and Materials SARS-CoV-2 variants Viral stocks of SARS-CoV-2 Delta and Omicron BA.1, BA.2 and BA.5 have been produced after isolation from left over of clinical samples collected at University Hospitals of Geneva as described previously 32 . All virus isolations, viral stocks production and titrations have been performed in Vero-E6 derived cells overexpressing TMPRSS2 33 . Trophoblast cells isolation and culture This study has been approved by the Cantonal Commission for research ethics -ID 2022 − 00873. Informed written consent to participate in the study was obtained from patients who voluntarily decided to interrupt their pregnancy (9–11 weeks of gestation) at the outpatient gynecology consultations of Geneva University Hospitals. First trimester CTBs were isolated as previously described 33 . Briefly, trophoblast tissues were isolated and enzymatically digested with a Difco Trypsin solution (BD, Le pont de Claix, France). After separation in a Percoll gradient (GE Healthcare, Uppsala, Sweden), CTBs were immunopurified using monoclonal mouse anti-human CD45 immobilized antibodies (Dako, Glostrup, Denmark). After immune-purification cells were washed and resuspended in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Invitrogen, Basel, Switzerland) supplemented with 10% FBS and 0.05 mg/ml gentamycin. Cells were then seeded either in 96-well (CTB, 1.10 5 cells/well) or 24-well (STB, 1.5.10 6 cells/well) plates and incubated 24h (for CTB infections) or 72h to allow cell fusion (for STB infection) at 37°C and 5% CO 2 before virus infection. Virus infection of trophoblastic cells Infections of trophoblast with SARS-CoV-2 were performed at 37°C and 5% CO 2 and an approximative MOI of 0.1–0.2. As shown in Fig. 1 , STB and CTB were inoculated with 100ul of virus diluted in 100ul of culture medium, DMEM supplemented with 2%FBS (Gibco), 2mM L-glutamine (Gibco), 1% penicillin-streptomycin (Gibco). They were washed one hour later, overlaid with 500ul (STB) or 200ul (CTB) with the same medium and incubated at 37°C, 5% CO2. For STB infections, supernatant was collected at 24 or 96 hours post infection (hpi) and cells were lysed using the lysis buffer of NucliSens easyMAG (BioMérieux) at 96hpi. Two days after CTB infections, supernatant was collected, and cells were fixed using 6% paraformaldehyde at least 1h at room temperature (RT). Virus replication Virus replication was assessed by viral RNA quantification and virus titration. Viral RNA was extracted from the infection supernatant collected at different time points and used for quantitative real time PCR (RT-qPCR) using SuperScript™ III Platinum™ One-Step qRT-PCR Kit (Invitrogen) in a CFX96 Thermo Cycler (BIORAD) and E-gene targeting primers and probe (30). Data was collected and analyzed using Bio-Rad CFX maestro software (BIORAD). Host response Induction of interferons (IFN-α, IFN-βand IFN-λ), ISG15 (Interferon stimulated gene 15) and virus entry factors ACE-2 and TMPRSS2 were assessed from SARS-CoV-2 infected STB lysates collected at 96hpi. After RNA extraction, mRNA was semi-quantified by real-time RT-PCR using commercially available gene expression assay kits (Life Technology). Gene induction in infected tissues was represented in log10 fold change (FC) relative to mock-infected cells from the same donor and normalized to a housekeeping gene, RNAseP (Life Technology). Immunofluorescence Mock- and SARS-CoV-2 infected STB were PFA-fixed at 96hpi, washed with PBS, permeabilized and co-stained with antibodies targeting Cytokeratin 7 (CK7; clone OV-TL, DAKO), as a trophoblast cell marker, and NP SARS-CoV-2 (Rockland 200-401-50) to detect infected cells. Nuclei were stained with DAPI (4', 6-diamino-2-phenylindole). Images were acquired using Zeiss LSM700/LSM800 Meta confocal microscope with a 63.6/1.4 objective. Fusion index Fusion index (FI) was determined by immunocytochemistry from infected CTB at 48hpi. After Mayer’s Hemalun staining, images were required using EVOS microscope and syncytia were counted from triplicates of each infection condition. Non-infected CTB were used as controls. The percentage of FI was calculated as follow: FI = 100X [(number of nuclei in syncytia- number of syncytia)/ total number of nuclei] and represented in percentage relative to FI in non-infected cells from the same donor. Staining of SARS-CoV-2 positive placentae and determination of SK Placentas from three non-vaccinated COVID19 positive patients with immunohistochemical confirmation of placental SARS-CoV-2 infection were assessed with the agreement of our local ethical committee. In absence of available material from the first trimester, placentas from the early second trimester of pregnancy (18 + 1, 21 + 1, and 24 + 6 gestational weeks respectively) were included. All three pregnancies resulted with intrauterine fetal demise. Expulsion took place between November 2020 and December 2021, corresponding to waves where Alpha (end of 2020-mid2021), then Delta (mid-2021-early 2022) variants were dominant. Placental histology was consistent with SARS-CoV-2 placentitis, showing massive perivillous fibrin deposition, with fibrin encasing more than 80% of the villi (Fig. 1 ). The clinical characteristics and main placental findings are summarized in Table 1 . For SK counts, 5 serial 3-µm thick sections were prepared from one selected formalin-fixed paraffin-embedded block from each of the three second trimester placentas. Immunohistochemistry was performed on the first and on the last sections, using a SARS-CoV-2 nucleocapsid antibody (Bio SB, clone BSB-134). Serial sections 2, 3 and 4 were stained with hematoxylin-eosin (H&E) (Fig. 1 S). SK counts were performed on the serial H&E placental sections. SK were defined as sessile aggregations of nuclei appearing and disappearing through the serial sections and seen in two or more sections. The analysis of SK release was then categorized on individual villi depending on the SARS-CoV-2 staining. Negative: villi showing no reactivity to SARS-CoV-2 nucleocapsid; mild: focal or weak SARS-CoV-2 staining; Positive: circumferential strong SARS-CoV-2 positivity. SK results are reported in Table 2 and compared to reference numbers 35 when feasible (reference data unavailable at 18 weeks of gestation). Results Permissiveness of STB to SARS-CoV-2 Delta and Omicron variants in vitro To test the susceptibility of first trimester STB to SARS-CoV-2 variants, CTB were cultured 3 days to allow cell fusion and differentiation before being infected with SARS-CoV-2 variants. As shown in Fig. 2 A, all donors were permissive to infection by SARS-CoV-2 Delta, with an increase of log10 viral RNA copies/mL (vRNAc/mL) varying from 7.9 to 8.6 at 24hpi and from 7.7 to 9.1 at 96hpi. An increase of viral replication of Omicron variants was shown in the majority of, but not all donors, with log10 viral RNA copies/mL (vRNAc/mL) for Omicron BA.1 ranging from 7.4 to 8.9 log10 vRNAc/mL at 24hpi and 6.5 to 9.5 log10 vRNAc/mL at 96hpi, for Omicron BA.2 from 7.4 to 9.5 log10 vRNAc/mL at 24hpi and 7.1 to 8.5 log10 vRNAc/mL at 96hpi, and for Omicron BA.5 from 7.6 to 9.6 log10 vRNAc/mL at 24hpi and 7.4 to 9.5 log10 vRNAc/mL at 96hpi. These results also showed inter-donor variability regarding SARS-CoV-2 infections. Donors 21,11 and 12 consistently showed high replication, while donors 14 and 6 consistently showed low replication in terms of production of viral RNA 24 and 96 h after infection. Viral replication was also confirmed by immunofluorescence at 96hpi that showed the localization of SARS-CoV-2 in STB (Fig. 2 B). However, only few STB were infected (not shown). In summary, these results confirm that SARS-CoV-2 is able to infect and replicate in STB with replication efficiency dependent on SARS-CoV-2 variants and placenta donors. Assessment of the association between host factors and SARS-CoV-2 infection in STB We next assessed innate immune response induction by the determination of fold change expression of IFN-α and IFN-β, IFN-λ and ISG15 in infected, relative to non-infected STB. Upon STB infections with SARS-CoV-2, we observed a heterogenous host response, including innate immunity and entry host factors, of the trophoblast donors (Fig. 3 and S2). As commonly observed in respiratory virus infections 36 – 38 , type III interferon, IFN-λ showed a more pronounced response compared to type I, IFN-α and IFN-β (Fig. 3 ). SARS-CoV-2 Delta infection induced the highest level of interferon response followed by a slight increase of ISG15 expression, while in Omicron infection this response was barely observed. In addition, no correlation was observed between virus replication and the induction of host response after infection in STB (data not shown). We further investigated the induction of host factors involved in virus entry, including SARS-CoV-2 receptor, ACE-2, and the cellular protease involved in fusion, TMPRSS2, by RT-qPCR. Donor-dependent differences in baseline expression of TMPRSS2 were observed (mock infected cells from the corresponding donors) with expression significantly correlating with viral replication of Delta and BA.1 infection (Fig. 4 ). In contrast, the expression of ACE-2 does not correlate with virus replication. No altered expression of ACE-2 or TMPRSS2 was observed during infection (Figure S2 ). SARS-CoV-2 infection of CTB induces significant increase of syncytia formation compared to control cells in vitro We then investigated the effect of SARS-CoV-2 variants infection on trophoblast cell fusion (Fig. 5 ). CTB were infectable by SARS-CoV-2 with a significant increase in viral replication between 1h and 48hpi for all the variants, except BA.1 (Fig. 5 A), reaching an average of 9 log10 vRNAc/mL for Delta variants and averages varying from 8.2 to 8.5 log10 vRNAc/mL for Omicron variants at 2days pi. The fusion index (FI, Fig. 5 B) was also significant increased in infected cells. As shown in Fig. 6 C, this FI enhancement positively correlated with viral replication (R 2 = 0.5466, p-value = 0.0047). In conclusion, we here demonstrated an efficient infection of SARS-CoV-2 in CTB leading to an increase in cell fusion, which corroborates the noted inter-donor variability. SARS-CoV-2 placenta in vivo is associated with an early release of syncytial knots An increased SK release was previously reported in third trimester placenta of COVID-19 infected patients suggesting that SARS-CoV-2 infection could alter STB turnover 26 , 39 . To check whether SARS-CoV-2 infection could alter STB turnover during earlier stage of pregnancy in vivo , we retrospectively evaluated SK release of three early second trimester placenta from COVID19 + pregnant patients (see Table 1 ). Detection of SK was compared in 3 different areas of the sections, depending on reactivity to the anti-SARS-CoV-2-N (Fig. 6 B). As shown in Fig. 6 C, a significant increase in SK in SARS-CoV-2-positive areas of placenta was observed compared to SARS-CoV-2- negative areas from the same placenta, suggesting an association between SARS-CoV-2 infection and SK early release. The percentage of villi showing one or more SK was also much higher than expected during the second trimester (35% vs. reference numbers of 5.5% at 21 GW; 43% vs. 8.6% at 24 GW; 34%, no reference data available at 18 GW). SK tended to be seen more frequently on villi encased by fibrin (Fig. 6 A). Discussion Placental formation, development and function are crucial for both maternal and fetal health during pregnancy. SARS-CoV-2 infections during pregnancy have been associated with placental injury and clinical adverse outcomes 12 , 40 , 41 . Recent trending SARS-CoV-2 studies were mainly focused on vaccination, management, pregnancy outcomes, and transmission to the fetus (7). Studies on the fetus’ susceptibility to SARS-CoV-2 predominantly targeted later stages of