Coronavirus Disease 2019 (COVID-19) During Pregnancy: Prevalence of Seroconversion, Effect on Maternal and Perinatal Outcomes and Risk of Vertical Transmission (COVID-MAP)
Conditions: COVID-19
Sponsor: Chinese University of Hong Kong

 
 
 
 
Study Protocol 
 
Official title: 
Coronavirus disease 2019 (COVID-19) 
during pregnancy: prevalence of 
seroconversion, effect on maternal and 
perinatal outcomes and risk of vertical 
transmission 
 
NCT number: TBC 
 
Document Date: 02 Jul 2020 
 

Title of Project 
Coronavirus disease 2019 (COVID-19) during pregnancy: prevalence of seroconversion, effect 
on maternal and perinatal outcomes and risk of vertical transmission 
Keywords: 1) COVID-19; 2) pregnancy; 3) vertical transmission; 4) pregnancy loss; 5) ACE2 
TMPRSS2 
Project objectives 
1) To determine the rate of SARS-CoV-2 seroconversion in unselected pregnant women in Hong 
Kong. 
2) To determine the rate of SARS-CoV-2 infection in women presenting with miscarriage and 
stillbirth. 
3) To follow the pregnancy course and perinatal outcome of confirmed COVID-19-infected 
pregnant cases. 
4) To determine the risk and characteristics of vertical transmission. 
5) To evaluate the placental barrier, immune response and fetal damage in vertical transmission 
of SARS-CoV-2. 
 Background of research and expected project commencement date 
Emerging infections have been shown to have an important impact on pregnant women and their 
fetuses1 . Recent examples include the 2009 pandemic H1N1 influenza virus 2 and the Zika virus3-
5. The emergence of a novel coronavirus not previously seen in humans, namely severe acute 
respiratory syndrome coronavirus 2 (SARS-CoV-2), was first reported in Wuhan, China, on 
December 31, 2019. The coronavirus disease 2019 (COVID-19), caused by the SARS-CoV-2, was 
declared by the World Health Organization (WHO) a pandemic on 11 March 2020. Since then, the 
number of reported cases has increased rapidly, with more than 10 million cases and 500000 
deaths as of 1 July, 2020. 
 
Coronaviruses are single-stranded RNA, non-segmented, enveloped viruses, which cause illness 
ranging in severity from a common cold to severe and fatal illness. The term coronavirus derives 
from the Latin word corona, which means crown or halo; the designation arises from the 
appearance of coronavirus virions viewed by electron microscopy (EM), in which the virus 
particles display a crown-like fringe typically referred to as spikes. In the past 2 decades, two 
notable coronaviruses, severe acute respiratory syndrome coronavirus (SARS-CoV) and the 
Middle East respiratory syndrome coronavirus (MERS-CoV), have caused severe respiratory 
illness in humans. Within a short period of time, the third coronavirus, SARS-CoV-2, has infected 
far more people than were reported for MERS-CoV and SARS-CoV combined. 
 
It is recognized that pregnant women are at an increased risk of acquiring viral respiratory 
infection and developing severe pneumonia due to the physiologic changes in their immune and 
cardiopulmonary systems6,7. It is important to determine the effect of the SARS-CoV-2 infection 

and its disease (COVID-19) on pregnant women and their fetuses as careful risk-benefit analysis 
and decisions regarding treatment and interventions during pregnancy are crucial. It is critical to 
acquire high-quality data on maternal and perinatal outcomes and risk of vertical transmission. 
The first study describing the clinical characteristics and investigating the possibility of vertical 
transmission of SARS-CoV-2 in 9 pregnant women with laboratory-confirmed COVID-19, 
demonstrated that the severity of COVID-19 in pregnant women was similar to that in non-
pregnant adults and that there was no evidence of vertical transmission when the infection 
manifests during the third trimester of pregnancy as SARS-CoV-2 was not detected in amniotic 
fluid, cord blood and neonatal throat swab samples in six cases8. However, there remain many 
unanswered questions concerning mother-to-child-transmission of SARS-CoV-2.  
 
