Intrauterine SARS-CoV-2 infection – a case conrming transplacental transmission followed by divergence of the viral genome

Pregnant women and their newborns may be at particular risk for poor outcomes due to the coronavirus (COVID-19) pandemic. Intrauterine SARS-CoV-2 transmission has been suggested but most reports have not ruled out postpartum infection. We demonstrate a conrmed case of transplacental transmission of SARS-CoV-2 in a neonate born to a mother infected in the third trimester. Comprehensive virological, pathological and genetic investigations establish that intrauterine SARS-CoV-2 transmission occurred via the following chain of events; I) maternal viremia in a seemingly mildly symptomatic patient, II) high viral load in the placenta with massive perivillous brin deposition, acute intervillositis in areas with strong positivity for SARS-CoV-2 and chorangiosis in the areas less affected by infection and inammation, III) intrauterine fetal distress with pathological cardiotocography and acidemia in validated umbilical cord blood gases and IV) mild neonatal COVID-19. Whole genome sequencing of isolates from the mother and placenta revealed a single variant of the virus. Interestingly, the neonate displayed a mixed SARS-CoV-2 population, harboring both an identical strain to the mother as well as a population with one single-nucleotide polymorphism difference, indicating intrapatient genetic drift. of the viral genome. difference, indicating intrapatient genetic drift in the unique setting of intrauterine transmission. a phylogenetic tree, a whole-genome by multiple alignment fast transform (MAFFT) 43 and tree construction by IQ-tree 44


Introduction
The pandemic caused by the emergence of the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), causing a novel infection  in humans, has come at a tremendous cost to society and the healthcare sector in particular 1 . Pregnant women are more likely to need intensive care treatment related to COVID-19 as compared to non-pregnant women of reproductive age 2 .
COVID-19 infection has also been associated with a higher rate of preterm birth, preeclampsia, Cesarean section (CS), and perinatal death 3 . Neonatal COVID-19 infection are rarely reported and the possibility of a maternal-fetal transmission has previously not been clearly shown. A number of reports have suggested postpartum transmission 4 , but to the best of our knowledge, only Vivanti et al. 5 have convincingly reported a case of transplacental SARS-CoV-2 transmission. SARS-CoV-2 may be physically blocked by the placental barrier defense mechanisms, combated by immune-regulatory molecular pathways or, in the case of placental infection, mitigate a erce immune response that may potentially reduce the risk for fetal SARS-CoV-2 transmission 6 . The placenta is therefore of key interest in understanding perinatal transmission.
We present a comprehensive case study demonstrating the transplacental transmission of SARS-CoV-2 with rapid placental dysfunction and fetal distress, followed by con rmed neonatal COVID-19.