pregnancy (5,8–9). While of strong interest, questions still remain regarding the susceptibility of fetuses to SARS-CoV-2 at the earliest stages of pregnancy. This is mainly due to the difficulty in obtaining clinical samples, from infected SARS-CoV-2 donors and who voluntarily interrupt their pregnancies at this stage, to participate in in vivo or in vitro studies respectively. Alternatively, stem cell-based system was used to evaluate the effects of SARS-CoV-2 infection in early gestation 16 , 30 , 42 . STB derived from human embryonic stem cells (hESC), human expanded potential stem cells or induced (i) or placenta-derived trophoblast stem cells (TSC) express the entry host factors ACE2 and TMPRSS2 and support replicative infection by SARS-CoV-2 16,30,42 . TSC-derived STB infected with SARS-CoV-2 also elicits an interferon-mediated immune response 42 suggesting that placental development could be altered by early SARS-CoV-2 infection. By using a relevant 2D first trimester trophoblast model and clinical viral isolates, we demonstrated that first trimester CTB and STB are permissive to SARS-CoV-2 variants. Supporting assumptions that Delta variant contributes to adverse pregnancy outcomes 8 , our study findings confirmed the high increase in viral replication of the Delta variant shown in most donors. Omicron variants BA.1, BA.2 and BA.5 also showed an increase in viral replication, yet not as substantial as the Delta variant. This is consistent with the clinical observation reporting higher rate of hospitalization in Delta- compared to Omicron-dominant waves of SARS-CoV-2 43 , which could indicate intrinsic higher pathogenicity of Delta. It remains to be considered though that also population immunity was lower in times of Delta virus circulation versus Omicron. In contrast to the observation made on term placenta 15 , trophoblast expression of ACE2 does not correlate with SARS-CoV-2 replication in first trimester STB derived from iTSCs 16 . In contrast, TMPRSS2 expression significantly correlates with Delta and BA.1 replication. We observed the same tendency with BA.5 replication, but not with BA.2 replication. This observation suggests that TMPRSS2 may play an important role in some SARS-CoV-2 variants entry to the STB. Since TMPRSS2 was previously shown to accelerate SARS-CoV‐2‐mediated cell-cell fusion 44 , we next investigated the effect of SARS-CoV-2 infection on trophoblast cell fusion. An increase in trophoblast cell fusion was observed in trophoblast cells infected with SARS-CoV-2 and correlates with viral replication. The increase in cell fusion is higher in cells infected with Delta variant compared to cells infected with BA.1 variant, suggesting a lower fusogenicity of Omicron BA.1 than Delta, as already described 45 – 47 . These results also suggest that the SARS-CoV-2 infection could accelerate trophoblast cell fusion and STB turnover. This hypothesis corroborated clinical analysis of early placenta infected by SARS-CoV-2 showing higher detection of SK release in SARS-CoV-2- positive, compared to negative, areas from the same placenta. A number of studies had previously reported an association between SK increase and adverse obstetrical outcomes 48 . The higher trophoblast cell fusion observed in vitro is coherent, not only with the pathological analysis of SARS-CoV-2 infected placenta (Fig. 4 ), but also with previous patients’ reports 26 – 28 . However, these observations are not in accordance with previous studies using human trophoblast stem cells (hTSC) and determining the inhibitory effect of SARS-CoV-2 infection on STB differentiation 16 , 30 . These discrepancies could be due to the different trophoblast models used for in vitro infection and syncytialisation studies 31 . Primary first trimester trophoblasts are widely recognized as a robust model for investigating trophoblast fusion. Trophoblast stem cell-derived placental model is interesting in studying infection during earlier stages. Mezzano et al. identified early STB (eSTB) with the TSC-based system model, which is undetectable in first trimester primary trophoblast but more susceptible to SARS-CoV-2 infection than STB 30 . In this model, eSTB infected with SARS-CoV-2 may decrease STB maturation. In any cases, it has been shown that SARS-CoV-2 infections could alter the STB renewal and/or turnover and consequently the integrity and function of STB, which in turn may impact the fetal-maternal exchanges as well as hormones secretion during pregnancy. Altogether, infecting 2D first trimester trophoblast and analyzing early second trimester infected placenta from fetal demise, filled gaps in the understanding of SARS-CoV-2 placenta infections and potential mechanisms involved in complications related to COVID-19 during pregnancy. The strength of our findings is the concordance between in vitro and clinical findings, that highlights the relevance of our model system when studying the effect of viral infection on trophoblastic cell fusion and virus host interactions. Such a model does not only serve to better understand SARS-CoV-2 impact in pregnancy but could also be used as an in vitro risk assessment tool for other emerging viruses and their impact during early pregnancy. Declarations Conflict of interest statement The authors declare no conflicts of interest. Ethical approval The study was approved by local ethical Committee (2022 − 00873). Author contributions Conceptualization and project administration: M.E.L, M.C and I.E; Cell experiments: M.C and M.E.L; RT-qPCR and immunofluorescence: Me.C, C.A, P.S.R, M.B, K.A, Y.S; Recruitment of patients and collect of tissues: M.Y, Me.C, C.W, M.C. Data analysis: M.E.L, M.C, A.L.R; Validation: M.E.L, M.C, A.L.R, I.E; Draft Preparation: M.E.L and M.C; All authors have read and agreed to the published version of the manuscript. Acknowledgements We acknowledge the HUG Private Foundation and the Pictet Foundation for their financial support. Data availability The data that support the findings of this study are available on request from the corresponding author. References Male V. SARS-CoV-2 infection and COVID-19 vaccination in pregnancy. Nat Rev Immunol 2022; : 1–6. Schwartz DA, Mulkey SB, Roberts DJ. SARS-CoV-2 placentitis, stillbirth, and maternal COVID-19 vaccination: clinical-pathologic correlations. Am J Obstet Gynecol 2023; 228 : 261–269. Zambrano LD, Ellington S, Strid P, Galang RR, Oduyebo T, Tong VT et al. Update: Characteristics of Symptomatic Women of Reproductive Age with Laboratory-Confirmed SARS-CoV-2 Infection by Pregnancy Status - United States, January 22-October 3, 2020. MMWR Morb Mortal Wkly Rep 2020; 69 : 1641–1647. Wong YP, Khong TY, Tan GC. The Effects of COVID-19 on Placenta and Pregnancy: What Do We Know So Far? Diagnostics (Basel) 2021; 11 : 94. Manirambona E, Okesanya OJ, Olaleke NO, Oso TA, Lucero-Prisno DE. Evolution and implications of SARS-CoV-2 variants in the post-pandemic era. Discov Public Health 2024; 21 : 16. Samara A, Khalil A, O’Brien P, Herlenius E. The effect of the delta SARS-CoV-2 variant on maternal infection and pregnancy. iScience 2022; 25 : 104295. Vousden N, Ramakrishnan R, Bunch K, Morris E, Simpson NAB, Gale C et al. Severity of maternal infection and perinatal outcomes during periods of SARS-CoV-2 wildtype, alpha, and delta variant dominance in the UK: prospective cohort study. BMJ Med 2022; 1 : e000053. Favre G, Maisonneuve E, Pomar L, Daire C, Poncelet C, Quibel T et al. Maternal and perinatal outcomes following pre-Delta, Delta, and Omicron SARS-CoV-2 variants infection among unvaccinated pregnant women in France and Switzerland: a prospective cohort study using the COVI-PREG registry. Lancet Reg Health Eur 2023; 26 : 100569. Garcia-Flores V, Romero R, Xu Y, Theis KR, Arenas-Hernandez M, Miller D et al. Maternal-fetal immune responses in pregnant women infected with SARS-CoV-2. Nat Commun 2022; 13 : 320. Group BMJP. Update to living systematic review on SARS-CoV-2 positivity in offspring and timing of mother-to-child transmission. BMJ 2024; 384 : p2929. Patanè L, Morotti D, Giunta MR, Sigismondi C, Piccoli MG, Frigerio L et al. Vertical transmission of coronavirus disease 2019: severe acute respiratory syndrome coronavirus 2 RNA on the fetal side of the placenta in pregnancies with coronavirus disease 2019–positive mothers and neonates at birth. Am J Obstet Gynecol MFM 2020; 2 : 100145. Tosto V, Meyyazhagan A, Alqasem M, Tsibizova V, Di Renzo GC. SARS-CoV-2 Footprints in the Placenta: What We Know after Three Years of the Pandemic. J Pers Med 2023; 13 : 699. Jackson CB, Farzan M, Chen B, Choe H. Mechanisms of SARS-CoV-2 entry into cells. Nat Rev Mol Cell Biol 2022; 23 : 3–20. Tallarek A-C, Urbschat C, Fonseca Brito L, Stanelle-Bertram S, Krasemann S, Frascaroli G et al. Inefficient Placental Virus Replication and Absence of Neonatal Cell-Specific Immunity Upon Sars-CoV-2 Infection During Pregnancy. Front Immunol 2021; 12 : 698578. Fahmi A, Brügger M, Démoulins T, Zumkehr B, Oliveira Esteves BI, Bracher L et al. SARS-CoV-2 can infect and propagate in human placenta explants. Cell Rep Med 2021; 2 : 100456. Chen J, Neil JA, Tan JP, Rudraraju R, Mohenska M, Sun YBY et al. A placental model of SARS-CoV-2 infection reveals ACE2-dependent susceptibility and differentiation impairment in syncytiotrophoblasts. Nat Cell Biol 2023; 25 : 1223–1234. Al-Nasiry S, Spitz B, Hanssens M, Luyten C, Pijnenborg R. Differential effects of inducers of syncytialization and apoptosis on BeWo and JEG-3 choriocarcinoma cells. Hum Reprod 2006; 21 : 193–201. Pollheimer J, Knöfler M. Signalling pathways regulating the invasive differentiation of human trophoblasts: a review. Placenta 2005; 26 Suppl A : S21-30. Ferretti C, Bruni L, Dangles-Marie V, Pecking AP, Bellet D. Molecular circuits shared by placental and cancer cells, and their implications in the proliferative, invasive and migratory capacities of trophoblasts. Hum Reprod Update 2007; 13 : 121–141. Goswami D, Tannetta DS, Magee LA, Fuchisawa A, Redman CWG, Sargent IL et al. Excess syncytiotrophoblast microparticle shedding is a feature of early-onset pre-eclampsia, but not normotensive intrauterine growth restriction. Placenta 2006; 27 : 56–61. Tannetta DS, Dragovic RA, Gardiner C, Redman CW, Sargent IL. Characterisation of syncytiotrophoblast vesicles in normal pregnancy and pre-eclampsia: expression of Flt-1 and endoglin. PLoS One 2013; 8 : e56754. Schuster J, Cheng S-B, Padbury J, Sharma S. Placental extracellular vesicles and pre-eclampsia. Am J Reprod Immunol 2021; 85 : e13297. Kazemi NY, Gendrot B, Berishvili E, Markovic SN, Cohen M. The Role and Clinical Interest of Extracellular Vesicles in Pregnancy and Ovarian Cancer. Biomedicines 2021; 9 : 1257. Hernandez PV, Chen L, Zhang R, Jackups R, Nelson DM, He M. The effects of preconception and early gestation SARS-CoV-2 infection on pregnancy outcomes and placental pathology. Annals of Diagnostic Pathology 2023; 62 : 152076. Shanes ED, Miller ES, Otero S, Ebbott R, Aggarwal R, Willnow AS et al. Placental Pathology After SARS-CoV-2 Infection in the Pre-Variant of Concern, Alpha / Gamma, Delta, or Omicron Eras. Int J Surg Pathol 2023; 31 : 387–397. Gao L, Ren J, Xu L, Ke X, Xiong L, Tian X et al. Placental pathology of the third trimester pregnant women from COVID-19. Diagn Pathol 2021; 16 : 8. Garg R, Agarwal R, Yadav D, Singh S, Kumar H, Bhardwaj R. Histopathological Changes in Placenta of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-Cov-2) Infection and Maternal and Perinatal Outcome in COVID-19. J Obstet Gynaecol India 2023; 73 : 44–50. Shchegolev AI, Kulikova GV, Lyapin VM, Shmakov RG, Sukhikh GT. The Number of Syncytial Knots and VEGF Expression in Placental Villi in Parturient Woman with COVID-19 Depends on the Disease Severity. Bull Exp Biol Med 2021; 171 : 399–403. Agostinis C, Toffoli M, Spazzapan M, Balduit A, Zito G, Mangogna A et al. SARS-CoV-2 modulates virus receptor expression in placenta and can induce trophoblast fusion, inflammation and endothelial permeability. Frontiers in Immunology 2022; 13 .https://www.frontiersin.org/articles/10.3389/fimmu.2022.957224 (accessed 1 Sep2023). Ruan D, Ye Z-W, Yuan S, Li Z, Zhang W, Ong CP et al. Human early syncytiotrophoblasts are highly susceptible to SARS-CoV-2 infection. Cell Rep Med 2022; 3 : 100849. Li X, Li Z-H, Wang Y-X, Liu T-H. A comprehensive review of human trophoblast fusion models: recent developments and challenges. Cell Death Discov 2023; 9 : 1–14. Bekliz M, Adea K, Vetter P, Eberhardt CS, Hosszu-Fellous K, Vu D-L et al. Neutralization capacity of antibodies elicited through homologous or heterologous infection or vaccination against SARS-CoV-2 VOCs. Nat Commun 2022; 13 : 3840. Bekliz M, Essaidi-Laziosi M, Adea K, Hosszu-Fellous K, Alvarez C, Bellon M et al. Immune escape of Omicron lineages BA.1, BA.2, BA.5.1, BQ.1, XBB.1.5, EG.5.1 and JN.1.1 after vaccination, infection and hybrid immunity. 