Several studies have attempted to determine the potential risk of vertical transmission, however, 
relevant biological samples have not been collected in a systematic manner9-18. Paired maternal-
perinatal testing i.e. with vaginal, cervical or rectal swabs, has been performed to detect genital 
tract viral shedding during vaginal delivery in 22.5% of the case-series. Breast milk has also been 
tested for SARS-CoV-2 using reverse transcription polymerase chain reaction (RT-PCR) in 22.5% 
of the studies. Five studies have reported immunoglobulin (Ig)G and IgM serology in the mothers 
and the neonates10,12,19,20with additional cytokine assays19,21. Several studies have evaluated the 
placenta for infectious pathology. Twenty-five percent of these reports have documented 
neonatal SARS-CoV-2 infection10,16,19,20,22-25. Reports from Italy, Peru and Iran attributed neonatal 
infection to horizontal transmission from RT-PCR positive mothers via breastfeeding without 
masks (respiratory droplets) and vaginal delivery (exposure to genital secretions).16,20,24 
 
Two reports suggested possible transplacental infection due to the presence of neonatal IgM 
antibodies and abnormal cytokine levels after birth, since IgM is not transmitted transplacentally 
and may have been produced by the fetus in response to in utero exposure to SARS-CoV-210,19. 
In both reports, RT-PCR of neonatal nasopharyngeal swab and maternal vaginal secretions were 
negative. Specifically, in the study of Dong et al., the observed decline of anti-SARS-CoV-2 IgG 
and IgM levels from 140.32 AU/mL (normal range is 0–10 AU/mL) and 45.83 AU/mL (normal 
range is 0–10 AU/mL), respectively, at 2 hours after birth to 69.94 AU/mL and 11.75 AU/mL, 
respectively, at 14 days of age, is not consistent with the typical profile of the body's antibody 
response to acute viral infection10. The rapid decline of anti-SARS-CoV-2 IgG antibody level in the 
infant within 14 days, in addition to the decline in IgM antibody level, strongly indicates that the 
neonatal anti-SARS-CoV-2 IgG antibodies were derived transplacentally from the mother and 
their production was not actively induced by the presumed neonatal infection.  
 
Nevertheless, there are several limitations in addressing the issue of vertical transmission as most 
studies reported infections in the third trimester where the interval between infection and 
delivery is relatively short. The risks of transplacental infection in the first and second trimesters, 
with a potentially longer fetal exposure time, are unclear. While most reported cases have been 
delivered by Cesarean section, the evidence does not suggest a clear association between 
neonatal SARS-CoV-2 and vaginal delivery.  
 
With regard to whether SARS-CoV-2 infection or its disease is associated with pregnancy loss, 

existing evidence is unclear. Limited data on SARS-CoV (2003) reported a high first trimester 
miscarriage risk of 50%26. A recent high-quality systematic review evaluating the effect of COVID-
19 on perinatal outcomes reported that there were only four reported cases of miscarriage 
among 295 pregnancies affected by COVID-1915, suggesting that the miscarriage risk is not 
significantly increased by COVID-19. However, it is likely that cases of miscarriage associated with 
COVID-19 have been missed or underreported because pregnancy symptoms can mask COVID-
19 symptoms, such as myalgia and fatigue. There was a case report of second trimester 
miscarriage associated with SARS-CoV-2 infection, where the virus was isolated in the placenta 
but not in the abortus9. This case raised the possibility of placental infection with SARS-CoV-2. 
Though there was no evidence of vertical transmission, absence of the virus in the abortus was 
not surprising given the short interval between maternal infection and miscarriage. Whether 
SARS-CoV-2 crosses the placental barrier warrants further investigation. In a non-consecutive 
case series of 9 cases of severe COVID-19 there were two pregnant women with intrauterine fetal 
death27. Further, latest evidence has demonstrated that placentas from pregnant women 
affected by COVID-19 show patterns of placental injury reflecting abnormalities in oxygenation 
within the intervillous space associated with adverse perinatal outcomes, such as stillbirth, fetal 
growth restriction (FGR)28. Based on data from the SARS-CoV (2003) case-series, 80% patients 
who presented after 24 weeks had preterm birth and 2 mothers recovered without delivery, but 
their ongoing pregnancies were complicated by FGR. It is important to determine if SARS-CoV-2 
infection increases the risk of pregnancy loss, preterm birth, FGR or other pregnancy 
complications26.   
 
Emerging evidence suggests that a significant proportion of SARS-CoV-2 carriers are 
asymptomatic29-31. This poses significant challenges in regard to the management of pregnancy 
and childbirth during the COVID-19 pandemic.  Pregnancy symptoms can mask COVID-19 
symptoms, especially if they are mild. Using the approach of targeted testing based on a 
significant epidemiological history or symptoms, a cohort study of 116 pregnant women with 
COVID-19 in China demonstrated that 23.3% of cases were asymptomatic32. Using the approach 
of universal testing by RT-PCR in women admitted for delivery, the rates of positive SARS-CoV-2 
result in asymptomatic women were 13.5% and 2.9% in and outside highly endemic regions in 
the United States, respectively, and 3.8% in Tokyo, Japan31. 
 
In this proposal, we plan to perform a systematic assessment of seroconversion, maternal and 
perinatal outcome and risk of vertical transmission of SARS-CoV-2 infection during pregnancy.  
 
