key: cord-0843493-nphzghk6 authors: Valdespino‐Vázquez, María Y.; Helguera‐Repetto, Cecilia A.; León‐Juárez, Moises; Villavicencio‐Carrisoza, Oscar; Flores‐Pliego, Arturo; Moreno‐Verduzco, Elsa R.; Díaz‐Pérez, Diana L.; Villegas‐Mota, Isabel; Carrasco‐Ramírez, Elba; López‐Martínez, Irma E.; Giraldo‐Gómez, David M.; Lira, Rosalia; Yocupicio‐Monroy, Martha; Rodríguez‐Bosch, Mario; Sevilla‐Reyes, Edgar E.; Cortés‐Bonilla, Manuel; Acevedo‐Gallegos, Sandra; Merchant‐Larios, Horacio; Cardona‐Pérez, Jorge Arturo; Irles, Claudine title: Fetal and placental infection with SARS‐CoV‐2 in early pregnancy date: 2021-04-06 journal: J Med Virol DOI: 10.1002/jmv.26965 sha: 9e2562244ed3e979cad2a76bfaf5827b460ea1d4 doc_id: 843493 cord_uid: nphzghk6 To date, mother‐to‐fetus transmission of severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2), responsible for the coronavirus disease 2019 (COVID‐19) pandemic, remains controversial. Although placental COVID‐19 infection has been documented in some cases during the second‐ and third‐trimesters, no reports are available for the first trimester of pregnancy, and no SARS‐CoV‐2 protein has been found in fetal tissues. We studied the placenta and fetal organs from an early pregnancy miscarriage in a COVID‐19 maternal infection by immunohistochemical, reverse transcription quantitative real‐time polymerase chain reaction, immunofluorescence, and electron microscopy methods. SARS‐CoV‐2 nucleocapsid protein, viral RNA, and particles consistent with coronavirus were found in the placenta and fetal tissues, accompanied by RNA replication revealed by double‐stranded RNA (dsRNA) positive immunostain. Prominent damage of the placenta and fetal organs were associated with a hyperinflammatory process identified by histological examination and immunohistochemistry. The findings provided in this study document that congenital SARS‐CoV‐2 infection is possible during the first trimester of pregnancy and that fetal organs, such as lung and kidney, are targets for coronavirus. The infection and multi‐organic fetal inflammation produced by SARS‐CoV‐2 during early pregnancy should alert clinicians in the assessment and management of pregnant women for possible fetal consequences and adverse perinatal outcomes. transcription quantitative real-time polymerase chain reaction, immunofluorescence, and electron microscopy methods. SARS-CoV-2 nucleocapsid protein, viral RNA, and particles consistent with coronavirus were found in the placenta and fetal tissues, accompanied by RNA replication revealed by double-stranded RNA (dsRNA) positive immunostain. Prominent damage of the placenta and fetal organs were associated with a hyperinflammatory process identified by histological examination and immunohistochemistry. The findings provided in this study document that congenital SARS-CoV-2 infection is possible during the first trimester of pregnancy and that fetal organs, such as lung and kidney, are targets for coronavirus. The infection and multiorganic fetal inflammation produced by SARS-CoV-2 during early pregnancy should alert clinicians in the assessment and management of pregnant women for possible fetal consequences and adverse perinatal outcomes. COVID-19, fetus, first trimester, miscarriage, placenta, pregnancy The coronavirus disease 2019 (COVID- 19) pandemic is a global public health emergency caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). 1 SARS-CoV-2 transmission from the mother to the fetus (termed vertical transmission) remains controversial [2] [3] [4] ; even though some cases have documented perinatal transmission assessed by serological analysis of positive neonates. [5] [6] [7] [8] [9] Accumulating evidence supports the transplacental transmission of SARS-CoV-2, with the detection of coronavirus presence in the maternal and fetal sides of the placenta [10] [11] [12] [13] [14] [15] (recently reviewed by Kotlyar et al. 16 ). To date, the most highly suggestive evidence for vertical transmission of SARS-CoV-2 is three case reports showing viral RNA in the placenta, fetal-intravascular mononuclear cells, 14 neonatal blood 9 and recently, SARS-CoV-2 RNA in fetal lung samples obtained by needle aspiration. 17 But no SARS-CoV-2 protein or virus particles have been detected in fetal tissues. Here, we present a case of fetal infection with SARS-CoV-2 during the first trimester of pregnancy with virus detection by polymerase chain reaction (PCR), immunofluorescence, and viral replication in fetal organs and placenta, associated with a hyperinflammatory process highly consistent with an in utero transmission of SARS-CoV-2. Tissues previously stored in RNAlater solution were disrupted through mechanical lysis and RNA was purified using the RNeasy Qiagen commercial kit (Qiagen). The SARS-CoV-2 RNA was detected following La Charité, Berlin protocol, 18 using TaqPath 1 step RT-PCR master Mix, CG commercial kit (Thermo Fisher Scientific), and probes and primers designed for RdRP and E viral genes. RNase P human gene was used as RNA isolation control. RT-qPCR was performed on a StepO-nePlus instrument (Applied Biosystems/Thermo Fisher Scientific). Each 20 µl RT-PCR reaction contained 5 µl enzyme mix, primers, and probes as indicated previously (La Charité) and 5 µl of RNA. Conditions at the thermocycler were previously reported. 