2024; : 2024.02.14.579654. Arnaudeau S, Arboit P, Bischof P, Shin-ya K, Tomida A, Tsuruo T et al. Glucose-regulated protein 78: A new partner of p53 in trophoblast. PROTEOMICS 2009; 9 : 5316–5327. Loukeris K, Sela R, Baergen RN. Syncytial knots as a reflection of placental maturity: reference values for 20 to 40 weeks’ gestational age. Pediatr Dev Pathol 2010; 13 : 305–309. Essaidi-Laziosi M, Royston L, Boda B, Pérez-Rodriguez FJ, Piuz I, Hulo N et al. Altered cell function and increased replication of rhinoviruses and EV-D68 in airway epithelia of asthma patients. Front Microbiol 2023; 14 : 1106945. Essaidi-Laziosi M, Alvarez C, Puhach O, Sattonnet-Roche P, Torriani G, Tapparel C et al. Sequential infections with rhinovirus and influenza modulate the replicative capacity of SARS-CoV-2 in the upper respiratory tract. Emerg Microbes Infect 2022; 11 : 412–423. Essaidi-Laziosi M, Brito F, Benaoudia S, Royston L, Cagno V, Fernandes-Rocha M et al. Propagation of respiratory viruses in human airway epithelia reveals persistent virus-specific signatures. J Allergy Clin Immunol 2018; 141 : 2074–2084. Singh N, Buckley T, Shertz W. Placental Pathology in COVID-19: Case Series in a Community Hospital Setting. Cureus 2021; 13 : e12522. Pomorski M, Trzeszcz M, Matera-Witkiewicz A, Krupińska M, Fuchs T, Zimmer M et al. SARS-CoV-2 Infection and Pregnancy: Maternal and Neonatal Outcomes and Placental Pathology Correlations. Viruses 2022; 14 : 2043. Zaigham M, Gisselsson D, Sand A, Wikström A-K, von Wowern E, Schwartz DA et al. Clinical-pathological features in placentas of pregnancies with SARS-CoV-2 infection and adverse outcome: case series with and without congenital transmission. BJOG 2022; 129 : 1361–1374. Kallol S, Martin-Sancho L, Morey R, Aisagbonhi O, Pizzo D, Meads M et al. Activation of the Interferon Pathway in Trophoblast Cells Productively Infected with SARS-CoV-2. Stem Cells Dev 2023; 32 : 225–236. Stock SJ, Moore E, Calvert C, Carruthers J, Denny C, Donaghy J et al. Pregnancy outcomes after SARS-CoV-2 infection in periods dominated by delta and omicron variants in Scotland: a population-based cohort study. Lancet Respir Med 2022; 10 : 1129–1136. Buchrieser J, Dufloo J, Hubert M, Monel B, Planas D, Rajah MM et al. Syncytia formation by SARS-CoV-2-infected cells. EMBO J 2020; 39 : e106267. Suzuki R, Yamasoba D, Kimura I, Wang L, Kishimoto M, Ito J et al. Attenuated fusogenicity and pathogenicity of SARS-CoV-2 Omicron variant. Nature 2022; 603 : 700–705. Meng B, Abdullahi A, Ferreira IATM, Goonawardane N, Saito A, Kimura I et al. Altered TMPRSS2 usage by SARS-CoV-2 Omicron impacts infectivity and fusogenicity. Nature 2022; 603 : 706–714. Kushwaha N, Dwivedi A, Tiwari S, Mishra P, Verma SK. Comprehensive analytics for virus-cell and cell-cell multinucleation system. Biochemical and Biophysical Research Communications 2024; 726 : 150281. Parks WT. Increased Syncytial Knot Formation. In: Khong TY, Mooney EE, Nikkels PGJ, Morgan TK, Gordijn SJ (eds). Pathology of the Placenta: A Practical Guide . Springer International Publishing: Cham, 2019, pp 131–137. Tables Tables 1 and 2 are available in the Supplementary Files section. Additional Declarations (Not answered) Supplementary Files FigureS1.tif Figure S1 FigureS2.jpg Figure S2 Table1.docx Table2.docx Cite Share Download PDF Status: Published Journal Publication published 07 Oct, 2025 Read the published version in Cell Death & Disease → Version 1 posted Editorial decision: revise 07 May, 2025 Review # 1 received at journal 15 Apr, 2025 Reviewer # 1 agreed at journal 31 Mar, 2025 Reviewers invited by journal 31 Mar, 2025 Submission checks completed at journal 18 Mar, 2025 First submitted to journal 17 Mar, 2025 Editor assigned by journal 17 Mar, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6246871","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":436424854,"identity":"942ef57d-36bb-404b-96ee-8efa92ce96dd","order_by":0,"name":"Marie-Benoite Cohen","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA8UlEQVRIiWNgGAWjYBACgwMMDEBkx2DAwMD4gIFBAiTIhleLJURLMkgLswFRWuwPgKmDIC1sElBB/FrMjp99eOADwwE5c/beY9W8Oyzs+hmYnz3Aq+VMusHBGQwHjC17zqXd5j0jkTyzgc3cAK+WA2kMh3kYDiRuuJFjdpu3TSLZ4AAP3IVYgcH5ZwyH/4C03H9jVgzSYk9Qyw2gLUDvA23hMWMGarEzYCCo5RnDwR6DZGODMznGknPbJBIkDrOZEXBYGvOHHxV2cgbHzxh+eNtWZ8/f3vwMrxaoRgQzsYGZsHpUYE+qhlEwCkbBKBj+AABaLEpXFuH4pQAAAABJRU5ErkJggg==","orcid":"","institution":"University of Geneva","correspondingAuthor":true,"prefix":"","firstName":"Marie-Benoite","middleName":"","lastName":"Cohen","suffix":""},{"id":436424855,"identity":"78d1885a-ef30-4be7-8097-821278369203","order_by":1,"name":"Manel Essaidi-Laziosi","email":"","orcid":"https://orcid.org/0000-0003-1643-4965","institution":"University of Geneva, Faculty of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Manel","middleName":"","lastName":"Essaidi-Laziosi","suffix":""},{"id":436424856,"identity":"a3667c2a-103a-4959-8d5e-8ca037ebf733","order_by":2,"name":"catia alvarez","email":"","orcid":"","institution":"University of Geneva","correspondingAuthor":false,"prefix":"","firstName":"catia","middleName":"","lastName":"alvarez","suffix":""},{"id":436424857,"identity":"4847ae25-8ac2-4e24-ae61-40f4c1e6756f","order_by":3,"name":"Michal Yaron","email":"","orcid":"","institution":"Geneva University Hospitals","correspondingAuthor":false,"prefix":"","firstName":"Michal","middleName":"","lastName":"Yaron","suffix":""},{"id":436424858,"identity":"7d4c8a96-b131-4307-a8f8-4e89c069c436","order_by":4,"name":"pascale satonnet-roche","email":"","orcid":"","institution":"University of Geneva","correspondingAuthor":false,"prefix":"","firstName":"pascale","middleName":"","lastName":"satonnet-roche","suffix":""},{"id":436424859,"identity":"488ab6a0-947d-45db-a459-8a8945c78e0a","order_by":5,"name":"melanie cornut","email":"","orcid":"","institution":"University of Geneva","correspondingAuthor":false,"prefix":"","firstName":"melanie","middleName":"","lastName":"cornut","suffix":""},{"id":436424860,"identity":"bcdf0f43-0062-46f3-839c-fa77ac8578b7","order_by":6,"name":"Christine Wuillemin","email":"","orcid":"","institution":"University of Geneva","correspondingAuthor":false,"prefix":"","firstName":"Christine","middleName":"","lastName":"Wuillemin","suffix":""},{"id":436424861,"identity":"945967d2-127f-41aa-9ac3-0669f433a971","order_by":7,"name":"Meriem Bekliz","email":"","orcid":"","institution":"University of Geneva","correspondingAuthor":false,"prefix":"","firstName":"Meriem","middleName":"","lastName":"Bekliz","suffix":""},{"id":436424862,"identity":"030e04cb-db13-487e-9347-af130d37abf2","order_by":8,"name":"Kenneth Adea","email":"","orcid":"","institution":"University of Geneva","correspondingAuthor":false,"prefix":"","firstName":"Kenneth","middleName":"","lastName":"Adea","suffix":""},{"id":436424863,"identity":"06df55f7-68ce-45fd-a7ff-9d58261ebe98","order_by":9,"name":"Yoann Sarmiento","email":"","orcid":"","institution":"University of Geneva","correspondingAuthor":false,"prefix":"","firstName":"Yoann","middleName":"","lastName":"Sarmiento","suffix":""},{"id":436424864,"identity":"3b23674d-d4c9-405e-a7b3-112bf25bb8d6","order_by":10,"name":"Chloe gibson","email":"","orcid":"","institution":"University of Geneva","correspondingAuthor":false,"prefix":"","firstName":"Chloe","middleName":"","lastName":"gibson","suffix":""},{"id":436424865,"identity":"89374da4-ede1-4d45-ba4c-f2c5fc3a3a63","order_by":11,"name":"Laurent Kaiser","email":"","orcid":"","institution":"Geneva University Hospitals and Faculty of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Laurent","middleName":"","lastName":"Kaiser","suffix":""},{"id":436424866,"identity":"26d08a33-b0eb-43d5-9229-93b45c19dc32","order_by":12,"name":"Anne-Laure Rougemont","email":"","orcid":"https://orcid.org/0000-0002-1785-792X","institution":"Geneva University Hospitals and Faculty of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Anne-Laure","middleName":"","lastName":"Rougemont","suffix":""},{"id":436424867,"identity":"5dfa4bb8-0e25-4444-a090-a1293049b6a2","order_by":13,"name":"isabella Eckerle","email":"","orcid":"","institution":"Geneva University Hospitals and Faculty of Medicine","correspondingAuthor":false,"prefix":"","firstName":"isabella","middleName":"","lastName":"Eckerle","suffix":""}],"badges":[],"createdAt":"2025-03-17 17:50:12","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6246871/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6246871/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41419-025-08016-x","type":"published","date":"2025-10-07T04:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":81037819,"identity":"e9252e05-5c09-4364-9578-d4ad56381d2b","added_by":"auto","created_at":"2025-04-21 12:44:52","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1712006,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMain histological findings (H\u0026amp;E).\u003c/strong\u003e The three early second trimester placentas with SARS-CoV-2 placentitis showed MPFD, together with trophoblast necrosis in Case #2 and CHI in Case #3 (H\u0026amp;E; Case #1 x20, Case #2 x250, Case #3 x250).\u003c/p\u003e","description":"","filename":"Figure1.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6246871/v1/fda690990dc14acb3dc3b38e.jpg"},{"id":81038064,"identity":"239be2a0-7b46-4351-b103-96073594c2d7","added_by":"auto","created_at":"2025-04-21 12:52:52","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1013271,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSARS-CoV-2 Delta and Omicron variants is propagating in first trimester STB. \u003c/strong\u003e\u003cem\u003eCTBs were isolated from first trimester trophoblasts. After which they were cultured for 96h to allow STB formation before being inoculated using clinical isolates of SARS-CoV-2 Delta or Omicron BA.1, BA.2 and BA.5 variants at multiplicity of infection (MOI) of respectively 0.1 and 0.2. After 1h of adsorption, cells were then washed and incubated at 37°C. (A) SARS-CoV-2 replication. For each time point, the supernatant was collected and replaced by fresh media. Viral RNA was quantified by real time RT-PCR. Statistically significant increase was calculated using 2-way ANOVA **P \u0026lt; 0.01\u003c/em\u003e. Each symbol represents an individual donor.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e(A) SARS-CoV-2 replication. For each time point, the supernatant was collected and replaced by fresh media. Viral RNA was quantified by real time RT-PCR. Statistically significant increase was calculated using 2-way ANOVA **P \u0026lt; 0.01\u003c/em\u003e. Each symbol represents an individual donor.