 Research plan and methodology 
 
The research project will engage in collaborative research across obstetricians/maternal-fetal 
medicine (MFM) subspecialists (Prof Poon, Prof Leung, Prof Yang, Dr Gil, Dr Chaemsaithong), 
gynecologist (Prof Chung), infectious disease specialists (Prof Chan, Prof Ng), pediatrician (Prof 
S 
Lam), 
developmental 
and 
molecular 
biologists 
(Prof 
Wang, 
Dr 
Leung) 
and 
biostatistician/bioinformatician (Prof Sahota) between the CUHK, China and Spain.  
Objective 1: Seroconversion during pregnancy 

Methodology: a longitudinal study. Inclusion criteria: (1) pregnant women who attended for 
Down syndrome screening (DSS) at 11-13 weeks and had a serum sample (A) taken between 1 
November 2019 and 1 June 2020; (2) consented for stored serum for future research; and (3) 
intended to deliver at the booking hospital. Ethics approval at Prince of Wales Hospital (PWH) 
has been acquired (CREC Ref 2020.214). A list of eligible patients has been identified from our 
DSS database. We have started recruiting eligible patients through telephone call. An alert is set 
within the Hospital Authority Clinical Management System and therefore when the participant 
attends for delivery, we will confirm informed consent and collect the second serum sample (B), 
which will be stored at -80oC for subsequent testing. Ethics approval at Kwong Wah Hospital 
(KWH) has been submitted. The research team believes that it is critical to utilize stored serum 
samples from women who attended for routine DSS between November 2019 and June 2020 as 
this will capture the seroconversion data from the start of the outbreak until after the peak of 
the outbreak. Assuming a worst-case scenario of SARS-CoV-2 penetrance of 1% we estimate that 
2400 women will need to be recruited from amongst the 4800 women who attended for DSS 
during the study period. For all consented participants, paired blood samples A and B will be 
analyzed for anti-SARS-CoV-2 specific antibodies (Elecsys Anti-SARS-CoV-2, cobas e411 analyzer, 
Roche Diagnostics GmbH, Mannheim, Germany). Results on the rate of seroconversion will be 
summarized in counts and percentages. Target achievement: we aim to recruit 2400 unselected 
pregnant women to quantify the rate of seroconversion of SARS-CoV-2 during pregnancy from 
the start until after the peak of the COVID-19 outbreak in Hong Kong. This will allow us to 
determine the rate of women of childbearing age who have potentially been infected with SARS-
CoV-2, but have remained asymptomatic or mildly symptomatic and undetected. 
 
Objective 2: SARS-CoV-2 and pregnancy loss  
Methodology: a cross-sectional study. We plan to recruit pregnant women presenting with first 
and second trimester miscarriage in Hong Kong and Spain, as well as those with stillbirth during 
periods of 6 and 12 months, respectively. The overall rates of clinical first trimester miscarriage, 
second trimester miscarriage and stillbirth are approximately 10%, 1% and 0.1-0.3%, respectively. 
We plan to recruit first trimester miscarriage cases undergoing surgical management in order to 
minimize the risk of contamination. Regarding second trimester miscarriage, a mixture of 
spontaneous, medical and surgical management cases will be recruited as the latter is not 
frequently performed due to the recognized increased risk of uterine perforation and cervical 
laceration. For cases of miscarriage, we anticipate a 50% uptake and accounting for women 
seeking private care (70%), we aim to recruit approximately 500 cases. Regarding stillbirth, during 
a 1-year period we anticipate a 50% uptake and aim to recruit 5 cases. Biological samples to be 
collected and tested are listed in Table 1. For first trimester products of conception (POC), 
specimen will be washed in cold saline or PBS to remove blood, which will then be dissected into 
small pieces and stored in micro-tubes that will be frozen by snap freezing in liquid nitrogen or 
dry ice and subsequently stored at -80oC. For second trimester POC and stillborn baby, placenta 
will be washed in cold saline or PBS to remove blood. Dissect the placenta into 5 full-thickness 
samples (1 x 1 cm) from 5 random sampling sites, away from cord insertion site and as far apart 
from each other as possible. One of these samples will be stored in 10% buffer-formalin and 
another 1 of them will be stored in EM fixative. For the remaining 3 samples, they will be 
dissected into 3 parts: chorionic plate, villous and maternal decidua; and store these samples into 

