Transplacental transmission of SARS-CoV-2 infection

SARS-CoV-2 outbreak is the first pandemic of the century. SARS-CoV-2 infection is transmitted through droplets; other transmission routes are hypothesized but not confirmed. So far, it is unclear whether and how SARS-CoV-2 can be transmitted from the mother to the fetus. We demonstrate the transplacental transmission of SARS-CoV-2 in a neonate born to a mother infected in the last trimester and presenting with neurological compromise. The transmission is confirmed by comprehensive virological and pathological investigations. In detail, SARS-CoV-2 causes: (1) maternal viremia, (2) placental infection demonstrated by immunohistochemistry and very high viral load; placental inflammation, as shown by histological examination and immunohistochemistry, and (3) neonatal viremia following placental infection. The neonate is studied clinically, through imaging, and followed up. The neonate presented with neurological manifestations, similar to those described in adult patients.


5.
What was the reason for Apgar score going down from 4 to 2 if resuscitation was performed according to guidelines? 6. What were the reasons for POC echo and lung ultrasound examination? 7. What is the results of CSF RT PCR? What is detailed CSF report? 8. What is meant by placenta was extremely positive -was it swabs? Was it maternal side or fetal side? 9. How do authors explain signs of chronic intervillositis? If mother was infected only 5 days prior to birth?
2. There is a report of presence of viral genes in amniotic fluid, so this needs to be corrected. https://obgyn.onlinelibrary.wiley.com/doi/abs/10.1002/pd.5713.
3. I would refrain from using term "vertical" or "Horizontal" as it should be more or less mechanism based rather than direction based. 4. BAL methods reference is author's own paper where the method is not the investigation target. (reference 3 in online methods). 5. Before confirming the case as "confirmed" case there are several clarifications needed to be fulfilled a. What is the false positive test rate of this testing? Above cycle threshold of 30, what is the false positive rate? b. Crushed sample of placenta -suggest possibility of contamination by maternal blood -how can this be differentiated? c. Newborn blood and maternal vaginal swab has very high cycle threshold (to the level which could be considered even negative) -please elaborate more. d. Figure 3 has 3 Newborn NP swabs -which one is on which day? e. Confirmed case would require clinical features of infection as well -for which clinical phenotype is still unknown and what neurological findings baby had could be an association with possible birth asphyxia picture (low Apgar, need for resuscitation, need for respiratory support) etc.
6. Emphasis is given on transplacental transmission -which indicate that it look like peripartum transmission. Is this what authors intend to say? 7. Before neurological findings are ascribed to possibility of association with SARS-CoV-2 further information is needed a. What was baby's first blood gas and what recovery occurred in blood gas over time?
b. What was baby's first blood count? c. What was baby's clinical neurological examination at birth and immediately thereafter? d. What sedation/muscle relaxants baby received on first day? e. Were virological studies conducted on CSF sample? f. Please provide full CSF report for better interpretation.
8. Figure 4, panel A -arrow is not seen and how is it possible to see fibrin deposition macroscopically?
9. The placental histopathology revealed chronic histiocytic intervillositis, which is a chronic immunologic disorder associated with intrauterine growth retardation and miscarriage or stillbirth. How authors thinks it is associated with congenital infections especially viral infection. Where are the histiocytes located? Around the chorionic villi or elsewhere?
10. The significance of the cycle threshold (Ct) results for the PCR tests performed on the mother and child is not explained in the manuscript. First of all, for clinicians, it would be important to explain the inverse relationship of Ct number to viral load.
11. It will be useful in appendix to have table of investigations with cycle threshold values and an explanation that the viral loads (as estimated from Ct values) were correlate to ……etc.
12. Reference 16 is suggested for diagnosis of SARS-CoV-2 in neonates from China. Authors mention that their study meets this definition. Could you please clarify further. The paper indicates that baby should meet all four criteria; one of which includes "chest radiographs showing abnormalities, including unilateral or bilateral milled glass opacities". Please report any chest x-ray findings after intubation or soften that conclusion.
13. In the absence of any other organ involvement, too much emphasis is placed on MRI findings. This needs to be softened to an association.
14. What is inherent reliability of testing as indicated by manufacturer and field testing for this particular test and it need to be reported.
15. What treatment did baby receive? Did baby receive any antivirals?
16. What follow up data are available for baby and mother?
17. Would authors suggest change in timeline for testing of newborns born to mother who is SARS-CoV-2 positive, which currently is around 24 hours of age? based on their findings? General comment: 1. Change all reference to isolation of "SARS-CoV-2 gene target isolation" by RT-PCR and not "virus isolation".
2. Figure 1 -does not add much to paper and can be removed.
3. Table of laboratory findings may be more useful.