\u003c/p\u003e\n\u003cp\u003e(B) \u003cem\u003eImmunofluorescence at 4 days post-infection (pi). Mock and SARS-CoV2 infected cells were fixed, permeabilized and co-stained using antibodies against SARS-CoV-2 nucleoprotein (SARS-CoV-2 N in green) and cytokeratin 7 (CK7 in pink), a trophoblast cells’ marker. The nuclei were stained by DAPI (blue). Images were acquired using Zeiss LSM 700 Meta confocal microscope with a 63.6/1.4 objective. a-mock; b- delta; c-BA.1; d- BA.2; e- BA.5.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6246871/v1/9a0fd51ed62f2bb1bd4205eb.jpg"},{"id":81038063,"identity":"0fcef548-8554-416e-80f0-3070dc39a0f3","added_by":"auto","created_at":"2025-04-21 12:52:51","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":339189,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eSTB responses following SARS-CoV-2 Delta and Omicron variants infection. \u003c/strong\u003e\u003c/em\u003e\u003cem\u003eInduction of IFN-α, IFN-β, IFN-λ and ISG15 were semi-quantified by real time RT-PCR and represented in fold change relative to non-infected cells and normalized to RNAseP.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6246871/v1/6844b1e0ed36546d869db092.jpg"},{"id":81037815,"identity":"a78e4a43-4ec3-4ea6-839a-f24b55af7e34","added_by":"auto","created_at":"2025-04-21 12:44:52","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":539153,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCorrelation of SARS-CoV-2 variants replication 96 h post infection of STB and ACE2 and TMPRSS2 mRNA levels. \u003c/strong\u003eAssociations were tested using the Spearman rank correlation test. Each symbol represents an individual donor\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Figure4.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6246871/v1/420edf94eebc56e210b54c18.jpg"},{"id":81038065,"identity":"9b894fc5-9602-4060-8875-6b04c9936c28","added_by":"auto","created_at":"2025-04-21 12:52:52","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":102357,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSARS-CoV-2 Delta and Omicron variants replication in first trimester cytotrophoblast cell and effects on cell fusion. \u003c/strong\u003e\u003cem\u003eCTBs were isolated from first trimester trophoblasts. 16 to 24h later, they were inoculated using clinical isolates of SARS-CoV-2 Delta or Omicron BA.1, BA.2 and BA.5 variants at multiplicity of infection (MOI) of respectively 0.1 and 0.2. After 1h of adsorption, cells were then washed and incubated at 37°C for 48h.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e \u003cem\u003eSARS-CoV-2 replication. For each time point, the supernatant was collected and replaced by fresh media. Viral RNA was quantified by real time RT-PCR. A statistically significant increase was calculated using 2-way ANOVA **P \u0026lt; 0.01\u003c/em\u003e. Each color represents an individual donor. Each value corresponds to the average of 3 replicates from the same donor.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(B)\u003c/strong\u003eNuclei and syncytia were counted and fusion index was calculated\u003cem\u003e. A statistically significant increase was calculated using 2-way ANOVA. *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001 and ****P \u0026lt; 0.0001\u003c/em\u003e. Each color represents an individual donor. Each value corresponds to the average of 3 replicates from the same donor.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(C)\u003c/strong\u003e\u003cem\u003e SARS-CoV-2 replication was plotted as a function of relative fusion index \u003c/em\u003eAssociations were tested using the Spearman rank correlation test. Hpi: hours post infection\u003c/p\u003e","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6246871/v1/6280f7c3f488431ce5891be2.jpg"},{"id":81038069,"identity":"d81c376c-5dce-490e-8ee2-8878c0898786","added_by":"auto","created_at":"2025-04-21 12:52:52","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":271782,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSyncytial knots evaluation depending on reactivity to the SARS-CoV-2 nucleocapsid in 3 early second trimester placenta.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTissue was stained using antibodies against SARS-CoV-2 nucleocapsid (clone BSB-134). SK evaluation was performed on serial H\u0026amp;E placental sections. Syncytial knots were defined as sessile aggregations of nuclei appearing and disappearing through the serial sections and seen in two or more sections.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e SK tended to be more numerous in villi encased by fibrin (Case #1, H\u0026amp;E, x200).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(B)\u003c/strong\u003e SK release was categorized depending on the SARS-CoV-2 staining on the first and the last serial sections as follows: negative: no reactivity to SARS-CoV-2 NC; mild: focal or weak SARS-CoV-2 staining; positive: SARS-CoV-2 staining is positive throughout the area assessed. Depicted are SK counts in a positive villous showing 5 SK (in circles), and in a negative villous with no SK (Case #3, H\u0026amp;E and SARS-CoV-2 NC, x130).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(C)\u003c/strong\u003e The analysis of SK release was then categorized depending on the SARS-CoV-2 staining. Negative: no reactivity to SARS-CoV-2 nucleocapsid; mild: focal or weak SARS-CoV-2 staining; Positive: SARS-CoV-2 staining is positive throughout the area assessed. \u003cem\u003eA statistically significant increase was calculated using one-way ANOVA test. *P \u0026lt; 0.05.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Figure6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6246871/v1/28e04e6c2b9b0580bdb095ac.jpg"},{"id":93009233,"identity":"ec038bfd-475f-4064-8be8-c521c8059dd5","added_by":"auto","created_at":"2025-10-08 07:08:11","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5107577,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6246871/v1/2dd94acf-d440-46d0-a0b2-27bcc25ed26c.pdf"},{"id":81038726,"identity":"6df12198-7e92-41d2-9998-235fab70ee64","added_by":"auto","created_at":"2025-04-21 13:00:52","extension":"tif","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1083440,"visible":true,"origin":"","legend":"\u003cp\u003eFigure S1\u003c/p\u003e","description":"","filename":"FigureS1.tif","url":"https://assets-eu.researchsquare.com/files/rs-6246871/v1/645b08ec39db8583eb861a42.tif"},{"id":81038071,"identity":"e2dafd8c-5faa-4e44-8ca0-205f2d0e9611","added_by":"auto","created_at":"2025-04-21 12:52:52","extension":"jpg","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":196279,"visible":true,"origin":"","legend":"\u003cp\u003eFigure S2\u003c/p\u003e","description":"","filename":"FigureS2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6246871/v1/bea99bb1ceab800313173745.jpg"},{"id":81037810,"identity":"64cb0c70-19db-4e0d-809e-2923aa65f075","added_by":"auto","created_at":"2025-04-21 12:44:51","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":17039,"visible":true,"origin":"","legend":"","description":"","filename":"Table1.docx","url":"https://assets-eu.researchsquare.com/files/rs-6246871/v1/cbbfc9c08a435140bf235128.docx"},{"id":81038725,"identity":"6540e55a-4e95-4b7e-be7b-f0fad620d3e3","added_by":"auto","created_at":"2025-04-21 13:00:52","extension":"docx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":28401,"visible":true,"origin":"","legend":"","description":"","filename":"Table2.docx","url":"https://assets-eu.researchsquare.com/files/rs-6246871/v1/a0d7ad7fd3cefabacae3666a.docx"}],"financialInterests":"(Not answered)","formattedTitle":"SARS-CoV-2 Delta and Omicron Variants alter Trophoblast Cell Fusion and Syncytiotrophoblast Dynamics: New Insights into Placental Vulnerability","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAssociation between COVID-19 infections and wide range of clinical adverse outcomes during gestation and post-partum period has been reported, as reviewed in \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e, affecting (i) the fetus, such as miscarriage, (ii) neonate like stillbirth, and (iii) the mother, with higher incidence of intensive care admission and death compared to age-matched non-pregnant women \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e, as well as higher risk of premature delivery and pre-eclampsia. SARS-CoV-2 placentitis may affect both symptomatic and asymptomatic infected pregnant women, and combines chronic histiocytic intervillositis, massive perivillous fibrin deposition, and trophoblast necrosis \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eSARS-CoV-2 considerably evolved genetically since its emergence, giving rise to a range of variants with different disease severity and transmission capacities \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Across the waves of variants of concerns, several studies have reported a progressive increase in hospitalization of pregnant patients infected with the Alpha variant compared to the ancestral virus, which further increased for the Delta variant that followed the Alpha variant in 2021 \u003csup\u003e6\u0026ndash;8\u003c/sup\u003e. In contrast, the risk of preterm birth and admission to maternal critical care has decreased during the Omicron- versus the Delta- dominant period \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Intrinsic phenotypic differences were observed between viruses, such as Delta and Omicron BA.2 and BA.5, but not BA.1, showed an enhanced fusogenicity compared to the ancestral virus. At the same time, with the emergence of Omicron, underlying population immunity has increased after the emergence of Omicron. Both changes in viral properties and immunity impact virus transmissibility and pathogenicity.\u003c/p\u003e \u003cp\u003eThe exact mechanisms responsible for pregnancy complications due to SARS-CoV-2 remain unclear. However, enhanced inflammatory responses are thought to be a major driver of the placenta pathologies observed during SARS-CoV-2 maternal infections. Massive infiltration of immune cells and high induction of pro-inflammatory cytokines could be found at the maternal-fetal interface in SARS-CoV-2 infected pregnant women even in the absence of placenta or fetus infections \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Although less common (maximum 8.5% according to a recent systematic review \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e), vertical transmission has been reported. Viral material could be detected in placental tissue and were localized mainly in syncytiotrophoblast (STB), but also in Hofbauer and stroma cells \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Placental infections have been associated with higher risk of adverse obstetrical outcomes \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. Although not surprising considering the critical role of this maternal-fetal interface organ in protecting, oxygenizing, and nourishing the developing fetus, a better understanding of the cause of these adverse clinical outcomes is still needed.