Results
Case presentation A 27-year-old woman (gravida 2, para 1) was transported with ambulance to the regional university hospital in gestational week (GW) 34 + 4 due to a three-day history of fever, abdominal pain and reduced fetal movements. She had developed a dry cough one day prior to the admission.
The woman, an immigrant of Turkish descent did not speak Swedish, was slightly overweight (BMI 27 kg/m 2 ) but otherwise healthy. She had normal antenatal controls during the current pregnancy. In 2016, she had a normal vaginal delivery in Turkey which was complicated by postpartum hemorrhage. During the current pregnancy, an obstetric ultrasound at GW 32 + 2 showed a normal fetal weight deviation of +8% 7 .
At admission, the patient was promptly isolated in a negative pressure room at the delivery unit and standard operating procedures and personal protective equipment (PPE) were used 8 . A combined nasopharynx (NPH) throat swab for SARS-CoV-2 real time reverse transcriptase quantitative polymerase chain reaction (RT qPCR) was obtained and normal vital parameters (apart from fever 38.3 degrees Celsius) were registered.
The admission cardiotocograph (CTG) test showed reduced baseline variability, absence of accelerations with recurrent prolonged, and late decelerations ( Figure 1). In light of the pathological CTG pattern, the obstetric team made the prompt decision to terminate the pregnancy by an immediate-emergency CS. Within eight minutes, the patient was anesthetized and intubated. An uncomplicated CS was performed in an operating theatre with negative pressure in line with the international recommendations for COVID-19 8 . The total blood loss was 200 mL. The amniotic uid was of normal amount and there were no signs of meconium staining, premature rupture of the amniotic membranes or blood in the amniotic uid. The placenta was easily detached.
The neonate was immediately transported to a separate room by a designated midwife equipped with sterile clothing and gloves. The neonate showed no initial signs of spontaneous breathing and was ventilated by neonatal staff. A maximum of 80% supplemental oxygen was needed to maintain adequate saturation. At six minutes of age, the neonate established spontaneous breathing and continuous positive airway pressure (5 cm H 2 O) was maintained for an additional 24 minutes, whereafter further ventilatory support was not needed. At one minute of age, the neonate had an Apgar score of 1 (heart rate = 1, remaining items = 0), at ve minutes of age Apgar 4 (heart rate = 2, muscular tonus = 1, re ex irritability = 1, remaining items = 0), and at ten minutes of age Apgar 8 (heart rate = 2, respiratory activity = 2, skin color = 1, muscular tonus = 2, re ex irritability = 1). Validated umbilical cord blood gases 9 from both cord artery and vein were retrieved showing a cord arterial pH of 7.20 and lactate 11 mmol/L (Supplementary table 2). The birth weight was appropriate for gestational age. Figure 2 illustrates the timeline of events for mother and child.

Maternal clinical course
After the CS, the mother was isolated in the postpartum ward and the NPH/throat swab taken upon admission returned positive for SARS-CoV-2. Analysis of maternal blood was also RT qPCR positive for SARS-CoV-2 (Table 1). Serology from the day of delivery revealed that the mother was weakly positive for immunoglobulin (Ig) M and negative for IgG (Table 2). Other maternal laboratory tests are shown in Supplementary table 1. Along with lymphocytopenia (0.7 x10 9 /L) and thrombocytopenia (98 x10 9 /L); in ammatory markers including c-reactive protein (36 mg/L), ferritin (340 µmol/L) and lactate dehydrogenase (9.5 µkat/L) were found to be elevated. The clinical condition of the mother improved and she was discharged four days after delivery with thromboprophylaxis (Tinzaparin 4500 IE subcutaneously once daily) for a total six weeks postpartum. A NPH/throat swab taken at day 9 postpartum returned negative for SARS-CoV-2 (Table 1). By day 11 postpartum, the mother was seropositive for anti-SARS-CoV-2 IgM and IgG (Table 2). Breast milk analyzed day 14 postpartum was RT qPCR negative for SARS-CoV-2 (Table 1), and further, at day 35 postpartum, negative for anti-SARS-CoV-2 total immunoglobulin ( Table 2).

Infection control measures
According to current national Swedish guidelines for newborns admitted to neonatal ward and with COVID-19 within the family 10 , a neonate can be cared for together with the parent if the required care and isolation measures are possible to provide. Due to a lack of isolation rooms, the neonate in the current case had no contact with any family member, including the mother, during the rst 60 hours of life. Since neither skin-to-skin care nor any other contact with the mother occurred, the neonate was regarded as noninfected, and neonatal staff did not use PPE. In accordance with national guidelines 10 , the neonate was tested for COVID-19 using a NPH swab 48 hours after delivery. This test returned positive for SARS-CoV-2 (Table 1) and the neonate was then regarded as contagious. Infection control routines were initiated to investigate a potential COVID-19 breakout at the neonatal ward but also to rule out the possibility of postpartum transmission. All staff that had tended to the neonate (n=27) and all nearby patients (n=4) were tested for COVID-19. The NPH/throat swabs for SARS-CoV-2 RT qPCR returned negative in all cases (data not shown).
Symptom surveillance in this group was continued for a further 14 days but no COVID-19 positive cases were discovered during this time.

