Confirmatory evidence of the visualization of severe acute respiratory syndrome coronavirus 2 invading the human placenta using electron microscopy

To the Editors:

In May 2020, our article reporting on the visualization of the invasion of the human placenta by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) using electron microscopy, was published online in the American Journal of Obstetrics and Gynecology.¹ In a subsequent Reply to a Letter to the Editors, we provided further evidence to support the findings of our original article, proving that it was indeed SARS-CoV-2 virions that were visualized.² The evidence we have presented includes the following: (1) the virus was visualized with transmission electron microscopy showing that the extracellular structures that were visualized were identical to those seen within the cells, thus indicating that these were not clathrin-coated vesicles; (2) immunohistochemical analysis of the placental samples was positive for SARS-CoV-2 glycoprotein using a specific antibody in conjunction with positive and negative controls; (3) the virus was immunolocalized using immunogold electron microscopy; and (4) the virus was detected in the placenta using real-time polymerase chain reaction (RT-PCR)–specific primers. Our aforementioned findings are very consistent with subsequent reports that also documented the visualization of SARS-CoV-2.^{3 , 4}

With the current letter, we would like to provide additional information regarding the detection of the virus in our placental sample. A fresh piece of the placenta from our case was tested for SARS-CoV-2 by RT-PCR by our neonatal research team, led by Dr. Nazeeh Hanna, who is also conducting coronavirus disease 2019 related research, funded by the National Institute of Child Health and Human Development (NICHD), R01 HD098258-01A1. Fresh placental tissues were collected immediately after birth. The total RNA was isolated with a miRNeasy mini kit (QIAGEN Sciences Inc, Germantown, MD). The SARS-CoV-2 nucleocapsid (N) 1 and N2 genes were assayed for by a 2-step RT-PCR test. Reverse transcription was carried out using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA), with primers specific for the N1, N2, and RNase P genes.⁵ The presence of the viral RNA was assayed for using a 2019-nCoV RUO Kit (Integrated DNA Technologies, Coralville, IA) and a sample with a cycle threshold value less than 40 was considered positive. The results were positive for the presence of viral RNA in the placenta of our case report.¹

In conclusion, we want to emphasize that literature published after our case report¹ has shown unequivocally that there is scientific and clinical evidence that placental invasion by SARS-CoV-2 is valid.^{3 ,} 6, 7, 8 As a matter of fact, when vertical transplacental transmission occurs, the viral load in the placenta is severalfold higher than in the other maternal and fetal compartments.⁸ For all the reasons outlined in this letter, we are convinced that we have successfully reported on the visualization of SARS-CoV-2 in the human placenta.¹