separate micro-tubes that will be frozen by snap freezing in liquid nitrogen or dry ice and 
subsequently stored at -80oC. Abortus/stillborn baby will be washed in cold saline or PBS to 
remove blood. Dissect the abortus/stillborn baby into different body parts, e.g. lungs, cartilage, 
skull, brain, muscle, intestines, and store them into separate micro-tubes that will be frozen by 
snap freezing in liquid nitrogen or dry ice and subsequently stored at -80oC.  Stillborn baby will 
also undergo routine autopsy and histopathological examination (HPE) of placenta. All placentas 
will be photographed against a scale bar. In addition, hematoxylin-eosin, myeloperoxidase 
immunohistochemistry, Gram, and periodic acid-Schiff colorations will be performed on POC and 
placenta that are tested positive for SARS-CoV-2 to exclude coexisting bacterial infection.  Results 
on the rate of SARS-CoV-2 infection in women with pregnancy loss will be summarized in counts 
and percentages. Target achievement: we aim to recruit 500 and 5 pregnant women with 
miscarriage and stillbirth, respectively, to determine the rate of SARS-CoV-2 infection in women 
presenting with early and late pregnancy loss, and in cases with positive SARS-CoV-2, matched 
with negative cases, whether vertical transmission has contributed to these complications (see 
details in objective 5).  
 
Objective 3: Pregnancy course and perinatal outcome  
Methodology: a longitudinal study. For pregnant women affected by COVID-19, following 
recovery, 4-weekly ultrasound scan for assessing fetal size, amniotic fluid volume, fetal Dopplers 
(umbilical artery, middle cerebral artery, ductus venosus), uterine artery Doppler and placental 
morphology (echogenicity, thickness, appearance) and function will be done. For first and early 
second trimester infected cases, a detailed fetal morphological scan will be undertaken to 
determine the risk of structural malformation. The following information will be collected: (i) 
maternal demographics including: age, weight, height, racial origin, smoking, medical history of 
chronic hypertension, cardiovascular disease, diabetes mellitus and respiratory disease, drug 
history, method of conception, parity, past obstetric history; (ii) delivery outcome: gestational 
age at delivery, mode of delivery, complications including stillbirth, FGR, preterm birth, 
preeclampsia; (iii) neonatal data: birthweight, sex, Apgar scores, cord arterial and venous pH and 
base excess, neonatal outcome, admission to neonatal intensive care unit (NICU), complications 
such as sepsis, anemia, intraventricular hemorrhage, necrotizing enterocolitis, respiratory 
distress syndrome, need for respiratory support, laboratory findings; (iv) COVID-19 specific data: 
gestational age at clinical manifestation; symptoms and signs; epidemiological history; laboratory 
and radiological findings; treatment measures; COVID-19 severity and outcomes; and intervals 
from onset of disease to hospital admission, delivery and recovery will be recorded. Results on 
maternal and perinatal outcomes will be summarized in counts and percentages and median and 
ranges. Target achievement: To date, there are 5 reported pregnant cases affected by COVID-19 
in Hong Kong. We anticipate that between Hong Kong, China and Spain, we will recruit 40-50 
pregnancies affected by COVID-19 with complete data according to protocol. We will learn a 
complete picture of the pregnancy course and perinatal outcome of cases affected by COVID-19. 
Such data will form the basis of counselling regarding the impact of coronavirus on pregnancy and 
perinatal outcome.  
 
 
 

