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\begin{document}

\title{Follow-up study on the outcomes of recovered pregnant women who had
previous COVID-19 during the first and second trimester: a retrospective
cohort study}



\author[1]{Yin Zhao}%
\author[2]{BangXing Huang}%
\author[2]{Hui Ma}%
\author[2]{You Shang}%
\author[2]{Xiu Nie}%
\author[3]{Li Zou}%
\affil[1]{Union Hospital of TongJi Medical College of Huazhong University of Science and Techonology}%
\affil[2]{Union Hospital of TongJi Medical College of Huazhong University of Science and Techonolog}%
\affil[3]{Huazhong University of Science and Technology Tongji Medical College}%


\vspace{-1em}



  
  \date{June 15, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
Objective: The research aimed to study the pregnancy outcomes and
determine the safety of continued pregnancy until the third trimester
after being infected with SARS-CoV-2 during early pregnancy.
Design:Retrospective cohort study. Setting: Wuhan, Hubei,
China.Population:Pregnant women in recovery who had a history of
COVID-19 during early pregnancy at the Union Hospital from 15 Jan 2020
to 30 April 2020 were included. Methods:The clinical manifestation,
laboratory examinations, treatment, pregnancy outcomes, maternal
SARS-CoV-2 throat swab RT-PCR test results, and SARS-CoV-2 antibody
tests in neonates were detected. The placental pathology, ACE2
expression and SARS-CoV-2 presence was also studied. Main outcome
measures: Maternal and neonatal clinical and laboratory status. Results:
We included cases in our study. Three cases were diagnosed with COVID-19
during early pregnancy (Case A to C), and two cases were serum IgG
positive asymptomatic cases (Case D and E). Case A showed complete
recovery after severe COVID-19. Case C was infected at 6 weeks during
the first trimester and had induced medical abortion at 12 weeks. No
pneumonia was found in all neonates, the nucleic acid test and serum IgM
were negative, and IgG were positive. All placental samples were
negative for SARS-CoV-2 nucleic acid test. Infants followed-up at 1-3
months were healthy and asymptomatic. Placental pathology showed chronic
ischemia performance.Conclusions COVID-19 during pregnancy did not
significantly affect pregnancy outcomes. No adverse outcomes were
observed in all the neonates. The inflammatory state of COVID-19 may
cause placental injury. The placenta does not appear be a target organ
for SARS-CoV-2.%
\end{abstract}%



\sloppy


\textbf{Tweetable abstract:}

Pregnant women with COVID-19 during early pregnancy were assessed to
determine the pregnancy outcomes.

\textbf{INTRODUCTION}

The novel coronavirus infection (COVID-19) was prevalent in Wuhan since
December 2019, including pregnant women\textsuperscript{1}. In Wuhan,
these patients represented 0.24\% of all reported cases with
COVID-19\textsuperscript{2}. COVID-19 affects the entire pregnancy;
however, current research is mostly about pregnancy during the third
trimester and peripartum\textsuperscript{2-4}. In New York, of the 215
women screened for severe acute respiratory syndrome coronavirus 2
(SARS-CoV-2) on admission using nasopharyngeal swabs, 33 women (15.4\%)
were found positive. Of these positive cases, four had fever or other
symptoms of COVID-19, while 29 were asymptomatic\textsuperscript{3}.

Currently, there are a few reports on the outcome of COVID-19 in
pregnant women during the first and second trimester, as well
post-recovery. The effect of COVID-19 on pregnancy remains unclear,
which obstetricians and neonatologists. Recent researches indicated that
there is no clear evidence of intrauterine infection in pregnancy with
COVID-19 during the third trimester\textsuperscript{2,5}. However, both
SARS-CoV-2 IgM and IgG were detected in newborns of infected mothers,
which question the possibility of intrauterine
infection\textsuperscript{6,7}. Some studies claimed that a positive IgM
test result could not be regarded as the definite evidence of vertical
transmission\textsuperscript{8}. Our previous study reported the
clinical characteristics and placental pathological analysis of three
peripartum women with COVID-19\textsuperscript{9}. We noted no severe
adverse pregnancy outcomes or morphological changes related to
infection. All samples were negative for the SARS-CoV-2 nucleic
acid\textsuperscript{9}. We concluded that COVID-19 intrauterine
infection does not occur during the third trimester.

The aim of our current study is to determine the safety and postpartum
outcomes in pregnant patients infected from early pregnancy (first
and/or second trimester) with COVID-19.