Neonatal clinical course
Isolation was carried out as presented above. The neonate was admitted to the neonatal ward at 30 minutes of age due to transient asphyxia and prematurity. Apart from the initial resuscitation, no ventilatory support or supplementary oxygen was needed. During the rst day of life (DOL) blood gas analyses were performed mainly to monitor blood glucose levels (Supplementary table 3). Mild hypoglycemia (2.4 mmol/L) was found at 6 hours of life and it was managed successfully with oral glucose gel and intensi ed feeding of formula milk via a nasogastric tube.
As the routine RT qPCR test obtained at 48 hours of age returned positive for SARS-CoV-2 and since the clinical condition of the neonate was stable, the neonate was transferred and united with the mother at the postpartum ward isolation room at DOL 3 (60 hours after birth). Breastfeeding was thereafter initiated and the neonate did not receive any breastmilk before this time point. When the mother was later discharged from the postpartum ward at DOL 4, the neonate was transferred to an isolation room at the pediatric infectious disease ward. The mother was still present and breastfeeding was continued. During the entire hospitalization period, the neonate displayed no upper respiratory tract symptoms nor any signs of neonatal COVID-19. At DOL 5, it was observed that the neonate was undernourished and clinical examination revealed moderate signs of hypovolemia, slight ventilatory distress and a mild systolic heart murmur. Pulmonary auscultatory ndings were normal. A chest X-ray revealed no abnormalities (Supplementary gure 1). Blood tests including coagulation, liver and hematological analyses were found to be normal for the gestational age. C-reactive protein was not elevated (Supplementary table 3). Following intensi ed breastfeeding, the hypovolemia, heart murmur and respiratory distress subsided by DOL 7. The neonate was discharged at DOL 15 and was being fully breastfed. A follow-up control at DOL 35 con rmed that the neonate had no respiratory or feeding problems. The physical examination was normal apart from a small, left-sided, asymptomatic, inguinal hernia and additional blood tests revealed no abnormalities (Supplementary table 3).
Regarding the neonatal COVID-19 infection, repeated RT qPCR analyses showed the lowest CT-value at DOL 5 where after a gradual increase was seen. By DOL 20, SARS-CoV-2 was not detectable in NPH or throat swabs (Table 1). Further, serology revealed that the neonate was anti-SARS-CoV-2 IgG negative at DOL 7 (IgM not analysed due to lack of material). At DOL 14, IgM was positive and IgG still negative and at DOL 20, the neonate was both IgM and IgG seropositive ( Table 2).

Viral genome sequencing
To determine the genetic clade and to fully investigate the viral genetic similarities, virus isolates from the mother (NPH/throat swab obtained on the day of delivery), and neonate (NPH swab obtained at 48 hours of age, labelled DOL 2, and further at DOL 5) as well as from placental tissue, were sent to the Public Health Agency of Sweden for whole-genome sequencing. Next-generation sequencing of samples produced several full length 29 903bp, SARS-CoV-2 genomes, all belonging to the genetic clade 20B/GR/B.1.1 11 . All four sequences showed high identity, which is illustrated in a phylogenetic tree (Supplementary gure 2). Further sequencing data analysis identi ed 12 variant positions in the sequences from isolates of the mother and placenta compared to the SARS-CoV-2 reference genome (NC_045512). These variants were also present in the sequences of the neonate isolates (Table 3). Notably, an additional variant, A107G, was identi ed in the neonate samples but only present in 67 and 80%, respectively, of the sequences.

Placental pathology
The placenta was easily detached from the uterus during the CS. The remaining umbilical cord stump had a central insertion, was 9 cm long with a diameter of 1 × 1.5 cm and contained three vessels. The membranes had normal color without signs of meconium staining. The trimmed weight of the placental disc was 342 grams, within the 10 th to 90 th percentile for GW 34+0 to 34+6 12 . At gross sectioning, brin depositions were evident as glistening white-grey-pink con uent lesions, encompassing approximately 50% of the total placental volume ( In summary, three main histopathological features were found; I) massive perivillous brin deposition, II) acute intervillositis in areas with strong positivity for SARS-CoV-2 and III) chorangiosis in the areas less affected by infection and in ammation.