Reviewer #1
We thank the Reviewer for the thoughtful comment which was also shared by the Editor. As suggested, we have provided results of placental immunostaining for viral proteins (now added as Figure 5) and updated the main text and the online methods, accordingly. We feel that, thanks to this suggestion and the other details provided to Reviewer 2, we made a stronger case of transplacental transmission, the manuscript is much improved and we are very grateful to the Reviewer. Changes are provided in red font in the manuscript. We are willing to provide further clarification/amendments if needed.
1.Major point. As suggested, we performed immunostaining and we provide results in the main text, pag.5, 1 st par and in the new figure 5, pag.16. We also added all the viral loads for each analyzed specimen in figure 3.
All these data are consistent and support our conclusions. In fact, 1) maternal viremia occurred and the virus reached the placenta; 2) virus was found in placental tissue by immunostaining and the viral load in the placenta is much higher than elsewhere, that is, the virus is actively replicating in placental tissue with inflammatory reaction as seen by histological exam and immunohistochemistry; 3) neonatal viremia occurred following placental infection. Of note RT-PCR curves of NP swabs at 3 (while the baby was in full isolation in a negative pressure room) and 18 days of life are higher than that at the first day: this is also another confirmation that we observed an actual vertical infection, rather than a contamination.
Some of these considerations have been added in the main text, case study, end of pag.4-beginning of pag.5 and in discussion, end of pag.5-beginning pf pag.6 2.Minor point: Figures 2 and 4: arrows should be added to the figures, as indicated in the legends. We are sorry, arrows were already there but, for a technical problem, they were not visible when the figure was embedded in the word file. Problem solved now.

Reviewer #2
We thank the Reviewer for the thoughtful comments. We followed all his/her suggestions and modified the manuscript accordingly.
We took the liberty to detail the diagnostic process, as expert neonatologists, to explain while the observed neurological manifestations cannot be ascribed to any other disorder than SARS-CoV-2 infection; we anyway refer to these symptoms as "associated" without a direct cause-effect relationship, as suggested. We also provided all the required details of both clinical care and virological testing. Moreover, as suggested by the Editor, we have provided results of placental immunostaining for viral proteins (now added as Figure 5) and updated the main text and the online methods, accordingly.
• Absent baseline FHR variability and any of the following: -Recurrent late decelerations -Recurrent variable decelerations -Bradycardia