\u003c/p\u003e \u003cp\u003eWhile the presence of SARS-CoV-2 was demonstrated in placenta of infected women, it is still unclear if it could efficiently replicate in this tissue. During the early steps of SARS-CoV-2 infection, the interplay between virus and placenta response might be determinant for viral replication and overall outcome of the pregnancy. This includes innate immune response mediated by the activation of interferon (IFN) pathways and host factors involved in virus entry such as Angiotensin-converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2) \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. Tallarek et al. failed to demonstrate replication of SARS-CoV-2 in placental explants \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. However, Fahmi et al. reported that SARS-CoV-2 could infect and propagate in term placental section explants in association with the expression of ACE2, but not with TMPRSS2 \u003csup\u003e15\u003c/sup\u003e. As Maternal-fetal virus transmission and pathogenesis might be dependent on gestational age, similar studies from earlier stage of gestation are relevant but limited by the restricted accessibility of placenta before term. Alternatively, trophoblast stem cells were recently used to generate an \u003cem\u003ein vitro\u003c/em\u003e SARS-CoV-2 infection model of placenta \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003ePlacental formation and development are complex multistep processes, which start with blastocyst implantation in the endometrium. At the feto-maternal interface, trophoblast cells differentiate according the villous or the extravillous pathway \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. In this pathway, extravillous cytotrophoblast (CTB) proliferates and differentiates into an invasive phenotype. These cells invade decidual stromal compartments as well as spiral arteries of the decidua and the proximal third of the myometrium \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. In the villous pathway, villous CTB (vCTB) remains in the fetal compartment, fuses and differentiates to form the syncytiotrophoblast (STB) \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. Then, vCTB fuses with overlying STB to bring fresh cellular components to the STB. To maintain homeostasis, apoptotic material of STB is packed into syncytial knots (SK) and then released into maternal circulation. Controlled CTB-STB fusion and STB turnover are thus crucial to maintain the integrity of the placental barrier. An increased number of SK has been suggested to induce an inflammatory response by the mother and may lead to adverse obstetrical outcomes such as preeclampsia, a hypertensive disorder of pregnancy and a major cause of maternal mortality and morbidity \u003csup\u003e\u003cspan additionalcitationids=\"CR21 CR22\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Examination of placenta obtained from patients with and without SARS-CoV-2 infection in pregnancy showed an association between SARS-CoV-2 infection and features of maternal vascular malperfusion and accelerated villous maturation \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e,\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. The strongest associations were found with infection in the eras of the Delta and Omicron variants \u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e, and early in gestation \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. In line with an accelerated villous maturation, several studies also reported an increase of SK formation in placenta in late stage of pregnancy analysed from SARS-CoV-2 infected pregnant women \u003csup\u003e\u003cspan additionalcitationids=\"CR27\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. These findings suggest that SARS-CoV-2 infection could alter STB turnover and are supported by a recent \u003cem\u003ein vitro\u003c/em\u003e study demonstrating that SARS-CoV-2 can induce trophoblast fusion and apoptosis \u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. However, studies using human trophoblast stem cells (hTSC) to determine the effect of SARS-CoV-2 infection on STB differentiation observed a decrease in cell fusion with SARS-CoV-2 infection \u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. These discrepancies could be due to the different trophoblast models used for infection and cell fusion studies. Indeed, immortalized trophoblast cell lines, especially the BeWo cell line, are often considered as a model for syncytialisation \u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. However, these models require biochemical reagents to induce cell fusion, which may differ from the spontaneous fusion of trophoblast observed \u003cem\u003ein vivo\u003c/em\u003e or \u003cem\u003ein vitro\u003c/em\u003e. hTSCs is another interesting model where the cells differentiate into different trophoblast cells, including STB \u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. Nevertheless, they have not undergone rigorous comparison with \u003cem\u003ein vivo\u003c/em\u003e or \u003cem\u003ein vitro\u003c/em\u003e trophoblast cells.\u003c/p\u003e \u003cp\u003eCTBs isolated from trophoblast where cells at different gestational stage spontaneously fuse and differentiate into STB, with a fusion index of 90% within 72h are considered to be a relevant model for studying trophoblast fusion \u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. This model is also considered as a valuable model in placental infection studies. However, the usage of first trimester CTB is limited by the difficulty to obtain patient samples.\u003c/p\u003e \u003cp\u003eIn this work, we evaluate replicative capacity of SARS-CoV-2 Delta variant and Omicron BA.1, BA.2 and BA.5 in primary first trimester CTB and STB. We also investigated host response induction in the context of infection and the effect of SARS-CoV-2 infection on trophoblast cell fusion.\u003c/p\u003e"},{"header":"Methods and Materials","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eSARS-CoV-2 variants\u003c/h2\u003e \u003cp\u003eViral stocks of SARS-CoV-2 Delta and Omicron BA.1, BA.2 and BA.5 have been produced after isolation from left over of clinical samples collected at University Hospitals of Geneva as described previously \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. All virus isolations, viral stocks production and titrations have been performed in Vero-E6 derived cells overexpressing TMPRSS2 \u003csup\u003e33\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eTrophoblast cells isolation and culture\u003c/h3\u003e\n\u003cp\u003e This study has been approved by the Cantonal Commission for research ethics -ID 2022\u0026thinsp;\u0026minus;\u0026thinsp;00873. Informed written consent to participate in the study was obtained from patients who voluntarily decided to interrupt their pregnancy (9\u0026ndash;11 weeks of gestation) at the outpatient gynecology consultations of Geneva University Hospitals. First trimester CTBs were isolated as previously described \u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. Briefly, trophoblast tissues were isolated and enzymatically digested with a Difco Trypsin solution (BD, Le pont de Claix, France). After separation in a Percoll gradient (GE Healthcare, Uppsala, Sweden), CTBs were immunopurified using monoclonal mouse anti-human CD45 immobilized antibodies (Dako, Glostrup, Denmark). After immune-purification cells were washed and resuspended in Dulbecco\u0026rsquo;s modified Eagle\u0026rsquo;s medium (DMEM; Gibco, Invitrogen, Basel, Switzerland) supplemented with 10% FBS and 0.05 mg/ml gentamycin. Cells were then seeded either in 96-well (CTB, 1.10\u003csup\u003e5\u003c/sup\u003e cells/well) or 24-well (STB, 1.5.10\u003csup\u003e6\u003c/sup\u003e cells/well) plates and incubated 24h (for CTB infections) or 72h to allow cell fusion (for STB infection) at 37\u0026deg;C and 5% CO\u003csub\u003e2\u003c/sub\u003e before virus infection.\u003c/p\u003e\n\u003ch3\u003eVirus infection of trophoblastic cells\u003c/h3\u003e\n\u003cp\u003eInfections of trophoblast with SARS-CoV-2 were performed at 37\u0026deg;C and 5% CO\u003csub\u003e2\u003c/sub\u003e and an approximative MOI of 0.1\u0026ndash;0.2. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, STB and CTB were inoculated with 100ul of virus diluted in 100ul of culture medium, DMEM supplemented with 2%FBS (Gibco), 2mM L-glutamine (Gibco), 1% penicillin-streptomycin (Gibco). They were washed one hour later, overlaid with 500ul (STB) or 200ul (CTB) with the same medium and incubated at 37\u0026deg;C, 5% CO2. For STB infections, supernatant was collected at 24 or 96 hours post infection (hpi) and cells were lysed using the lysis buffer of NucliSens easyMAG (BioM\u0026eacute;rieux) at 96hpi. Two days after CTB infections, supernatant was collected, and cells were fixed using 6% paraformaldehyde at least 1h at room temperature (RT).\u003c/p\u003e\n\u003ch3\u003eVirus replication\u003c/h3\u003e\n\u003cp\u003eVirus replication was assessed by viral RNA quantification and virus titration. Viral RNA was extracted from the infection supernatant collected at different time points and used for quantitative real time PCR (RT-qPCR) using SuperScript\u0026trade; III Platinum\u0026trade; One-Step qRT-PCR Kit (Invitrogen) in a CFX96 Thermo Cycler (BIORAD) and E-gene targeting primers and probe (30). Data was collected and analyzed using Bio-Rad CFX maestro software (BIORAD).\u003c/p\u003e\n\u003ch3\u003eHost response\u003c/h3\u003e\n\u003cp\u003eInduction of interferons (IFN-α, IFN-βand IFN-λ), ISG15 (Interferon stimulated gene 15) and virus entry factors ACE-2 and TMPRSS2 were assessed from SARS-CoV-2 infected STB lysates collected at 96hpi. After RNA extraction, mRNA was semi-quantified by real-time RT-PCR using commercially available gene expression assay kits (Life Technology). Gene induction in infected tissues was represented in log10 fold change (FC) relative to mock-infected cells from the same donor and normalized to a housekeeping gene, RNAseP (Life Technology).\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eImmunofluorescence\u003c/h2\u003e \u003cp\u003eMock- and SARS-CoV-2 infected STB were PFA-fixed at 96hpi, washed with PBS, permeabilized and co-stained with antibodies targeting Cytokeratin 7 (CK7; clone OV-TL, DAKO), as a trophoblast cell marker, and NP SARS-CoV-2 (Rockland 200-401-50) to detect infected cells. Nuclei were stained with DAPI (4', 6-diamino-2-phenylindole). Images were acquired using Zeiss LSM700/LSM800 Meta confocal microscope with a 63.6/1.4 objective.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eFusion index\u003c/h3\u003e\n\u003cp\u003eFusion index (FI) was determined by immunocytochemistry from infected CTB at 48hpi. After Mayer\u0026rsquo;s Hemalun staining, images were required using EVOS microscope and syncytia were counted from triplicates of each infection condition. Non-infected CTB were used as controls. The percentage of FI was calculated as follow: FI\u0026thinsp;=\u0026thinsp;100X [(number of nuclei in syncytia- number of syncytia)/ total number of nuclei] and represented in percentage relative to FI in non-infected cells from the same donor.\u003c/p\u003e \u003cp\u003e\u003cb\u003eStaining of SARS-CoV-2 positive placentae and determination of SK\u003c/b\u003e Placentas from three non-vaccinated COVID19 positive patients with immunohistochemical confirmation of placental SARS-CoV-2 infection were assessed with the agreement of our local ethical committee. In absence of available material from the first trimester, placentas from the early second trimester of pregnancy (18\u0026thinsp;+\u0026thinsp;1, 21\u0026thinsp;+\u0026thinsp;1, and 24\u0026thinsp;+\u0026thinsp;6 gestational weeks respectively) were included. All three pregnancies resulted with intrauterine fetal demise. Expulsion took place between November 2020 and December 2021, corresponding to waves where Alpha (end of 2020-mid2021), then Delta (mid-2021-early 2022) variants were dominant. Placental histology was consistent with SARS-CoV-2 placentitis, showing massive perivillous fibrin deposition, with fibrin encasing more than 80% of the villi (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe clinical characteristics and main placental findings are summarized in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003eFor SK counts, 5 serial 3-\u0026micro;m thick sections were prepared from one selected formalin-fixed paraffin-embedded block from each of the three second trimester placentas. Immunohistochemistry was performed on the first and on the last sections, using a SARS-CoV-2 nucleocapsid antibody (Bio SB, clone BSB-134). Serial sections 2, 3 and 4 were stained with hematoxylin-eosin (H\u0026amp;E) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eS).