Objective 4: Vertical transmission 
Methodology: a cross-sectional and longitudinal study. Appropriate biological samples (Table 2) 
will be collected using aseptic technique and tested from consented pregnant women affected 
by COVID-19 from above objectives (Table 3). When the biological samples that have been 
collected immediately after birth are negative for SARS-CoV-2, but the IgM and IgG antibodies 
are positive in the newborn, longitudinal follow-up of the IgG antibody concentrations in the 
infant is required. If the IgG antibodies in the infant become negative within 6 months, the 
possibility of intrauterine infection can be ruled out, and if the IgG antibodies in the infant persist 
till the age of 18 months or beyond, the diagnosis of congenital infection can be confirmed after 
exclusion of infection during infancy. Results on the rate of positive results will be summarized in 
counts and percentages. Target achievement: Relevant testing (RT-PCR and/or anti-SARS-CoV-2 
antibodies testing) of a comprehensive collection of biological samples from the mothers and the 
newborns will allow evaluation of whether vertical transmission has occurred.  
Objective 5: Potential mechanisms for vertical transmission 
Currently there is much controversy regarding the possibility of vertical transmission of SARS-
CoV-2 infection. There is a need for a systematic approach to characterize the transmission 
potential of SARS-CoV-2. In order to successfully establish an infection and transmission of the 
virus in the target tissues, i.e. placenta and developing fetus, SARS-CoV-2 has to be able to dock 
and invade the placental barrier as well as to overcome the host immunologic response at the 
site of infection. In order to have a comprehensive investigation of these underlying mechanisms, 
laboratory tests will be divided into 3 parts to evaluate: 1) placental barrier, 2) immune response 
and 3) fetal damage of vertical transmission and mechanism in SARS-CoV-2 infection. 
Methodology: For the placenta collected from pregnant women affected by COVID-19 (objective 
4), dissect it into 10 full-thickness samples (1 x 1 cm) from 10 random sampling sites, away from 
cord insertion site and far apart from each other. Three of these samples will be stored in 10% 
buffer-formalin and 2 of them will be stored in EM fixative. For the remaining 5 samples, they 
will be dissected into 3 parts: chorionic plate, villous and maternal decidua; and store these 
samples into separate micro-tubes that will be frozen by snap freezing in liquid nitrogen or dry 
ice and subsequently stored at -80oC.  
Placental barrier: we will look into localization, infection, replication and resistance of the viral 
infection in decidua, amnion, maternal-fetal interface, syncytium and placenta. Tissues and cells 
from COVID-19 affected pregnancies with live birth (objective 4) and pregnancy loss (objective 2) 
will be collected and isolated for the study; unaffected pregnancies with live birth and pregnancy 
loss will also be recruited as control for comparison. Standard scanning EM (SEM) and 
transmission EM (TEM) will be employed. SEM could provide detailed images of the surfaces of 
cells, so that the distribution and site of infection of the virion in the maternal, interface and fetal 
compartments could be located. While TEM provides details of interior structures within the cell, 
and thus any dysplasia or reconfiguration of cellular organelles due to viral infection could be 
studied.  

Immune response: during viral infection, the virus has to employ host cell surface molecules as 
specific adhesive receptor. This interaction between the virus and the host cells induces 
conformational changes on both the viral protein as well as the host plasma membrane, and in 
turns, promotes viral entry. Viral entry could generally fall into two categories: 1) direct infection 
of the placenta or 2) indirect infection via transcytosis. Whether SARS-CoV-2 employs direct 
infection and/or indirect infection through virion transcytosis synctium will be determined. 
Results will also correlate with the viral +sense ssRNA load and histologic changes in the 
corresponding cells. Meanwhile, it has been confirmed that the SARS-CoV-2 invades human cells 
by utilizing two protein molecules: receptor of angiotensin-converting enzyme 2 (ACE2) which 
aids in host cell attachment, and a transmembrane serine protease (TRPRSS2) which further 
assists cell entry. The relative abundancy, distribution and co-localization of these cell surface 
proteins in different epithelial, stroma, endothelial, trophoblast, fibroblast and various immune 
cells will be measured and compared between COVID-19 cases and controls. Efficiency and 
resistance infection will be measured by viral replication in various human trophoblast primary 
cells and tissue explants using viral-specific neutralizing antibody titers. Tissue-specific spread, 
cell-to-cell spread, and bypass of the viral infection will be determined. Nonetheless, not all cells 
displaying these required receptors are permissive to productive infection. The presence or 
absence of specific host proteins may also play a critical role during viral infection. Little is known 
about how these intracellular host factors may influence successful SARS-CoV-2 infection. In 
order to address this question, we will look into the immunologic reaction of the cells in the 
decidua, amnion, maternal-fetal interface, syncytium, placental villi core and maternal and 
cord/fetal blood. Single cell RNA sequencing (scRNAseq) of maternal-fetal interface, maternal 
blood and cord blood will be conducted in the affected pregnancies with live birth and pregnancy 
loss. Gene expression of viral infection cascades (e.g. HLA-G, FcRn, TLR, NOD, defensin, etc) and 
surface molecules (e.g. ICAM, Cadherin, gap junction, etc) in epithelial, stroma, endothelial, 
trophoblast, fibroblast and various immune cells will be measured and compared with controls. 
Cell-specific mechanism of immune evasion and defense of SARS-CoV-2 infection for vertical 
transmission will be determined. 
For fetal damage, we will look into the fetal barrier and immune response of the viral infection 
in the affected fetuses. POC and stillborn babies of affected pregnancies will be collected and 
isolated for SEM and TEM, ssRNA load qualification, and viral replication to confirm the 
localization, infection, replication and immunologic reaction as above. If the fetuses have any 
developmental structural abnormalities identified during antenatal ultrasound examinations, the 
affected fetal organs, tissues and cells will be specifically collected and studied as above. The 
results will be correlated with the pathology of the fetuses and compared with the POC and 
stillborn fetuses of unaffected pregnancies.  
Target achievement: We aim to collect 5-10 affected cases with live birth and 5-10 affected cases 
with pregnancy loss, compared with gestational-age matched controls. We anticipate to 
characterize and reveal underlying mechanisms for vertical transmission associated with 
pregnancy loss and fetal complications. 