• Sinusoidal pattern
The FHR of our cases showed tachycardia, absent baseline variability, absence of accelerations and recurrent late decelerations and, thus, it fulfilled the criteria for category III FHR tracings, which is strongly associated with adverse neonatal outcome.
According to the classification of the Royal College of Obstetricians-Gynecologists and Anesthetists (new reference 8 in the manuscript), a C-section is defined as category-II if it is an urgent delivery because of maternal of fetal compromise which is not immediately life-threatening (see classification below): Therefore, we performed an urgent C-section based on the abnormal FHR tracing.
We described better this issue and provided some of these data in the main text, case study, pag.3 and in legend of Figure 1 (pag.11) which is an illustrative image taken from the FHR monitoring of our case, to help the average reader. We also added the reference to the classification of the Royal College of Obstetricians-Gynecologists and Anesthetists (now reference n.8 in the manuscript). We realize that these are very specialistic details that may be excessive or necessary according to the Reader's background. We are willing to add more detail if the Editor wish so.
4. Was the cesarean section performed due to maternal cough? It says maternal respiratory status but not indicated what was her status from respiratory perspectives? No. As explained above -see point 3-(and in the main text, case study, pag.3) the urgent C-section was indicated because of abnormal fetal heart rate tracing (altough the woman presented with severe cough and expectoration, for 5 days before the delivery). 5. What was the reason for maternal admission for 6 days after delivery? The mother remained hospitalized for six days after the delivery for surveillance of her clinical (particularly respiratory) conditions due to COVID19. Added in the main text, pag.3, 1 st par.
6. What was the reason for Apgar score going down from 4 to 2 if resuscitation was performed according to guidelines? The reason is related to the nature of Apgar score's items and the timing of the different resuscitation steps. The observed reduction is absolutely nothing strange and often occur. In detail: 1.The Apgar score is a classical tool evaluating 5 vital parameters (heart rate, respiratory activity, muscular tonus, skin color and grimace) giving 0-2 points for each item as shown in the table on the right: Our baby had an Apgar of 4 at 1 minute of life (in detail: heart rate=1, respiratory activity=1, skin color=1, muscular tonus=1, remaining items were coded zero). According to current guidelines (reference n.9 in the manuscript), non-invasive manual ventilation with face mask and T-piece resuscitator was started (with PEEP=5 cmH2O and peak pressure 25 cmH2O in room air, respiratory rate 40-45/min), while heart rate was monitored with ECG.
2. Our baby had an Apgar of 2 at 5 minutes (in detail: skin color=1, muscular tonus=1, remaining items were coded zero). This means that the non-invasive ventilation did not improve the situation, as the heart rate and the respiratory activity were no more evident. Thus, the neonatal fellow promptly intubated the baby (intubation verified by end-tidal CO2 measurement) and started invasive ventilation at 6 minutes of life. As usual, the invasive ventilation was very efficacious in resuscitating the baby (non-invasive ventilation does not always work for a number of reasons, such as patency of upper airways, difficult patient's positioning, lack of synchronization, leaks reducing pressure delivery…).
Thus, the Apgar at 5 minutes did not capture the effect of resuscitation as the main resuscitative maneuver (intubation and invasive ventilation) occurred at 6th minute. In fact, Apgar has been re-coded at 10 minutes of life and it was 7 (in detail: heart rate=2, respiratory activity=2, skin color=2, muscular tonus=1).
3. In its original formulation (still used worldwide) the Apgar score does not take any resuscitative maneuvers into consideration (although the American Academy of Pediatrics suggested to include negative points for each item depending on the type of resuscitative actions provided -AAP Position paper Pediatrics 2006; Rudiger M. NeoReview 2012). In other words, the classical Apgar score describes the newborn conditions regardless of any medical intervention. Thus, the Apgar score itself does not describe correctly the effect of neonatal resuscitation even when the resuscitative interventions are provided at the same moment of Apgar calculation.
We better described the Apgar scores of our baby and the resuscitation timing and details in the main text, cases study, pag.3, 2 nd par to help the average reader. We realize that these are very specialistic details that may be excessive or necessary according to the Reader's background. We are willing to add more detail if the Editor wish so.
7. What were the reasons for POC echo and lung ultrasound examination? According to our NICU clinical protocols, POC echo and lung ultrasound are routinely performed by attending/fellow neonatologists in all patients admitted to the NICU for cardio-respiratory compromise, suspected heart/lung malformation or for post-delivery room resuscitation care. Thus, our protocols fully comply with the "International evidence- In our case, this was obviously performed to detect any hemodynamic problem (impaired contractility, need for volume resuscitation, pulmonary pressure measurement) or respiratory disorders (post-resuscitation lung oedema, transient tachypnoea of the neonate, RDS, others…) A small explanation has been added in the main text, case study, pag.3, last par. and the international POCUS guidelines have been cited (reference 10) 8. What is the results of CSF RT PCR? What is detailed CSF report? Added as suggested (main text, case study, pag.4, 2 nd par. and in the new Table 1 reporting main laboratory findings) -see also below point 27,28 and 41 9. What is meant by placenta was extremely positive -was it swabs? Was it maternal side or fetal side? In the placenta, the terminal villi (composed of capillaries (fetal blood), fetal macrophages and fibroblasts and surrounded by trophoblast cells) are surrounded by the intervillous space containing the maternal blood. So RT-PCR performed on placental tissue specimens unavoidably contain both maternal and fetal components.
However, there was no placental swab, but a real sample of placental tissue was taken from the chorionic (fetal) side, crushed and homogenized to be analyzed by RT-PCR, as explained in online methods, pag.1, par.1 "A sample of placental tissues was taken from the chorionic side and crushed in 400 mL of RNAase-DNAase-free water". This ensure a fetal representative sampling.
Therefore, placental specimen used for RT-PCR techniques is collected from the chorionic (that is, fetal) side of the placenta, thus with predominance of fetal side. Moreover, the different viral load (viral load is extremely higher in the placenta than in maternal blood!) ensure that we did not observe a simple "contamination" but a true infection, as the virus replicates in the placental tissues.
We added some of these considerations in main text, case study, pag.4, last par and in Figure 3-Panel C and in Discussion, pag. 5-6 10. How do authors explain signs of chronic intervillositis? If mother was infected only 5 days prior to birth? Chronic intervillositis is not the only type of inflammation present in the placenta of our case. There is a polymorphonuclear cellular infiltrate composed of neutrophils, consisting with the acute phase of inflammation, associated with histiocytes appearing few days after the beginning of the process. As inflammation is a perpetual phenomenon it is common to observe an « active chronic inflammation » in placental tissues, such as here an « active chronic intervillitis ». Such phenomenon has been described in the placenta in case of viral infections, associated with recent infectious symptoms and is well known in feto-pathology. Of note, the mother was symptomatic for 5 days before the delivery but likely to be infected much earlier (impossible to know how long the period free from symptoms lasted).
We are willing to add some of these considerations in the main text if required by the Editor or the Reviewer.