\u003c/p\u003e \u003cp\u003eSK counts were performed on the serial H\u0026amp;E placental sections. SK were defined as sessile aggregations of nuclei appearing and disappearing through the serial sections and seen in two or more sections. The analysis of SK release was then categorized on individual villi depending on the SARS-CoV-2 staining. Negative: villi showing no reactivity to SARS-CoV-2 nucleocapsid; mild: focal or weak SARS-CoV-2 staining; Positive: circumferential strong SARS-CoV-2 positivity.\u003c/p\u003e \u003cp\u003eSK results are reported in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and compared to reference numbers \u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e when feasible (reference data unavailable at 18 weeks of gestation).\u003c/p\u003e "},{"header":"Results","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003ePermissiveness of STB to SARS-CoV-2 Delta and Omicron variants in vitro\u003c/h2\u003e \u003cp\u003eTo test the susceptibility of first trimester STB to SARS-CoV-2 variants, CTB were cultured 3 days to allow cell fusion and differentiation before being infected with SARS-CoV-2 variants. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, all donors were permissive to infection by SARS-CoV-2 Delta, with an increase of log10 viral RNA copies/mL (vRNAc/mL) varying from 7.9 to 8.6 at 24hpi and from 7.7 to 9.1 at 96hpi. An increase of viral replication of Omicron variants was shown in the majority of, but not all donors, with log10 viral RNA copies/mL (vRNAc/mL) for Omicron BA.1 ranging from 7.4 to 8.9 log10 vRNAc/mL at 24hpi and 6.5 to 9.5 log10 vRNAc/mL at 96hpi, for Omicron BA.2 from 7.4 to 9.5 log10 vRNAc/mL at 24hpi and 7.1 to 8.5 log10 vRNAc/mL at 96hpi, and for Omicron BA.5 from 7.6 to 9.6 log10 vRNAc/mL at 24hpi and 7.4 to 9.5 log10 vRNAc/mL at 96hpi. These results also showed inter-donor variability regarding SARS-CoV-2 infections. Donors 21,11 and 12 consistently showed high replication, while donors 14 and 6 consistently showed low replication in terms of production of viral RNA 24 and 96 h after infection.\u003c/p\u003e \u003cp\u003eViral replication was also confirmed by immunofluorescence at 96hpi that showed the localization of SARS-CoV-2 in STB (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). However, only few STB were infected (not shown).\u003c/p\u003e \u003cp\u003eIn summary, these results confirm that SARS-CoV-2 is able to infect and replicate in STB with replication efficiency dependent on SARS-CoV-2 variants and placenta donors.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eAssessment of the association between host factors and SARS-CoV-2 infection in STB\u003c/h2\u003e \u003cp\u003eWe next assessed innate immune response induction by the determination of fold change expression of IFN-α and IFN-β, IFN-λ and ISG15 in infected, relative to non-infected STB. Upon STB infections with SARS-CoV-2, we observed a heterogenous host response, including innate immunity and entry host factors, of the trophoblast donors (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e and S2). As commonly observed in respiratory virus infections \u003csup\u003e\u003cspan additionalcitationids=\"CR37\" citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e, type III interferon, IFN-λ showed a more pronounced response compared to type I, IFN-α and IFN-β (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSARS-CoV-2 Delta infection induced the highest level of interferon response followed by a slight increase of ISG15 expression, while in Omicron infection this response was barely observed. In addition, no correlation was observed between virus replication and the induction of host response after infection in STB (data not shown).\u003c/p\u003e \u003cp\u003eWe further investigated the induction of host factors involved in virus entry, including SARS-CoV-2 receptor, ACE-2, and the cellular protease involved in fusion, TMPRSS2, by RT-qPCR. Donor-dependent differences in baseline expression of TMPRSS2 were observed (mock infected cells from the corresponding donors) with expression significantly correlating with viral replication of Delta and BA.1 infection (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). In contrast, the expression of ACE-2 does not correlate with virus replication. No altered expression of ACE-2 or TMPRSS2 was observed during infection (Figure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cb\u003eSARS-CoV-2 infection of CTB induces significant increase of syncytia formation compared to control cells in vitro\u003c/b\u003e \u003c/p\u003e \u003cp\u003eWe then investigated the effect of SARS-CoV-2 variants infection on trophoblast cell fusion (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). CTB were infectable by SARS-CoV-2 with a significant increase in viral replication between 1h and 48hpi for all the variants, except BA.1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA), reaching an average of 9 log10 vRNAc/mL for Delta variants and averages varying from 8.2 to 8.5 log10 vRNAc/mL for Omicron variants at 2days pi. The fusion index (FI, Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB) was also significant increased in infected cells. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC, this FI enhancement positively correlated with viral replication (R\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.5466, p-value\u0026thinsp;=\u0026thinsp;0.0047). In conclusion, we here demonstrated an efficient infection of SARS-CoV-2 in CTB leading to an increase in cell fusion, which corroborates the noted inter-donor variability.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eSARS-CoV-2 placenta in vivo is associated with an early release of syncytial knots\u003c/h2\u003e \u003cp\u003eAn increased SK release was previously reported in third trimester placenta of COVID-19 infected patients suggesting that SARS-CoV-2 infection could alter STB turnover \u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e,\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. To check whether SARS-CoV-2 infection could alter STB turnover during earlier stage of pregnancy \u003cem\u003ein vivo\u003c/em\u003e, we retrospectively evaluated SK release of three early second trimester placenta from COVID19\u0026thinsp;+\u0026thinsp;pregnant patients (see Table \u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Detection of SK was compared in 3 different areas of the sections, depending on reactivity to the anti-SARS-CoV-2-N (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC, a significant increase in SK in SARS-CoV-2-positive areas of placenta was observed compared to SARS-CoV-2- negative areas from the same placenta, suggesting an association between SARS-CoV-2 infection and SK early release. The percentage of villi showing one or more SK was also much higher than expected during the second trimester (35% vs. reference numbers of 5.5% at 21 GW; 43% vs. 8.6% at 24 GW; 34%, no reference data available at 18 GW). SK tended to be seen more frequently on villi encased by fibrin (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA).\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003ePlacental formation, development and function are crucial for both maternal and fetal health during pregnancy. SARS-CoV-2 infections during pregnancy have been associated with placental injury and clinical adverse outcomes \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e,\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. Recent trending SARS-CoV-2 studies were mainly focused on vaccination, management, pregnancy outcomes, and transmission to the fetus (7). Studies on the fetus\u0026rsquo; susceptibility to SARS-CoV-2 predominantly targeted later stages of pregnancy (5,8\u0026ndash;9). While of strong interest, questions still remain regarding the susceptibility of fetuses to SARS-CoV-2 at the earliest stages of pregnancy. This is mainly due to the difficulty in obtaining clinical samples, from infected SARS-CoV-2 donors and who voluntarily interrupt their pregnancies at this stage, to participate in \u003cem\u003ein vivo\u003c/em\u003e or \u003cem\u003ein vitro\u003c/em\u003e studies respectively. Alternatively, stem cell-based system was used to evaluate the effects of SARS-CoV-2 infection in early gestation \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e,\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. STB derived from human embryonic stem cells (hESC), human expanded potential stem cells or induced (i) or placenta-derived trophoblast stem cells (TSC) express the entry host factors ACE2 and TMPRSS2 and support replicative infection by SARS-CoV-2\u003csup\u003e16,30,42\u003c/sup\u003e. TSC-derived STB infected with SARS-CoV-2 also elicits an interferon-mediated immune response \u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e suggesting that placental development could be altered by early SARS-CoV-2 infection.\u003c/p\u003e \u003cp\u003eBy using a relevant 2D first trimester trophoblast model and clinical viral isolates, we demonstrated that first trimester CTB and STB are permissive to SARS-CoV-2 variants. Supporting assumptions that Delta variant contributes to adverse pregnancy outcomes \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e, our study findings confirmed the high increase in viral replication of the Delta variant shown in most donors. Omicron variants BA.1, BA.2 and BA.5 also showed an increase in viral replication, yet not as substantial as the Delta variant. This is consistent with the clinical observation reporting higher rate of hospitalization in Delta- compared to Omicron-dominant waves of SARS-CoV-2 \u003csup\u003e43\u003c/sup\u003e, which could indicate intrinsic higher pathogenicity of Delta. It remains to be considered though that also population immunity was lower in times of Delta virus circulation versus Omicron.\u003c/p\u003e \u003cp\u003eIn contrast to the observation made on term placenta \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e, trophoblast expression of ACE2 does not correlate with SARS-CoV-2 replication in first trimester STB derived from iTSCs \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. In contrast, TMPRSS2 expression significantly correlates with Delta and BA.1 replication. We observed the same tendency with BA.5 replication, but not with BA.2 replication. This observation suggests that TMPRSS2 may play an important role in some SARS-CoV-2 variants entry to the STB. Since TMPRSS2 was previously shown to accelerate SARS-CoV‐2‐mediated cell-cell fusion \u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e, we next investigated the effect of SARS-CoV-2 infection on trophoblast cell fusion. An increase in trophoblast cell fusion was observed in trophoblast cells infected with SARS-CoV-2 and correlates with viral replication. The increase in cell fusion is higher in cells infected with Delta variant compared to cells infected with BA.1 variant, suggesting a lower fusogenicity of Omicron BA.1 than Delta, as already described \u003csup\u003e\u003cspan additionalcitationids=\"CR46\" citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e. These results also suggest that the SARS-CoV-2 infection could accelerate trophoblast cell fusion and STB turnover. This hypothesis corroborated clinical analysis of early placenta infected by SARS-CoV-2 showing higher detection of SK release in SARS-CoV-2- positive, compared to negative, areas from the same placenta. A number of studies had previously reported an association between SK increase and adverse obstetrical outcomes \u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e. The higher trophoblast cell fusion observed \u003cem\u003ein vitro\u003c/em\u003e is coherent, not only with the pathological analysis of SARS-CoV-2 infected placenta (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e), but also with previous patients\u0026rsquo; reports \u003csup\u003e\u003cspan additionalcitationids=\"CR27\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eHowever, these observations are not in accordance with previous studies using human trophoblast stem cells (hTSC) and determining the inhibitory effect of SARS-CoV-2 infection on STB differentiation \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. These discrepancies could be due to the different trophoblast models used for \u003cem\u003ein vitro\u003c/em\u003e infection and syncytialisation studies \u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. Primary first trimester trophoblasts are widely recognized as a robust model for investigating trophoblast fusion. Trophoblast stem cell-derived placental model is interesting in studying infection during earlier stages. Mezzano et al. identified early STB (eSTB) with the TSC-based system model, which is undetectable in first trimester primary trophoblast but more susceptible to SARS-CoV-2 infection than STB \u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. In this model, eSTB infected with SARS-CoV-2 may decrease STB maturation. In any cases, it has been shown that SARS-CoV-2 infections could alter the STB renewal and/or turnover and consequently the integrity and function of STB, which in turn may impact the fetal-maternal exchanges as well as hormones secretion during pregnancy.