\textbf{METHODS}

\textbf{Study design and participants}

This is a retrospective cohort study done at Union Hospital, China from
15 Jan 2020 to 30 April 2020. A total of five cases were included in
this study. This study included three fully-recovered pregnant patients
confirmed with COVID-19 during the first or second trimester
(\textless{}28 weeks gestation). COVID-19 recovery criteria were defined
according to the World Health Organization interim guidance
criteria\textsuperscript{10} and the Chinese Clinical Guidance for
COVID-19 Pneumonia Diagnosis and Treatment\textsuperscript{11}, which
are as follows: (1) normal or significantly improved chest computed
tomography (CT), (2) negative SARS-CoV-2 nucleic acid test, and (3)
negative SARS-CoV-2 serum IgM, and positive or negative IgG. Two
asymptomatic cases with positive IgG at the time of delivery were also
included. Case D and case E developed mild cold at 26 week and 27 weeks,
they did not seek medical attention. The symptoms improved after 1 week.
Clinical symptoms of COVID-19, laboratory results, CT imaging results,
treatment course, pregnancy outcomes, placental pathology, SARS-CoV-2
laboratory results, angiotensin-converting enzyme 2 (ACE2) expression,
and neonatal throat swabs and antibody tests were analysed.

We reviewed the medical records, laboratory results, and imaging
findings of the recruited cases from admission to postpartum. Laboratory
examination before admission includes SARS-CoV-2 pharyngeal swab and
serum antibody, and chest CT scan. Postpartum, neonatal throat swab,
umbilical cord blood antibody testing, and QT-PCR for SARS-CoV-2, and
placental pathology were performed. Laboratory confirmation of
SARS-CoV-2 infection was done. SARS-CoV-2 infection was confirmed using
real-time RT-PCR\textsuperscript{12}.

This study was approved by the Medical Ethics Committee of the Union
Hospital, TongJi Medical College of HuaZhong University of Science and
Technology {[}2020, NO. 0144{]}. Written informed consent was obtained
from the patients before enrolment and during data collection.

The Serum antibody test: SARS-CoV-2 IgG and IgM Colloidal gold method
(Vazyme, NanJing, China) on 20 \(\mu\)ml plasma.

Pathological examination: Placental tissue specimens (Case A, B, C, D)
were fixed using 3.7\% formaldehyde solution for 48 hours. Conventional
HE staining was performed after paraffin embedding, each slice was
4\selectlanguage{greek}μ\selectlanguage{english}m\selectlanguage{ngerman} thick. All HE sections were observed and reviewed by
one attending physician and one associate chief physician. Using RT-PCR
method to detect SARS-CoV-2 in placental tissue: three wax blocks were
selected for SARS-CoV-2 detection in each case, and the samples to be
tested included the placental villi (including maternal and foetal
surfaces), foetal membrane and umbilical cord as far as possible.
Formaldehyde-fixed paraffin-embedded sample RNA separation kit and
SARS-CoV-2 detection kit were used (Aide biological medicine technology
co., Ltd, XiaMen, China). ACE2 Recombinant rabbit monoclonal antibody
(Clone No. BP6153, Bailing (Biolynx) Biotechnology Co., Ltd. Hangzhou,
China) was used to study the expression of ACE2 in the placenta.

ACE2 evaluation criteria: the area quartering method was 1, 2, 3, and 4
points. The staining intensity was 1 (weak), 2 (medium), and 3 (strong),
respectively. The total score was multiplied by each other.

Funding

Funding was provided by the National Natural Science Foundation of
China, Hubei Provincial Natural Science Foundation of China.

\textbf{RESULTS}

Early pregnancy infections were noted in three cases who were infected
at 24 and 26 weeks and recovered after treatment for pneumonia. Before
delivery, CT imaging showed resolution of pneumonia. Throat swab
SARS-CoV-2 tests were done twice with negative results. Only the
SARS-CoV-2 IgG antibody was positive. Case A delivered at 40 weeks age
of gestation after administration of dinoprostone suppository improving
cervical ripening and oxytocin augmentation of uterine contraction. Case
B had premature rupture of membranes at 35 weeks with transverse
presentation and delivered via caesarean section. Case C was infected at
6 weeks who underwent an induced medical abortion at 12 weeks after
administration of mifepristone and misoprostol.

Additionally, two COVID-19 asymptomatic cases were (Case D and E) who
had serum SARS-CoV-2 IgG positive on routine screening before delivery,
chest CT scan and nucleic acid tests were unremarkable.