What was trophoblastic involvement?
On morphological microscopic examination on HES stains, peri-villous trophoblastic cells (cyto-and syncytiotrophoblastic cells) showed no morphological alterations except in the territories of diffuse perivillous fibrin deposition with infarction. However, on immunohistochemistry, in the cytoplasm of the trophoblastic cells a strong positive signal was observed with the SARS-CoV-2 N-protein antibody. Fig.5 and in main text, case study, pag.5, 1 st par and commented in Discussion, pag.6, 1 st par.

This, as suggested by the Editor, confirms the presence of the virus in these cells/tissue and has been added in
12. Was viral gene isolated from placental tissues? Yes, as shown in figure 3 both E (fig.3A) and S (fig.3B) viral genes were found in placental tissue. These were found in very high amount as indicated by number of PCR cycles and by the viral load that has now been added to this figure as Panel C. It is also reported in the main text, case study, pag.4, last par -See also point 9 above (RT-PCR was performed on predominantly fetal side of placenta).
13. Discussion: Remove "first". Done, as suggested 14. There is a report of presence of viral genes in amniotic fluid, so this needs to be corrected. https://obgyn.onlinelibrary.wiley.com/doi/abs/10.1002/pd.5713. Done, as suggested. This has also been added to the reference list. This manuscript does not reduce the interest of our work, since these authors (as other already cited) did not systematically test placenta, amniotic fluid and mother/newborn blood in every mother-infant pair.
15. I would refrain from using term "vertical" or "Horizontal" as it should be more or less mechanism based rather than direction based. The Reviewer is totally right. Corrected throughout the manuscript 16. BAL methods reference is author's own paper where the method is not the investigation target. (reference 3 in online methods).
The Reviewer is right, as that paper target was not the BAL method validation. However, it is an ideal citation as it describes in detail the neonatal BAL technique used in our unit. We will describe it more in detail directly in the main text or in the online methods if the Editor or the Reviewer wish so.
The technique in use is also the commonest one in neonatal critical care and is used in the majority of research projects in the field and able to retrieve more fluid from alveoli and distal airways than other simpler techniques (Dargaville P. Am J Resp Crit Care Med 1999 -DOI:10.1164/ajrccm.160.3.9811048). We shall also remember that no gold standard technique for neonatal BAL exists (as acknowledged by the European Respiratory Society guidelines on pediatric BALreference n.4 of online methods), thus the most important thing is to describe which technique is used and that the technique would be correctly standardized and performed.
17. Before confirming the case as "confirmed" case there are several clarifications needed to be fulfilled What is the false positive test rate of this testing? Above cycle threshold of 30, what is the false positive rate? PCR is highly specific. False positive results are extremely uncommon, because negative controls are included in each run as provided by the kit manufacturer.