18 A cycle threshold (C t ) value <38 was considered positive for SARS-CoV-2 RNA. C t values were collected using the threshold at 0.035 fluorescence level. Post-mortem fetal tissues were fixed in 10% formalin and embedded in paraffin for further histopathological examination by hematoxylin/ eosin stain and immunohistochemistry using monoclonal rabbit antibodies for CD163 (clone EP324, dilution 1:50-1:200) and CD68 (clone KP-1, dilution 1:100-1:500) (Bio SB Inc.). Tissue Sections (3 μm thick sections) prepared for antibody application were immersed in a citrate buffer and subjected to microwave irradiation. All primary antibodies were incubated with the tissue sections for 2 h at RT. Slides were then developed using an avidin-biotin-peroxidase method (Dako). Samples were fixed in 2.5% glutaraldehyde in the cacodylate buffer The case study was performed in agreement with the principles of the Declaration of Helsinki and following the CARE guidelines of the Equator Network (https://www.equator-network.org/reporting-guidelines/care/). The patient gave her written informed consent to the inclusion of material for publication, acknowledging they cannot be identified via this manuscript and that they are fully anonymized. gestational age), external habitus without congenital malformations, general edema, and congestive appearance. The weight of the lung was 1.08 g (according to gestational age) and of the heart was 20 mg (according to gestational age). Mild state of maceration. Fetus B appeared with a more evident macerated state. The placenta was bichorial, biamniotic. The placental weight was 25 g with measurements of 8 × 7 × 1 cm with parenchymal infarcts in 25% of the examined surface. Post-mortem fetal samples of lung, kidney, liver, heart, larynx, trachea, bowel, brain, and placenta were collected, and stored at −80°C until further analysis (several fetal and placental specimens were also stored in RNAlater solution). The woman had an adequate evolution and was discharged 24 h later. The patient was classified as a COVID-19 case according to a recent classification for maternal SARS-CoV-2 infection. 23 Furthermore, laboratory findings in the mother supported this diagnosis since the patient showed an 23 We next investigated the viral protein marker presence in three samples that suggest vertical infection: placenta, lung, and fetal kidney of fetus A. The analysis of confocal microscopy showed a positive signal to N viral protein of SARS-CoV-2 in the placenta and fetal tissues ( Figure 1A ). This result suggests that the virus accesses the tissues' cellular components. In an attempt to evaluate wether viral genome replication occurred in these tissues, we performed a stain to identify the replication intermediates of dsRNA viral using a monoclonal J2-dsRNA-specific antibody that has been widely used for replication studies, including SARS-CoV. 21, 22 Interestingly, the dsRNA signal was detected in the placenta, fetal lung, and kidney tissues indicating viral replication, and suggesting that virus genome synthesis take place in these tissues ( Figure 1B) . Immunofluorescence in other areas of the fetal organs analyzed revealed that some regions were negative for dsRNA or N protein staining, representing uninfected cells. We carried out the scanning electron microscopy with trans- We performed histological examination on paraffin-embedded tissues from fetus A organs and placenta A carried out with hematoxylin/eosin stain and immunohistochemistry to detect CD68+ and CD163+ inflammatory cells. The fetal heart showed striking evidence of endarteritis in small arteries (Figure 2A ) with CD68+ interstitial inflammatory mononuclear cells ( Figures 2B and 2E) , edema between cardiomyocytes and ischemic changes; the great vessels (pulmonary artery and aorta) showed no alterations. Both lungs were in the pseudoglandular stage, presenting reactive bronchial epithelium and hypercellularity composed of CD68+ inflammatory macrophages ( Figures 2C and 2F ). These were also seen in interstitium and pleura. Noteworthy, myositis was observed as a mononuclear inflammation between the fascicles of striated muscle cells of the neck, extremities and diaphragm, causing severe damage in fibers with apoptotic cells, atrophy, and intercellular edema ( Figure 2H ). Both kidneys were found intraluminal with scattered cellular detritus and mild interstitial inflammation