Study coordination 
Prof Poon has conceived and designed this project and will manage and coordinate all aspects of 
the study. For objective 1, together with Prof Sahota, who is the team bioinformatician, a list of 
eligible patients has been drawn from our DSS database. Dr Chaemsaithong is overseeing the 
recruitment of eligible patients for recruitment for objective 1. She will also collect and manage 
study data. For objective 3, there are currently 5 pregnant women in Hong Kong affected by 
COVID-19 and their pregnancy is ongoing. Prof Leung will provide clinical guidance for and 
oversee the follow up of the affected pregnant women. Prof Yang will contribute maternal and 
perinatal outcome data of the pregnant cases that had acquired SARS-CoV-2 infection during the 
first and second trimester of pregnancy from her existing cohort study in China. Dr Gil from Spain 
has started recruitment of patients affected by COVID-19 and collection of biological samples 
according to the proposed protocol (objective 4). Prof Chung is an academic gynecologist with 
an extensive research experience in pregnancy loss. She will organize for collection of POC and 
recruit stillbirth cases. Prof Wang is a clinician scientist and Dr Leung is a molecular biologist, both 
with a special interest in disease mechanism; together they will conduct all experiments for 
objective 5. Prof Yang will contribute and assist with the interpretation of data for objective 5.  
Prof Chan and Prof Ng are infectious disease specialists with extensive experience in COVID-19 
research. Their laboratory will be in charge of analyzing all biological samples for objectives 2,4. 
Prof Lam is an academic pediatrician with expertise in neonatal intensive care research. He will 
coordinate follow up of the neonates born to mothers with COVID-19. 
 
Work done by us  
The multi-disciplinary research team includes experts in the fields of MFM, gynecology, infectious 
disease, pediatric, developmental and molecular biology and biostatistics/bioinformatics. Since 
March 2020, the PC has published 11 papers on COVID-19 during pregnancy, including a 
consecutive case series of 116 pregnant women with COVID-19 (with Prof Yang), and a high 
quality systematic review of 324 pregnant women in evaluating the effects of COVID-19 on 
maternal and neonatal outcomes (with Prof Yang and Dr Gil). She has also authored three 
international guidelines on the management of pregnant women with COVID-19, one consensus 
statement on organization of routine and specialist obstetric ultrasound services in the context 
of COVID-19, and two safety position statements regarding the appropriate use of personal 
protective equipment and safe performance of obstetric and gynecological scans and equipment 
cleaning. 
 
Pregnancy outcome and vertical transmission 
In the consecutive case series of 116 pregnant women with COVID-19 in China32, the data were 
collected using a standardized methodology by a team of experienced clinicians, curated with 

customized data collection form and verified independently by two investigators. The median 
gestational age on admission was 38+0 (interquartile range 36+0-39+1) weeks. The most common 
symptoms were fever (50.9%) and cough (28.4%). Abnormal radiologic findings were found in 
96.3% cases. There were eight cases (6.9%) of severe pneumonia but no maternal deaths. One of 
eight patients (12.5%) that presented in the first and early second trimester had miscarriage. 
Ninety-eight pregnant women, including one with twin pregnancy, delivered their babies during 
hospitalization, 85 (86.7%) underwent Cesarean section. The Cesarean section was indicated for 
COVID-19 pneumonia in 38.8% of cases. The rates of preterm delivery before 34 weeks and 37 
weeks were 2.0% and 21.4%, respectively. Among the 21 preterm deliveries, 6 (28.6%) had 
preterm premature ruptured of membranes. The rate of spontaneous preterm birth before 37 
weeks was therefore 6.1% (6/98). There were no cases of fetal deaths but one case of neonatal 
death. 85/99 neonates were tested for SARS-CoV-2 on pharyngeal swab samples and the results 
were negative, of these nine neonates had paired amniotic fluid and cord blood samples that 
tested negative for COVID-19. At the time of publication, there were 17 ongoing pregnancies, of 
which 9 cases acquired SARS-CoV-2 during the first and second trimester.  
In our high-quality systematic review15, we have summarized data on clinical characteristics, 
including at least one maternal, perinatal or neonatal outcome, in 295 pregnant women affected 
by COVID-19 from 8 consecutive case series. Amongst these 295 cases, 210 cases originated from 
China. We have also identified a high number of possible duplicate reporting and excluded 327 
cases from China. The most common symptoms at presentation were fever, cough, 
dyspnea/shortness of breath, fatigue and myalgia. The rate of severe pneumonia reported 
amongst the case series ranged from 0 to 14%. Almost all cases from the case series had positive 
computer tomography chest findings. Only 4 cases of spontaneous miscarriage or abortion were 
reported. In the consecutive case series, 219/295 women had delivered at the time of reporting, 
and of these, 78.1% had Cesarean section. The gestational age at delivery ranged from 28 to 41 
weeks. Apgar scores at 1 and 5 min ranged from 7 to 10 and 7 to 10, respectively. Only eight 
(8/103; 7.8%) neonates had birth weight <2500 g and nearly one-third of cases (49/173; 28.3%) 
were transferred to the NICU. There was one case each of neonatal asphyxia and neonatal death. 
In 155 neonates that had nucleic-acid testing in throat swab, all, except three cases, were negative 
for SARS-CoV-2. Our systematic review has highlighted that despite the increasing number of 
published studies on COVID-19 in pregnancy, there are insufficient good-quality data to draw 
unbiased conclusions with regard to complications of COVID-19 in pregnant women as well as 
vertical transmission and perinatal complications. Our research to date will form the basis of our 
prospective studies described in this proposal. In addition, the PC is a project partner for a UK 
Research Institute (UKRI) and NIHR funded international registry of pregnancy and neonatal 
outcomes of COVID-19 (PAN-COVID; https://pan-covid.org). This registry is an international 
observational study with a web-based portal, collecting data on outcomes of pregnant women 
and their newborn babies who have suspected COVID-19 in pregnancy or confirmed SARS-CoV-2 
infection. The registry will focus on reporting the incidence of the following outcomes: 
miscarriage, FGR and stillbirth, preterm delivery, vertical transmission and early-onset neonatal 