Discussion
Vertical transmission is one of the major complications of viral diseases during pregnancy 13 . The previous coronavirus outbreaks of the SARS-CoV-1 and the Middle East respiratory syndrome coronavirus (MERS-CoV) have indicated that pregnant women and their fetuses are particularly susceptible to poor outcomes 14,15 . The current study presents clinical, genetic, and histopathological evidence of intrauterine SARS-CoV-2 transmission.
Several studies have found SARS-CoV-2 in placental tissue, amniotic uid and in cord blood [16][17][18] . To the best of our knowledge, intrauterine transmission has been conclusively proven in one case report by Vivanti et al. 5 , in which the placenta was shown to carry a high viral load and clinical data suggested placental dysfunction. Following delivery, the neonate was critically ill and SARS-CoV-2 infection could be identi ed by early invasive sampling 5 . In another study, Facchetti et al. 19 analyzed the placentas from 101 women and found SARS-CoV-2 proteins expressed in only of them. This placenta originated from a COVID-19 pregnant woman whose newborn tested positive for viral RNA soon after birth, however, it was unclear if postpartum neonatal infection had been ruled out. Similarly, severe placental pathology due to COVID-19 has been implicated in another case report that led to adverse neonatal outcome despite mild maternal disease 20 . Transplacental transmission of the virus can therefore have dire intrauterine consequences for the fetus in seemingly mildly symptomatic mothers secondary to masked placental dysfunction.
In the present case, apart from being overweight, the mother did not speak Swedish. Socio-economic risk factors have been associated with more severe COVID-19 due to a general higher prevalence of medical problems 21,22 . The mother presented with classic COVID-19 symptoms including fever and a dry cough 23 but abdominal pain and reduced fetal movements were also reported.
Similar to previous reports, we observed that the clinical condition of the mother improved rapidly after delivery 24,25 . Blood tests taken from the mother on the day of delivery revealed discreet liver injury. Transient elevation of serum aminotransferases has often been observed in severe COVID-19 cases 26,27 , however the exact mechanism for direct SARS-CoV-2 related hepatocyte injury is still unknown. The mother also presented with elevated concentrations of several acute phase proteins including ferritin, procalcitonin and c-reactive protein, indicating systemic in ammation 28 . In addition, at the time of delivery, SARS-CoV-2 RNA was found in the maternal blood and RT qPCR indicated the highest viral load within the placenta. RT qPCR does not produce an exact quanti cation of viral load as different materials are analyzed. However, the CT-values were clearly the lowest in the placental specimen and histopathological placental analyses indicated high levels of SARS-CoV-2. Viral protein was found in the villous cytotrophoblasts, in the syncytiotrophoblasts and massive perivillous brin deposits covered over 50% of the placenta. Viremia in the blood is rare.
According to Wang et al. 29 SARS-CoV-2 RNA was found in only 1% of blood samples taken from COVID-19 patients. The placental histopathological changes seen in this case are similar to several previous reports on SARS-CoV-2, as well as SARS-CoV-1 and MERS-CoV 5,15,19,20,30 .