Anyway, the only samples with cycles above 30 are the vaginal swab (but the baby has been delivered by Csection), the amniotic fluid (but this is slightly above 30 and the placenta is way more positive) and the newborn blood (but the baby had several other positive samples demonstrating that he was actually infected -see points n.12-18 and 20)
Going more deeply in this area, the false positive rate of our RT-PCR technique has been recently estimated to be within 0.8 and 4% of cases (Cohen AN et al, posted on MedRxiV on May 1, 2020doi.org/10.1101/2020.04.26.20080911), but these low rates have an impact only when testing is done when prevalence is low.

Our case was observed in the worst moment of the COVID19 outbreak, in the Parisian area (Ile de France) which is amongst the more hardly hit in Europe (see https://www.santepubliquefrance.fr/maladies-et-traumatismes/maladies-et-infectionsrespiratoires/infection-a-coronavirus/articles/infection-au-nouveau-coronavirus-sars-cov-2-covid-19-france-et-monde#block-238896)
and in a case presenting other 10 extremely positive samples (well below 30 cycles) and with a high suspicion index (clinical symptoms typical of COVID for both the mother and the neonate).

The US Center for Disease Control and Prevention (https://www.cdc.gov/sars/guidance/f-lab/assays.html) states that: 1) the most common cause of false-positive results is contamination. 2) Liberal use of negative control samples in each assay and a well-designed plan for confirmatory testing (with more than one gene) can help ensure that laboratory contamination is detected. 3) Testing should be limited to patients with a high index of suspicion for having COVID19.
We do respect all these three conditions (we have negative and positive controls; all samples are positive for two viral genes; we have a lot of consistently positive different specimens from our case; there is a high clinical suspicion index) as described both in the main text (case study) and in the online methods,2 nd par, thus we can consider the possibility of false positive virtually zero. We added some of these considerations in Discussion, pag.5, 3 rd par.
18. Crushed sample of placenta -suggest possibility of contamination by maternal blood -how can this be differentiated? As described above (points n.9 and 11 and point 1 of Reviewer 1) we provided enough data to exclude the simple contamination: 1) placenta was sampled mainly on the chorionic (that is, fetal) side; 2) viral load in the placenta is extremely higher than in maternal blood, thus there is an active viral replication in placental tissues; 3) immunostaining for viral proteins confirms the presence of the virus in placental cells; 4) neonatal viremia was observed and there was no other likely transmission route (as amniotic membranes were intact and the baby was delivered by C-section).
19. Newborn blood and maternal vaginal swab has very high cycle threshold (to the level which could be considered even negative) -please elaborate more. RT-PCR in newborn blood and maternal vaginal were not negative since they were below the cycle threshold value of 40 as described in the online methods, beginning of pag.2. There are several reasons to consider this as positive: 1

According to the European Centre for Disease Control (ECDC -https://www.ecdc.europa.eu/en/alltopics-z/coronavirus/threats-and-outbreaks/covid-19/laboratory-support/questions), a high CT value (e.g. > 35) for the E-gene RT-PCR should be confirmed by a second gene target (to exclude a contamination) and all our samples from both the mother and the neonate were positive for two genes (the "E" and the "S" gene of SARS-CoV-2). Thus, our results were confirmed and interpreted according to ECDC guidelines.
Some of these considerations have been added in the main text, discussion, pag.5, 3 rd par 20. Figure 3 has 3 Newborn NP swabs -which one is on which day? NP swabs at 1, 3 and 18 day of life are represented by the light orange, grey and green curves, respectively. This has been now specified in Figure 3 legend. Of note RT-PCR curves of NP swabs at 3 and 18 day of life are higher than that at the first day (while the baby was in full isolation in a negative pressure room): this is also another confirmation that we observed an actual vertical infection, rather than a contamination.