\u003c/p\u003e \u003cp\u003eAltogether, infecting 2D first trimester trophoblast and analyzing early second trimester infected placenta from fetal demise, filled gaps in the understanding of SARS-CoV-2 placenta infections and potential mechanisms involved in complications related to COVID-19 during pregnancy. The strength of our findings is the concordance between \u003cem\u003ein vitro\u003c/em\u003e and clinical findings, that highlights the relevance of our model system when studying the effect of viral infection on trophoblastic cell fusion and virus host interactions. Such a model does not only serve to better understand SARS-CoV-2 impact in pregnancy but could also be used as an in vitro risk assessment tool for other emerging viruses and their impact during early pregnancy.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eConflict of interest statement\u003c/h2\u003e \u003cp\u003eThe authors declare no conflicts of interest.\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eEthical approval\u003c/h2\u003e \u003cp\u003e The study was approved by local ethical Committee (2022\u0026thinsp;\u0026minus;\u0026thinsp;00873).\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eAuthor contributions\u003c/h2\u003e \u003cp\u003eConceptualization and project administration: M.E.L, M.C and I.E; Cell experiments: M.C and M.E.L; RT-qPCR and immunofluorescence: Me.C, C.A, P.S.R, M.B, K.A, Y.S; Recruitment of patients and collect of tissues: M.Y, Me.C, C.W, M.C. Data analysis: M.E.L, M.C, A.L.R; Validation: M.E.L, M.C, A.L.R, I.E; Draft Preparation: M.E.L and M.C; All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003eWe acknowledge the HUG Private Foundation and the Pictet Foundation for their financial support.\u003c/p\u003e\u003ch2\u003eData availability\u003c/h2\u003e \u003cp\u003eThe data that support the findings of this study are available on request from the corresponding author.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eMale V. SARS-CoV-2 infection and COVID-19 vaccination in pregnancy. \u003cem\u003eNat Rev Immunol\u003c/em\u003e 2022; : 1\u0026ndash;6.\u003c/li\u003e\n\u003cli\u003eSchwartz DA, Mulkey SB, Roberts DJ. SARS-CoV-2 placentitis, stillbirth, and maternal COVID-19 vaccination: clinical-pathologic correlations. \u003cem\u003eAm J Obstet Gynecol\u003c/em\u003e 2023; \u003cstrong\u003e228\u003c/strong\u003e: 261\u0026ndash;269.\u003c/li\u003e\n\u003cli\u003eZambrano LD, Ellington S, Strid P, Galang RR, Oduyebo T, Tong VT \u003cem\u003eet al.\u003c/em\u003e Update: Characteristics of Symptomatic Women of Reproductive Age with Laboratory-Confirmed SARS-CoV-2 Infection by Pregnancy Status - United States, January 22-October 3, 2020. \u003cem\u003eMMWR Morb Mortal Wkly Rep\u003c/em\u003e 2020; \u003cstrong\u003e69\u003c/strong\u003e: 1641\u0026ndash;1647.\u003c/li\u003e\n\u003cli\u003eWong YP, Khong TY, Tan GC. The Effects of COVID-19 on Placenta and Pregnancy: What Do We Know So Far? \u003cem\u003eDiagnostics (Basel)\u003c/em\u003e 2021; \u003cstrong\u003e11\u003c/strong\u003e: 94.\u003c/li\u003e\n\u003cli\u003eManirambona E, Okesanya OJ, Olaleke NO, Oso TA, Lucero-Prisno DE. Evolution and implications of SARS-CoV-2 variants in the post-pandemic era. \u003cem\u003eDiscov Public Health\u003c/em\u003e 2024; \u003cstrong\u003e21\u003c/strong\u003e: 16.\u003c/li\u003e\n\u003cli\u003eSamara A, Khalil A, O\u0026rsquo;Brien P, Herlenius E. The effect of the delta SARS-CoV-2 variant on maternal infection and pregnancy. \u003cem\u003eiScience\u003c/em\u003e 2022; \u003cstrong\u003e25\u003c/strong\u003e: 104295.\u003c/li\u003e\n\u003cli\u003eVousden N, Ramakrishnan R, Bunch K, Morris E, Simpson NAB, Gale C \u003cem\u003eet al.\u003c/em\u003e Severity of maternal infection and perinatal outcomes during periods of SARS-CoV-2 wildtype, alpha, and delta variant dominance in the UK: prospective cohort study. \u003cem\u003eBMJ Med\u003c/em\u003e 2022; \u003cstrong\u003e1\u003c/strong\u003e: e000053.\u003c/li\u003e\n\u003cli\u003eFavre G, Maisonneuve E, Pomar L, Daire C, Poncelet C, Quibel T \u003cem\u003eet al.\u003c/em\u003e Maternal and perinatal outcomes following pre-Delta, Delta, and Omicron SARS-CoV-2 variants infection among unvaccinated pregnant women in France and Switzerland: a prospective cohort study using the COVI-PREG registry. \u003cem\u003eLancet Reg Health Eur\u003c/em\u003e 2023; \u003cstrong\u003e26\u003c/strong\u003e: 100569.\u003c/li\u003e\n\u003cli\u003eGarcia-Flores V, Romero R, Xu Y, Theis KR, Arenas-Hernandez M, Miller D \u003cem\u003eet al.\u003c/em\u003e Maternal-fetal immune responses in pregnant women infected with SARS-CoV-2. \u003cem\u003eNat Commun\u003c/em\u003e 2022; \u003cstrong\u003e13\u003c/strong\u003e: 320.\u003c/li\u003e\n\u003cli\u003eGroup BMJP. Update to living systematic review on SARS-CoV-2 positivity in offspring and timing of mother-to-child transmission. \u003cem\u003eBMJ\u003c/em\u003e 2024; \u003cstrong\u003e384\u003c/strong\u003e: p2929.\u003c/li\u003e\n\u003cli\u003ePatan\u0026egrave; L, Morotti D, Giunta MR, Sigismondi C, Piccoli MG, Frigerio L \u003cem\u003eet al.\u003c/em\u003e Vertical transmission of coronavirus disease 2019: severe acute respiratory syndrome coronavirus 2 RNA on the fetal side of the placenta in pregnancies with coronavirus disease 2019\u0026ndash;positive mothers and neonates at birth. \u003cem\u003eAm J Obstet Gynecol MFM\u003c/em\u003e 2020; \u003cstrong\u003e2\u003c/strong\u003e: 100145.\u003c/li\u003e\n\u003cli\u003eTosto V, Meyyazhagan A, Alqasem M, Tsibizova V, Di Renzo GC. SARS-CoV-2 Footprints in the Placenta: What We Know after Three Years of the Pandemic. \u003cem\u003eJ Pers Med\u003c/em\u003e 2023; \u003cstrong\u003e13\u003c/strong\u003e: 699.\u003c/li\u003e\n\u003cli\u003eJackson CB, Farzan M, Chen B, Choe H. Mechanisms of SARS-CoV-2 entry into cells. \u003cem\u003eNat Rev Mol Cell Biol\u003c/em\u003e 2022; \u003cstrong\u003e23\u003c/strong\u003e: 3\u0026ndash;20.\u003c/li\u003e\n\u003cli\u003eTallarek A-C, Urbschat C, Fonseca Brito L, Stanelle-Bertram S, Krasemann S, Frascaroli G \u003cem\u003eet al.\u003c/em\u003e Inefficient Placental Virus Replication and Absence of Neonatal Cell-Specific Immunity Upon Sars-CoV-2 Infection During Pregnancy. \u003cem\u003eFront Immunol\u003c/em\u003e 2021; \u003cstrong\u003e12\u003c/strong\u003e: 698578.\u003c/li\u003e\n\u003cli\u003eFahmi A, Br\u0026uuml;gger M, D\u0026eacute;moulins T, Zumkehr B, Oliveira Esteves BI, Bracher L \u003cem\u003eet al.\u003c/em\u003e SARS-CoV-2 can infect and propagate in human placenta explants. \u003cem\u003eCell Rep Med\u003c/em\u003e 2021; \u003cstrong\u003e2\u003c/strong\u003e: 100456.\u003c/li\u003e\n\u003cli\u003eChen J, Neil JA, Tan JP, Rudraraju R, Mohenska M, Sun YBY \u003cem\u003eet al.\u003c/em\u003e A placental model of SARS-CoV-2 infection reveals ACE2-dependent susceptibility and differentiation impairment in syncytiotrophoblasts. \u003cem\u003eNat Cell Biol\u003c/em\u003e 2023; \u003cstrong\u003e25\u003c/strong\u003e: 1223\u0026ndash;1234.\u003c/li\u003e\n\u003cli\u003eAl-Nasiry S, Spitz B, Hanssens M, Luyten C, Pijnenborg R. Differential effects of inducers of syncytialization and apoptosis on BeWo and JEG-3 choriocarcinoma cells. \u003cem\u003eHum Reprod\u003c/em\u003e 2006; \u003cstrong\u003e21\u003c/strong\u003e: 193\u0026ndash;201.\u003c/li\u003e\n\u003cli\u003ePollheimer J, Kn\u0026ouml;fler M. Signalling pathways regulating the invasive differentiation of human trophoblasts: a review. \u003cem\u003ePlacenta\u003c/em\u003e 2005; \u003cstrong\u003e26 Suppl A\u003c/strong\u003e: S21-30.\u003c/li\u003e\n\u003cli\u003eFerretti C, Bruni L, Dangles-Marie V, Pecking AP, Bellet D. Molecular circuits shared by placental and cancer cells, and their implications in the proliferative, invasive and migratory capacities of trophoblasts. \u003cem\u003eHum Reprod Update\u003c/em\u003e 2007; \u003cstrong\u003e13\u003c/strong\u003e: 121\u0026ndash;141.\u003c/li\u003e\n\u003cli\u003eGoswami D, Tannetta DS, Magee LA, Fuchisawa A, Redman CWG, Sargent IL \u003cem\u003eet al.\u003c/em\u003e Excess syncytiotrophoblast microparticle shedding is a feature of early-onset pre-eclampsia, but not normotensive intrauterine growth restriction. \u003cem\u003ePlacenta\u003c/em\u003e 2006; \u003cstrong\u003e27\u003c/strong\u003e: 56\u0026ndash;61.\u003c/li\u003e\n\u003cli\u003eTannetta DS, Dragovic RA, Gardiner C, Redman CW, Sargent IL. Characterisation of syncytiotrophoblast vesicles in normal pregnancy and pre-eclampsia: expression of Flt-1 and endoglin. \u003cem\u003ePLoS One\u003c/em\u003e 2013; \u003cstrong\u003e8\u003c/strong\u003e: e56754.\u003c/li\u003e\n\u003cli\u003eSchuster J, Cheng S-B, Padbury J, Sharma S. Placental extracellular vesicles and pre-eclampsia. \u003cem\u003eAm J Reprod Immunol\u003c/em\u003e 2021; \u003cstrong\u003e85\u003c/strong\u003e: e13297.\u003c/li\u003e\n\u003cli\u003eKazemi NY, Gendrot B, Berishvili E, Markovic SN, Cohen M. The Role and Clinical Interest of Extracellular Vesicles in Pregnancy and Ovarian Cancer. \u003cem\u003eBiomedicines\u003c/em\u003e 2021; \u003cstrong\u003e9\u003c/strong\u003e: 1257.\u003c/li\u003e\n\u003cli\u003eHernandez PV, Chen L, Zhang R, Jackups R, Nelson DM, He M. The effects of preconception and early gestation SARS-CoV-2 infection on pregnancy outcomes and placental pathology. \u003cem\u003eAnnals of Diagnostic Pathology\u003c/em\u003e 2023; \u003cstrong\u003e62\u003c/strong\u003e: 152076.\u003c/li\u003e\n\u003cli\u003eShanes ED, Miller ES, Otero S, Ebbott R, Aggarwal R, Willnow AS \u003cem\u003eet al.\u003c/em\u003e Placental Pathology After SARS-CoV-2 Infection in the Pre-Variant of Concern, Alpha / Gamma, Delta, or Omicron Eras. \u003cem\u003eInt J Surg Pathol\u003c/em\u003e 2023; \u003cstrong\u003e31\u003c/strong\u003e: 387\u0026ndash;397.\u003c/li\u003e\n\u003cli\u003eGao L, Ren J, Xu L, Ke X, Xiong L, Tian X \u003cem\u003eet al.\u003c/em\u003e Placental pathology of the third trimester pregnant women from COVID-19. \u003cem\u003eDiagn Pathol\u003c/em\u003e 2021; \u003cstrong\u003e16\u003c/strong\u003e: 8.