The neonate in the current case suffered from transient asphyxia attributed to intrauterine hypoxia secondary to placental dysfunction. This was signaled by the pathological CTG registering and validated umbilical cord blood gases revealed a cord arterial and venous pH well below normal median reference values 9 , along with high cord lactate values 31 . Following initial resuscitation, only standard supportive care of prematurity was needed. No evident signs of COVID-19 were observed. The mild respiratory distress and systolic heart murmur observed at DOL 6 was attributed to hypovolemia and promptly relieved after intensi ed feeding. Blood tests found no abnormalities and repeated RT qPCR testing revealed the lowest CT-values at DOL 5, suggestive of the highest in the upper respiratory tract at this time point. The CT-values later increased and by DOL 20, SARS-CoV-2 RNA was not detectable. Consistent with the observed viral clearance, neonatal IgM and IgG seroconversion was found. Previous knowledge of immunoglobulin transfer during pregnancy along with new data from the current COVID-19 pandemic con rm that anti-SARS-CoV-2 IgG can pass through the placental barrier whilst IgM does not 32,33 . In the current case, maternal serum was weakly positive for IgM and negative for IgG at the day of delivery. Thus, transplacental transfer of anti-SARS-CoV-2 immunoglobulin was not likely and we therefore conclude that the neonate seroconverted by its own means. The possibility of the neonate acquiring COVID-19 postpartum was ruled out by vigorous testing of all staff that had been in contact with the neonate during the rst 48 hours of life, as well as surrounding patients and their attendees. Secondary symptom surveillance for two weeks revealed no new cases.
To fully determine viral genome similarities between the mother, neonate and the placenta, whole-genome sequencing was performed. All four isolates revealed 29 903bp SARS-CoV-2 genomes, belonging to the genetic clade 20B/GR/B.1.1. Further analysis of the sequencing data showed that the isolate from mother and placenta had 11 single-nucleotide polymorphisms (SNPs) and one multiple-nucleotide polymorphism (MNP) differences compared to the reference Wuhan genome of SARS-CoV-2. Interestingly, the two neonate isolates, from DOL 2 and DOL 5, both had a mixed population of the virus. In addition to a population of the virus with the same genotype as the isolates from the mother and placenta, the neonate isolates contained another population of virus (80% in DOL 2 and 67% in DOL 5) with an additional SNP, e.g. A107G. Intrapatient genetic variation has previously been described in both MERS-CoV and SARS-CoV-2 34,35 . To the best of our knowledge, this is the rst case of ongoing genetic change in neonatal COVID-19 in the unique setting of intrauterine transmission. Possibly, the observed genetic drift is a response to a change in the external environment for the virus. Overall however, all virus isolates from mother, child and the placenta, displayed a clear similarity and shared a majority of the SNP's. The SNPs at position 107, 9871, 23851 and 24292, respectively, are rare in the context of sequenced isolates, and have only been described occasionally. Speci cally, the SNP at position 9871 is rare among sequenced isolates in international databases, and has only been described in one additional isolate among data currently available 11,36 The phylogenetic representation of the viral sequences from mother and neonate show a separation, although not at more than a distance of 0.0003 (distance measured as substitutions per site). These ndings are in accordance with the ndings that the neonate had a mixed population, with a major population having acquired a single SNP compared to the mother. The closest related sequence (three SNP difference) is from a case collected in Lund, Sweden, during the summer, in July, placing similar but not identical sequences in the geographical vicinity of the patient 11 .
Given these genetic ndings and the series of events presented above, along with the marked placental pathology and the high viral load, it can therefore be concluded that the neonate was infected in utero. The two main clinical lessons that can be learnt from the current case are; I) COVID-19 during pregnancy may cause severe placental dysfunction and fetal compromise and II) intrauterine SARS-CoV-2 transmission may not necessarily lead to severe neonatal outcome.