SARS-CoV-2 infection. Data from our proposed project will have the opportunity to be compared 
with data collected through PAN-COVID. 
 
SARS-CoV-2 target cells 
Together with Prof Yang, we have conducted a pilot study and applied bioinformatic analysis on 
published scRNAseq datasets of early trophectoderm (TE) and first and second trimester 
placentas and identified the existence of ACE2 and TMPRSS2 expression in human TE and first 
and second trimester placentas (Figure 1). In human TE data, 54.4% of TE cells, 9.0% of 
cytotrophoblasts 
(CTBs), 
3.2% 
of 
extravillous 
trophoblasts 
(EVTs) 
and 
29.5% 
of 
syncytiotrophoblasts (STBs) were ACE2 positive. As for TMPRSS2, 90.7% of TE cells, 31.5% of CTBs, 
22.1% of EVTs and 70.8% of STBs were TMPRSS2 positive. Amongst the placental cells, 20.4% of 
CTBs, 44.1% of STBs, 3.4% of EVT at 8 weeks and 63% of EVT at 24 weeks were ACE2 positive. And 
1.6% of CTBs, 26.5% of STBs, 1.9% of EVT at 8 weeks and 20.1% of EVT at 24 weeks were TMPRSS2 
positive. Pathway analysis revealed associations to morphogenesis of branching structure, 
extracellular matrix interaction, oxygen binding and antioxidant activity in ACE2+TMPRSS2+ EVT 
at 24 weeks cells. The ACE2+TMPRSS2+ TE cells were correlated with an increased capacity of 
viral invasion, epithelial cell proliferation and cell adhesion. Based on immunohistochemical 
results, a higher expression level of ACE2 and TMPRSS2 in first and second trimester placentas 
was observed (Figure 2). Localization of virion in the maternal, interface and fetal compartments; 
and associated dysplasia or reconfiguration of cellular organelles will be examined by TEM as we 
have previously described33. 
 
Table 1. Biological samples from cases of pregnancy loss 
 
Table 2. Biological samples from pregnant cases affected by COVID-19 
Biological samples 
Tests to be done 
Before delivery: maternal blood*# 
At or after delivery: amniotic fluid*, cord blood*#, placental tissue*†, 
amnion-chorion interface swab*, neonatal throat swab* 
*RT-PCR; #IgG and IgM; 
†HPE  
Breast milk from both breasts for the duration of hospitalization 
RT-PCR, IgG and IgM 
Newborn throat swab at 24 hours of life 
RT-PCR 
Newborn stool samples for the duration of hospitalization 
RT-PCR 
Infant throat swab and stool sample at 7 days of age 
RT-PCR 
Infant blood at 14 days, 6 months & 18 months of age 
IgG and IgM 
 
Biological samples 
Tests to be done 
Miscarriage  POC*, maternal blood# 
*SARS-CoV-2 RT-PCR; #IgG; HPE;  
Stillbirth       Maternal vaginal swab†¶, amniotic fluid†¶, 
amnion-chorion interface swab†¶, 
placental tissue¶, fetal blood¶‡, fetal 
throat swab†¶, maternal blood‡  
†Culture+PCR; ¶SARS-CoV-2 RT-PCR; 
‡IgG 
 