Some of these considerations (together with those about the viral loads) have been added in the main text, discussion, beginning of pag.6
21. Confirmed case would require clinical features of infection as well -for which clinical phenotype is still unknown and what neurological findings baby had could be an association with possible birth asphyxia picture (low Apgar, need for resuscitation, need for respiratory support) etc. We respectfully disagree. Some of us are neonatologists with long experience and academic career (VZS and DLD), we can testify that the neurological findings of our baby are not linked to any other possible cause known so far. In detail the following were excluded: -Other infections (as LCR, blood and BAL were sterile for other bacteria, microbes and viruses).
- We added some of these clinical reasoning in the main text, case study, pag.4 and in Discussion, end of pag.6beginning of pag.7 also citing the statement of the Neurocritical Care Society (reference n.20 in the manuscript)

Consequences of perinatal asphyxia (since the baby was efficaciously resuscitated as proven by the first blood gas analysis showed normal pH and lactate. Moreover, Sarnat score, echocardiogram, blood test (including troponin, liver and kidney function were totally normal). Given these data the baby does NOT qualify 1) for the definition of significant perinatal asphyxia according to the American College of Obstetricians and Gynaecologists and the American Academy of Paediatrics (American Collge of Obstetricians and Gynecologists (ACOG). Neonatal Encephalopathy and Cerebral Palsy: Executive Summary. Obstet Gynecol 2004), nor for 2) nor for its hypothermic treatment (American Academy of
We anyway described these neurological manifestations as only "associated" to the SARS-CoV-2 infection, as suggested (that is, we refrained to indicate a direct causal link).