\u003c/li\u003e\n\u003cli\u003eGarg R, Agarwal R, Yadav D, Singh S, Kumar H, Bhardwaj R. Histopathological Changes in Placenta of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-Cov-2) Infection and Maternal and Perinatal Outcome in COVID-19. \u003cem\u003eJ Obstet Gynaecol India\u003c/em\u003e 2023; \u003cstrong\u003e73\u003c/strong\u003e: 44\u0026ndash;50.\u003c/li\u003e\n\u003cli\u003eShchegolev AI, Kulikova GV, Lyapin VM, Shmakov RG, Sukhikh GT. The Number of Syncytial Knots and VEGF Expression in Placental Villi in Parturient Woman with COVID-19 Depends on the Disease Severity. \u003cem\u003eBull Exp Biol Med\u003c/em\u003e 2021; \u003cstrong\u003e171\u003c/strong\u003e: 399\u0026ndash;403.\u003c/li\u003e\n\u003cli\u003eAgostinis C, Toffoli M, Spazzapan M, Balduit A, Zito G, Mangogna A \u003cem\u003eet al.\u003c/em\u003e SARS-CoV-2 modulates virus receptor expression in placenta and can induce trophoblast fusion, inflammation and endothelial permeability. \u003cem\u003eFrontiers in Immunology\u003c/em\u003e 2022; \u003cstrong\u003e13\u003c/strong\u003e.https://www.frontiersin.org/articles/10.3389/fimmu.2022.957224 (accessed 1 Sep2023).\u003c/li\u003e\n\u003cli\u003eRuan D, Ye Z-W, Yuan S, Li Z, Zhang W, Ong CP \u003cem\u003eet al.\u003c/em\u003e Human early syncytiotrophoblasts are highly susceptible to SARS-CoV-2 infection. \u003cem\u003eCell Rep Med\u003c/em\u003e 2022; \u003cstrong\u003e3\u003c/strong\u003e: 100849.\u003c/li\u003e\n\u003cli\u003eLi X, Li Z-H, Wang Y-X, Liu T-H. A comprehensive review of human trophoblast fusion models: recent developments and challenges. \u003cem\u003eCell Death Discov\u003c/em\u003e 2023; \u003cstrong\u003e9\u003c/strong\u003e: 1\u0026ndash;14.\u003c/li\u003e\n\u003cli\u003eBekliz M, Adea K, Vetter P, Eberhardt CS, Hosszu-Fellous K, Vu D-L \u003cem\u003eet al.\u003c/em\u003e Neutralization capacity of antibodies elicited through homologous or heterologous infection or vaccination against SARS-CoV-2 VOCs. \u003cem\u003eNat Commun\u003c/em\u003e 2022; \u003cstrong\u003e13\u003c/strong\u003e: 3840.\u003c/li\u003e\n\u003cli\u003eBekliz M, Essaidi-Laziosi M, Adea K, Hosszu-Fellous K, Alvarez C, Bellon M \u003cem\u003eet al.\u003c/em\u003e Immune escape of Omicron lineages BA.1, BA.2, BA.5.1, BQ.1, XBB.1.5, EG.5.1 and JN.1.1 after vaccination, infection and hybrid immunity. 2024; : 2024.02.14.579654.\u003c/li\u003e\n\u003cli\u003eArnaudeau S, Arboit P, Bischof P, Shin-ya K, Tomida A, Tsuruo T \u003cem\u003eet al.\u003c/em\u003e Glucose-regulated protein 78: A new partner of p53 in trophoblast. \u003cem\u003ePROTEOMICS\u003c/em\u003e 2009; \u003cstrong\u003e9\u003c/strong\u003e: 5316\u0026ndash;5327.\u003c/li\u003e\n\u003cli\u003eLoukeris K, Sela R, Baergen RN. Syncytial knots as a reflection of placental maturity: reference values for 20 to 40 weeks\u0026rsquo; gestational age. \u003cem\u003ePediatr Dev Pathol\u003c/em\u003e 2010; \u003cstrong\u003e13\u003c/strong\u003e: 305\u0026ndash;309.\u003c/li\u003e\n\u003cli\u003eEssaidi-Laziosi M, Royston L, Boda B, P\u0026eacute;rez-Rodriguez FJ, Piuz I, Hulo N \u003cem\u003eet al.\u003c/em\u003e Altered cell function and increased replication of rhinoviruses and EV-D68 in airway epithelia of asthma patients. \u003cem\u003eFront Microbiol\u003c/em\u003e 2023; \u003cstrong\u003e14\u003c/strong\u003e: 1106945.\u003c/li\u003e\n\u003cli\u003eEssaidi-Laziosi M, Alvarez C, Puhach O, Sattonnet-Roche P, Torriani G, Tapparel C \u003cem\u003eet al.\u003c/em\u003e Sequential infections with rhinovirus and influenza modulate the replicative capacity of SARS-CoV-2 in the upper respiratory tract. \u003cem\u003eEmerg Microbes Infect\u003c/em\u003e 2022; \u003cstrong\u003e11\u003c/strong\u003e: 412\u0026ndash;423.\u003c/li\u003e\n\u003cli\u003eEssaidi-Laziosi M, Brito F, Benaoudia S, Royston L, Cagno V, Fernandes-Rocha M \u003cem\u003eet al.\u003c/em\u003e Propagation of respiratory viruses in human airway epithelia reveals persistent virus-specific signatures. \u003cem\u003eJ Allergy Clin Immunol\u003c/em\u003e 2018; \u003cstrong\u003e141\u003c/strong\u003e: 2074\u0026ndash;2084.\u003c/li\u003e\n\u003cli\u003eSingh N, Buckley T, Shertz W. Placental Pathology in COVID-19: Case Series in a Community Hospital Setting. \u003cem\u003eCureus\u003c/em\u003e 2021; \u003cstrong\u003e13\u003c/strong\u003e: e12522.\u003c/li\u003e\n\u003cli\u003ePomorski M, Trzeszcz M, Matera-Witkiewicz A, Krupińska M, Fuchs T, Zimmer M \u003cem\u003eet al.\u003c/em\u003e SARS-CoV-2 Infection and Pregnancy: Maternal and Neonatal Outcomes and Placental Pathology Correlations. \u003cem\u003eViruses\u003c/em\u003e 2022; \u003cstrong\u003e14\u003c/strong\u003e: 2043.\u003c/li\u003e\n\u003cli\u003eZaigham M, Gisselsson D, Sand A, Wikstr\u0026ouml;m A-K, von Wowern E, Schwartz DA \u003cem\u003eet al.\u003c/em\u003e Clinical-pathological features in placentas of pregnancies with SARS-CoV-2 infection and adverse outcome: case series with and without congenital transmission. \u003cem\u003eBJOG\u003c/em\u003e 2022; \u003cstrong\u003e129\u003c/strong\u003e: 1361\u0026ndash;1374.\u003c/li\u003e\n\u003cli\u003eKallol S, Martin-Sancho L, Morey R, Aisagbonhi O, Pizzo D, Meads M \u003cem\u003eet al.\u003c/em\u003e Activation of the Interferon Pathway in Trophoblast Cells Productively Infected with SARS-CoV-2. \u003cem\u003eStem Cells Dev\u003c/em\u003e 2023; \u003cstrong\u003e32\u003c/strong\u003e: 225\u0026ndash;236.\u003c/li\u003e\n\u003cli\u003eStock SJ, Moore E, Calvert C, Carruthers J, Denny C, Donaghy J \u003cem\u003eet al.\u003c/em\u003e Pregnancy outcomes after SARS-CoV-2 infection in periods dominated by delta and omicron variants in Scotland: a population-based cohort study. \u003cem\u003eLancet Respir Med\u003c/em\u003e 2022; \u003cstrong\u003e10\u003c/strong\u003e: 1129\u0026ndash;1136.\u003c/li\u003e\n\u003cli\u003eBuchrieser J, Dufloo J, Hubert M, Monel B, Planas D, Rajah MM \u003cem\u003eet al.\u003c/em\u003e Syncytia formation by SARS-CoV-2-infected cells. \u003cem\u003eEMBO J\u003c/em\u003e 2020; \u003cstrong\u003e39\u003c/strong\u003e: e106267.\u003c/li\u003e\n\u003cli\u003eSuzuki R, Yamasoba D, Kimura I, Wang L, Kishimoto M, Ito J \u003cem\u003eet al.\u003c/em\u003e Attenuated fusogenicity and pathogenicity of SARS-CoV-2 Omicron variant. \u003cem\u003eNature\u003c/em\u003e 2022; \u003cstrong\u003e603\u003c/strong\u003e: 700\u0026ndash;705.\u003c/li\u003e\n\u003cli\u003eMeng B, Abdullahi A, Ferreira IATM, Goonawardane N, Saito A, Kimura I \u003cem\u003eet al.\u003c/em\u003e Altered TMPRSS2 usage by SARS-CoV-2 Omicron impacts infectivity and fusogenicity. \u003cem\u003eNature\u003c/em\u003e 2022; \u003cstrong\u003e603\u003c/strong\u003e: 706\u0026ndash;714.\u003c/li\u003e\n\u003cli\u003eKushwaha N, Dwivedi A, Tiwari S, Mishra P, Verma SK. Comprehensive analytics for virus-cell and cell-cell multinucleation system. \u003cem\u003eBiochemical and Biophysical Research Communications\u003c/em\u003e 2024; \u003cstrong\u003e726\u003c/strong\u003e: 150281.\u003c/li\u003e\n\u003cli\u003eParks WT. Increased Syncytial Knot Formation. In: Khong TY, Mooney EE, Nikkels PGJ, Morgan TK, Gordijn SJ (eds). \u003cem\u003ePathology of the Placenta: A Practical Guide\u003c/em\u003e. Springer International Publishing: Cham, 2019, pp 131\u0026ndash;137.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTables 1 and 2 are available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"cell-death-and-disease","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"cddis","sideBox":"Learn more about [Cell Death \u0026 Disease](http://www.nature.com/cddis/)","snPcode":"41419","submissionUrl":"https://mts-cddis.nature.com/cgi-bin/main.plex","title":"Cell Death \u0026 Disease","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"first trimester pregnancy, SARS-CoV-2 variants, cell fusion, syncytial knots, trophoblast","lastPublishedDoi":"10.21203/rs.3.rs-6246871/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6246871/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePregnancy constitutes an at-risk factor for severe COVID-19. Several clinical studies have previously suggested increased risks of adverse obstetrical outcomes and placental pathological changes in pregnant women infected with SARS-CoV-2. In this study, our goal was to assess the susceptibility of trophoblast to SARS-CoV-2 infection at early stage of pregnancy and its impact on trophoblast cell fusion \u003cem\u003ein vitro\u003c/em\u003e. We showed that first trimester cytotrophoblast (CTB) and syncytiotrophoblast (STB) are permissive to SARS-CoV-2 in variant- and donor-dependent manner. Delta variant showed a higher efficiency of replication in STB and CTB compared to Omicron BA.1, BA.2 and BA.5. In STB, despite a slight subsequent increase of type III IFN response, no correlation was observed between virus replication and the induction of the overall host response (including expression of entry receptors and immune response) after infection. In CTB, virus replication significantly correlated with the increased level of trophoblast cell fusion leading to syncytia formation. In line with increased STB formation \u003cem\u003ein vitro\u003c/em\u003e, we \u003cem\u003ein vivo\u003c/em\u003e observed an increase of syncytial knots release in early placenta infected by SARS-CoV-2 compared both to SARS-CoV-2- negative areas from the same placenta, and to age matched references. Altogether, our \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e data suggested that efficient replication of SARS-CoV-2 variants in placenta cells during early stage of pregnancy might alter STB turnover.\u003c/p\u003e","manuscriptTitle":"SARS-CoV-2 Delta and Omicron Variants alter Trophoblast Cell Fusion and Syncytiotrophoblast Dynamics: New Insights into Placental Vulnerability","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-21 12:44:47","doi":"10.21203/rs.3.rs-6246871/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"revise","date":"2025-05-07T10:17:34+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"This content is not available.","date":"2025-04-15T16:10:38+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2025-03-31T15:26:29+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewersInvited","content":"","date":"2025-03-31T15:19:13+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-03-18T11:25:33+00:00","index":"","fulltext":""},{"type":"submitted","content":"Cell Death \u0026 Disease","date":"2025-03-17T17:45:20+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-03-17T17:45:20+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"cell-death-and-disease","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"cddis","sideBox":"Learn more about [Cell Death \u0026 Disease](http://www.nature.com/cddis/)","snPcode":"41419","submissionUrl":"https://mts-cddis.nature.com/cgi-bin/main.plex","title":"Cell Death \u0026 Disease","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"5c18c83f-c9d4-4af3-ad2b-6c11faed8c33","owner":[],"postedDate":"April 21st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":46467974,"name":"Health sciences/Pathogenesis/Infection"},{"id":46467975,"name":"Health sciences/Medical research/Experimental models of disease"}],"tags":[],"updatedAt":"2025-10-08T07:08:04+00:00","versionOfRecord":{"articleIdentity":"rs-6246871","link":"https://doi.org/10.1038/s41419-025-08016-x","journal":{"identity":"cell-death-and-disease","isVorOnly":false,"title":"Cell Death \u0026 Disease"},"publishedOn":"2025-10-07 04:00:00","publishedOnDateReadable":"October 7th, 2025"},"versionCreatedAt":"2025-04-21 12:44:47","video":"","vorDoi":"10.1038/s41419-025-08016-x","vorDoiUrl":"https://doi.org/10.1038/s41419-025-08016-x","workflowStages":[]},"version":"v1","identity":"rs-6246871","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6246871","identity":"rs-6246871","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}