Table 3. COVID-19 pregnant cases with biological samples collected or to be collected (for 
objectives 4&5) 
 
Cases Age 
(yrs) 
Gestational 
age  
on 
admission 
(wks) 
COVID-19  
severity 
 
 
Clinical  
outcomes 
 
Pregnancy 
status 
 
Current 
gestational  
age (wks) 
HK 
1 
33 
16 
Mild 
Recovered Ongoing 
30 
2 
38 
19 
Mild 
Recovered Ongoing 
31 
3 
34 
25 
Mild 
Recovered Ongoing 
39 
4 
47 
33 
Asymptomatic In hospital 
Ongoing 
34 
Spain 
5 
30 
40 
Mild 
Recovered Delivered 
 
6 
22 
34 
Mild 
Recovered Delivered 
 
7 
35 
38 
Mild 
Recovered Delivered 
 
8 
41 
38 
Mild 
Recovered Delivered 
 
9 
18 
38 
Mild 
Recovered Delivered 
 
10 
28 
19 
Pneumonia 
Recovered Ongoing 
34 
11 
38 
24 
Asymptomatic Recovered Ongoing 
38 
12 
39 
40 
Mild 
Recovered Delivered 
 
13 
35 
21 
Mild 
Recovered Delivered 
 
14 
36 
25 
Mild 
Recovered Ongoing 
34 
15 
29 
8 
Pneumonia 
Recovered Ongoing 
20 
16 
40 
11 
Pneumonia 
Recovered Ongoing 
25 
17 
35 
14 
Mild 
Recovered Ongoing 
36 
18 
30 
10 
Mild 
Recovered Ongoing 
23 
19 
27 
39 
Asymptomatic Recovered Delivered 
 
20 
33 
37 
Asymptomatic Recovered Delivered 
 
21 
22 
17 
Pneumonia 
Recovered Ongoing 
32 
22 
31 
32 
Asymptomatic Recovered Ongoing 
36 
23 
24 
27 
Asymptomatic Recovered Ongoing 
31 
24 
33 
1 
Mild 
Recovered Ongoing 
15 
25 
34 
32 
Asymptomatic Recovered Ongoing 
36 
26 
34 
33 
Asymptomatic Recovered Ongoing 
36 
27 
29 
34 
Mild 
Recovered Ongoing 
38 
28 
36 
11 
Mild 
Recovered Ongoing 
20 
29 
37 
36 
Mild 
Recovered Ongoing 
41 
30 
37 
35 
Mild 
Recovered Ongoing 
37 
31 
37 
5 
Mild 
Recovered Ongoing 
19 
32 
31 
37 
Asymptomatic Recovered Delivered 
 
33 
32 
20 
Asymptomatic Recovered Ongoing 
23 
34 
43 
29 
Asymptomatic Recovered Ongoing 
34 
35 
32 
32 
Asymptomatic Recovered Delivered 
 

36 
31 
33 
Asymptomatic Recovered Ongoing 
38 
37 
39 
29 
Asymptomatic Recovered Ongoing 
34 
38 
39 
16 
Mild 
Recovered Ongoing 
29 
39 
28 
29 
Asymptomatic Recovered Ongoing 
35 
40 
36 
14 
Asymptomatic Recovered Ongoing 
24 
41 
40 
1 
Mild 
Recovered Ongoing 
14 
42 
40 
22 
Mild 
Recovered Ongoing 
37 
43 
30 
25 
Mild 
Recovered Ongoing 
36 
 
 
 
 
 
 
 
 
 
 
 
Figure 1. Single-cell RNA sequencing of SARS-CoV-2 related molecules in early trophectoderm and the first and second trimester 
placenta. UMAP distributions of ACE2 and TMPRSS2 in different placental cell types from embryonic TEs obtained 6-14 days after 
fertilization (upper panels) and T-SNE distributions of ACE2 and TMPRSS2 in 8 week and 24 weeks placentas (lower panels).  
Figure 2. Immunohistochemistry staining of ACE2 and TMPRSS2 in the first (upper 
panels), second (middle panels) and third (lower panels) trimesters of human 
placenta. KRT7 (marker of trophoblasts), HLA-G (marker of EVTs) and VIM (marker 
of stromal cells) are included as controls.
Figure 3. Transmission electron microscopy of 
trophoblast cells in placenta from normal and 
selective fetal growth restricted (sFGR) 
pregnancies. Damage of mitochondrial and 
endoplasmic reticulum are shown.   

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