Patient sampling
Blood samples, NPH and throat swabs from the mother and neonate were collected according to clinical praxis. Placental tissue for RT qPCR analysis was obtained by a triangulate section reaching the core of the placental disc and the remaining placenta was kept in formalin. Breastmilk was collected day 14 and 35 postpartum for RT qPCR and Ig analysis. NPH swabs were collected from 27 healthcare workers at the Neonatal ward Skåne University Hospital, Malmö, Sweden. Sample collection, processing and laboratory testing followed guidance from Skåne University Hospital, Department of Clinical Microbiology 37 and The Swedish Public Health Authority 38 . PCR SARS-CoV-2 RNA was detected by RT qPCR or using the Panther Fusion open access platform (Hologic, Belgium), in both cases with primers and probe targeting the envelope gene designed by Corman et al. 39 .The RT qPCR was performed with the following modi cations: the thermal cycling program was 48°C for 10 minutes and 95°C for 10 minutes followed by 45 cycles of 95°C for 15 seconds and 55°C for 45 seconds. All RT qPCR were performed using the Path-ID multiplex one-step RT PCR kit and the Applied Biosystems 7500 fast instrument (Thermo Fisher, USA).

Sequencing
Virus isolates from NPH/throat swab from the mother, NPH swabs from the neonate (obtained at DOL 2 and DOL 5), and placenta, were sent to the Public Health Agency of Sweden. Samples were extracted (MagLEAD system, Precision System Science, Japan) and subjected to RT qPCR analysis of the RdRp gene of SARS-CoV-2 genome 39 . cDNA was synthesized by SuperScript IV VILO Master Mix (Thermo Fisher Scienti c) and used as template for target ampli cation reactions using two primer pools included in the Ion AmpliSeq SARS-CoV-2 Research Panel (Thermo Fisher Scienti c) together with 12 spike-in primers for the panel. The targeted ampli cation and subsequent library preparation steps were performed automatically on an Ion Chef system (Thermo Fisher Scienti c) using Ion AmpliSeq Kit for Chef DL8 (Thermo Fisher Scienti c). The amplicon libraries were quanti ed as previously published 40 and used for template preparation and Ion 540 chip loading on an Ion Chef system and sequenced on Ion GeneStudio S5 Prime system (Thermo Fisher Scienti c). Extracted samples were also sequenced and analyzed on the Ion Genexus system (Thermo Fisher Scienti c) using the assay Ion Ampliseq SARS-CoV-2 Research Assay 2.0.

Sequence analysis
The resulting data from the sequencing was processed using the torrent mapping alignment program (TMAP), mapping the sequences towards the reference genome of SARS-CoV-2 (Accession no NC_045512) 41 . After initial mapping, a variant call was performed using the Torrent Variant Caller. The consensus sequence for the viruses was acquired by ltering major frequency alleles from the variant call le (VCF) and apply them to the reference sequence, using bcftool 42 . The COVID19AnnotateSnpEff plugin (Thermo Fisher Scienti c), was used to generate an annotated list of variants of the samples. The two sequences were compared using both the VCF results as well as phylogenetically, both by classi cation by Nextstrain, GSAID and Pangolin classi cation systems as well as by constructing a phylogenetic tree, using a whole-genome alignment by multiple alignment using fast fourier transform (MAFFT) 43 and tree construction by IQ-tree 44 .

Histopathology and immunohistochemistry
The placenta was subjected to histopathological examination, including careful dissection and macroscopic, and microscopic of the cord, membranes and placental discs. Hematoxylin-eosin staining was done according to routine automated procedures. Immunohistochemistry was by standard methods for myeloperoxidase using a polyclonal antibody (Catalog no. A0398, Dako) at 1:2500, for CD68 clone PG-m1 (M0786, Dako) at 1:100, for CD3 the clone 2GVG (760-4341, Ventana) prediluted, for CD20 the clone L26

COMPETING INTERESTS
The authors declare no competing interests.

ETHICS
The mother and father have provided written informed consent to publication, available upon request. The case study was performed in agreement with principles of the Declaration of Helsinki.
Variant analysis and annotation of whole-genome sequencing data from virus isolates obtained from the mother and placenta at delivery and from the neonate at day of life (DOL) 2 and DOL 5. The table shows all identi ed variants, single-nucleotide polymorphisms (SNP) or multiple-nucleotide polymorphisms (MNP), with corresponding nucleotide position in reference genome of for severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) (NC_045512), the frequency (%) of the variant in the sequencing data and their gene-based annotation.