Emphasis is given on transplacental transmission -which indicate that it looks like peripartum transmission. Is this what authors intend to say?
Not actually, we focused on transplacental transmission which is a maternal-fetal (congenital) transmission occurred during the pregnancy. This is totally different from the intra-partum transmission which is an infection acquired during vaginal birth and also different from the post(peri)partum transmission which means an infection transmitted from the mother during mother/infant bonding or newborn care or breastfeeding and/or transmitted from the environment.
The differences between these transmission routes are well described in the International Classification of for SARS-CoV-2 infection in pregnant women, fetuses, and neonates, that we apply in clinical care and that is cited in the manuscript (reference n.14). According to this classification criteria, our case is a transplacental (congenital) infection in a live born neonate, as we described in Discussion, pag.5, 3 rd par.
We focused on transplacental transmission (i.e.: congenital) transmission occurred during the pregnancy, because this is what is really new and never clearly demonstrated so far.
23. Before neurological findings are ascribed to possibility of association with SARS-CoV-2 further information is needed What was baby's first blood gas and what recovery occurred in blood gas over time? As we described above (point n.21), blood gas analyses were normal and the baby did not qualify neither for the definition of significant perinatal asphyxia (American College of Obstetricians and Gynecologists (ACOG 25. What was baby's clinical neurological examination at birth and immediately thereafter? As we already explained in the main text, case study, end of pag.3, the neurological exam at the birth and in the first hours, classified according to Sarnat score was totally normal and this is why the baby was almost immediately extubated upon NICU admission. Sarnat score is the classical diagnostic tool internationally used for the evaluation of neurological conditions of neonates after delivery room resuscitation (Sarnat H et al, Arch Neurol 1976-10.1001/archneur.1976 26. What sedation/muscle relaxants baby received on first day? Absolutely nothing. The neonate did not receive any sedation/analgesia. This is our internal protocol to achieve a more detailed neurological examination in babies after delivery room resuscitation and is also recommended by the American Academy of Pediatrics-Committee on Fetus and Newborn, Pediatrics 2014 -https://doi.org/10.1542/peds.2014-0899 for the "virgin" background evaluation of babies potentially candidate to therapeutic hypothermia for perinatal asphyxiainduced encephalopathy). We better specified this in the main text, case study, end of pag.3 27. Were virological studies conducted on CSF sample? Yes, as we already wrote in the main text, case study, pag.4, 2 nd par, RT-PCR was done on two LCR obtained on DOL3 (that is when neurological examination started) and DOL5 and both were negative for SARS-CoV-2. Conversely, the first showed signs of inflammation (300 leukocytes/mm 3 and slightly raised proteins (1.49 g/L)). We have now better detailed this in the main text, case study, pag.4, 2 nd par. See also above point 8 28. Please provide full CSF report for better interpretation. We have now better detailed this in the main text, case study, pag.4, 2 nd par and added a specific table (Table 1-pag13), resuming the main laboratory findings. See also points n.8, 23 and 24 above.
Please find below all the LCR test results:
We are sorry, arrows were already there but, for a technical problem, they were not visible when the figure was embedded in the word file. Problem solved now.
Massive perivillous fibrin deposits are classically visible on gross examination of the cut sections of the placenta as irregular strands or masses of pale yellow-white induration and so it is in our case. We added this description in the legend of Figure 4.
30. The placental histopathology revealed chronic histiocytic intervillositis, which is a chronic immunologic disorder associated with intrauterine growth retardation and miscarriage or stillbirth. How authors think it is associated with congenital infections especially viral infection. Where are the histiocytes located? Around the chorionic villi or elsewhere? Intervillitis, is also commonly seen in different conditions such as viral infections (and not only in immunologic disorders), as already reported above (points n.9-10). Histiocytes, as well as neutrophils, are present in the intervillous space and are maternal inflammatory cells. There was no sign of villitis either acute or chronic in our case. Anyway, we did not intend to indicate intervillositis as a direct effect of SARS-CoV-2 infection but just to report the associate findings.
We are willing to add some of these considerations in the text if required by the Editor or the Reviewer.
31. The significance of the cycle threshold (Ct) results for the PCR tests performed on the mother and child is not explained in the manuscript. First of all, for clinicians, it would be important to explain the inverse relationship of Ct number to viral load. We thank the Reviewer and we have added a brief explanation in Figure 3 legend.
32. It will be useful in appendix to have table of investigations with cycle threshold values and an explanation that the viral loads (as estimated from Ct values) were correlate to ……etc. We added all the viral loads as a table (Panel C)  This allows to understand that 1) maternal viremia occurred and the virus reached the placenta; 2) viral load in the placenta is much higher than elsewhere, that is the virus is actively replicating in placental tissue with inflammatory reaction as seen by histological exam and immunohistochemistry; 3) neonatal viremia occurred following placental infection. Some of these considerations have been added in the main text, discussion, pag.6, 1 st par.
33. Reference 16 is suggested for diagnosis of SARS-CoV-2 in neonates from China. Authors mention that their study meets this definition. Could you please clarify further. The paper indicates that baby should meet all four criteria; one of which includes "chest radiographs showing abnormalities, including unilateral or bilateral milled glass opacities". Please report any chest x-ray findings after intubation or soften that conclusion. We thank the Reviewer as this was an inaccuracy from our side. That Chinese manuscript is quite "old" in this quickly evolutive pandemic situation: it was published in February, when extrapulmonary manifestations of COVID19 were not well known. Thus, the criteria reported by these Chinese colleagues are not adapted to our case as our baby did not have any respiratory disorder. We deleted this part and also the reference, as suggested.
34. In the absence of any other organ involvement, too much emphasis is placed on MRI findings. This needs to be softened to an association. This has been softened and indicated only as an association through all the manuscript.
35. What is inherent reliability of testing as indicated by manufacturer and field testing for this particular test and it need to be reported.