with an enhanced infiltration of CD68+ cells ( Figure 2I ). The histological analysis of the placenta exhibited placental infarction, with diffuse perivillous fibrin, active chronic intervillositis, and subchorial inflammation ( Figure 2D ). The observed immunophenotypes were of CD163 positivity in the villous stroma and intervillous space ( Figure 2G ). We present novel evidence in support of intrauterine transmission of SARS-CoV-2 during early pregnancy (13 weeks), associated with miscarriage, from a COVID-19-infected woman. We detected SARS-CoV-2 N-protein and RNA as well as viral replication in fetal lungs and kidneys, and the placenta. Furthermore, viral particles consistent with coronavirus were observed by electron microscopy in fetal lung tissue adjacent to the alveolus region, within the cytosol of apoptotic and not well-preserved cells, and the placenta parenchyma. The size of these virions' structures was consistent with the size and appearance of SARS-CoV-2. 25, 26 As recently discussed, it is difficult to distinguish virus particles from normal subcellular organelles. Virions can be mistakenly identified with clathrin-coated vesicles or with heterogeneous vesicles in multivesicular bodies. 27 However, we are confident that the particle aggregates inside vesicles correspond to coronavirus as we have carefully compared them with normal cell organelles. In addition, histopathological findings of fetal and placenta tissues showed a hyperinflammatory process, which most likely led to miscarriage. Our findings at the histopathological level in the placenta are similar to those reported by Schwartz and Morotti. 28 The latter study defined that SARS-CoV-2 generates a common characteristic placental damage, which is due to the presence of a chronic histiocytic intervillositis (CHI). In this sense, a review by Wong et al. 29 30 Shah et al. 23 have proposed a classification of maternal, fetal, and neonatal SARS-CoV-2 infections taking into account maternal/perinatal tests. According to this classification, a confirmed congenital infection with intrauterine fetal death/stillbirth is defined by virus detection by PCR or viral particle detection by electron microscopy from fetal or placental tissue and requires that maternal status is either definitive or probable. Therefore, our Vertical infection by viruses is a process that depends on several factors, such as cellular permissiveness of the virus to the components of the placenta. 31 A study based on single-cell RNA-seq analysis suggests that the receptor of SARS-CoV-2, ACE2, is highly expressed in trophoblast and syncytiotrophoblast, which could promote the entry of the virus in these cell types, and facilitate the dissemination into the fetal circulation. 32 However, our result shows that replication events of the viral genome take place in the cells of the placenta, as we detect the presence of the replicative intermediate (dsRNA) that only occurs when the viral genome is synthesized. Therefore, it is likely that placental components are a region during the first trimester of pregnancy that allows the establishment of tissue areas of viral production, as observed with viruses such aszika or cytomegalovirus. 33 However, another possible placental transfer mechanism that has been described with other pathogens such as in Cytomegalovirus maternal infections, could be through neonatal Fc receptor-mediated transcytosis into syncytiotrophoblasts, cytotrophoblasts, and macrophages. 34 In our study, the most striking observation in the placenta was a prominent amount of macrophages (Hoffbauer cells). These cells are a normal component of the stroma of the chorionic villi of fetal origin, appearing in the chorionic villi at the 10th-18th days of gestation. An increased number of these cells is an abnormal condition that has been reported to occur as a result of a variety of pathological conditions associated with pregnancy, including ascending infections as well as maternal blood-borne infections that cause villitis, like syphilis or cytomegalovirus infections. 35 It is possible that proliferation of these cells occurs during infection with SARS-CoV-2, as observed with the Zika virus. 36 We could also hypothesize that these cells can be susceptible to infection with SARS-CoV-2, possibly allowing them to be a virus-transporting cell to regions of the placenta, and to the fetal capillaries for subsequent infection. 37 Evidence of macrophage responses activated in fetuses under stress (infections, pre-eclampsia), induced by maternal cytokines release, could explain the massive inflammatory response we observed in our study. 38 The evidence presented in this case report shows fetal organs are targets for SARS-CoV-2 and comply with a congenital infection associated with fetal death in early pregnancy. 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