\textbf{COVID-19 during pregnancy}

Following the WHO interim guidance criteria, three cases were diagnosed
with COVID-19: Case A was 24 weeks (+3), while Case B was 26 weeks (+1)
when they were diagnosed with COVID-19. They had no chronic disease.
Both presented with fever, fatigue, and dry cough. The descriptions are
listed in Table 1. Case A had respiratory distress and dyspnoea; Case B
presented with stuffy nose and respiratory distress. Case C had no
dyspnoea. All three cases had moderate anaemia. Case B had lymphopenia
(0.54 G/L), leukopenia (3.21 G/L), and neutropenia (1.65 G/L). Case C
presented with moderate leukopenia. Cases A, B, and C had elevated
C-reactive protein and IL-6 (Case A:CRP 6.9ng/L, IL-6 67.98 pg/ml,Case B
CRP 31.88mg/L, IL-6 not available; Case C: CRP 26.8 ng/L, 7.49 pg/ml);
and had decreased albumin and total protein (TP). Case A (TP 55.8 g/L,
ALB 27.9 g/L , ALT 510 U/L, AST 246U/L) , Case B (TP 57.1 g/L, ALB:31.4
g/L, AST 21 U/L, ALT 14 U/L) , Case C: (TP: 57.6 g/L, ALB:31.4 g/L, ALT
38U/L, AST 104U/L). Case A and C had positive nucleic acid tests. Case B
had two consecutive negative nucleic acid tests, however, she presented
with viral pneumonia. On readmission after 70 days, SARS-CoV-2 IgG was
positive confirming previous COVID-19 infection. Case A had a severe
type of infection and was admitted for 12 days. Case B and C were normal
type infections and were treated for 6 days and 10 days, respectively.
According to chest CT imaging, they all had viral pneumonia. CT imaging
of Case A showed multiple, membranous, ground-glass-patterns or dense
shadows surrounding the parenchyma of both lungs. The pulmonary lesions
showed resolution post-treatment. Case B and C show similar chest CT
findings (Fig. 1).

All cases received oxygen therapy using a nasal catheter. Case A had
severe dyspnoea, fatigue, and desaturations (SPO\textsubscript{2} 90\%
on room air). Case B and C had moderate distress
(SPO\textsubscript{2}95-96\%) (Table 1).

Case A received antiviral treatment, interferon (Recombinant human
interferon a-2b 5 million IU daily), atomisation inhalation for 7 days,
oral umifenovir tablets (200 mg three times daily for 7 days), and
azithromycin (0.25g bid for 3 days). Polyene phosphatidyl choline (458
mg thrice daily) and reduced glutathione (1.8 g daily) were used to
decrease the ALT and AST for 7 days. All cases were given antibiotic
treatment (Table 2.) They were discharged improved and stable. CT scan
showed resolution of pulmonary lesions, and the SARS-CoV-2 throat swab
showed two consecutive negative results. After discharge, Case A and C
were prescribed oral liver supplements and was quarantined for 14 days.
Repeat antibody testing before delivery showed positive serum IgG and
negative IgM antibodies in all three cases (see Table 2).

\textbf{Pregnancy outcome}

The interval period from discharge to delivery was 42 to 109 days for
all three cases. Case A was admitted at 40 weeks, with a cervical score
of 6. After dinoprostone administration and oxytocin, a 3650 g male was
delivered with an APGAR score of 8 at 1 minute and 9 at 5 minutes.
Epidural labour analgesia was administered when the cervix 3 cm dilated.
Case B had premature rupture of membranes at 35 weeks with transverse
presentation delivered using caesarean section to a 2800 g female baby.
APGAR score was 7 at 1 minute and 9 at 5 minutes.

The newborns of Case A and B had no pneumonia, negative COVID-19 throat
swab nucleic acid test, and positive serum IgG. Neonate A was roomed-in
and was breastfed. At 1 month follow-up, neonate A was healthy and
asymptomatic. Neonate B was transferred to NICU due to preterm labour.
The neonate remained asymptomatic during admission and was discharged
after a week following two consecutive negative nucleic acid test
results. IgG was positive. At 2 months follow-up, the infant was healthy
and mixed-feeding.

The neonates of Case D and E were healthy and asymptomatic. Both of them
showed negative SARS-CoV-2 throat swab nucleic acid test, and positive
serum IgG (Table 2).

\textbf{Placental examination}

The placental tissues of Case A to D were examined which showed varying
degrees of injury. Case A showed multiple placental calcifications and
increased syncytiotrophoblast knot. Case B showed placental chronic
infarct at the maternal side. The villous structure collapsed with
fibrin deposition. The middle of the placenta was normal. Case C showed
oedema in the villi and fibrosis in the 12-week-old placenta. No
definite thrombosis was found. No obvious chorioamnionitis or virus
inclusion body formation was observed in all cases. The umbilical cord
and amnion membranes were normal (Fig.2).

Immunohistochemical staining of ACE2 showed that both the placenta and
the decidual membrane expressed ACE2, which was located in the membrane
and cytoplasm of the syncytiotrophoblast and decidual cells. The
placental expression was strongest at 12 weeks of gestation (Fig. 3).

Placental SARS-CoV-2 nucleic acid test results of all the cases Case
A,B,C,D were negative (Table 1).

\textbf{DISCUSSION}

Main Findings

The current follow-up study showed that, pregnant women infected with
SARS-CoV-2 in the early pregnancy and recovered had good outcome. No
pneumonia was found in all neonates, the nucleic acid test and serum IgM
were negative, and IgG were positive. All placental samples were
negative for SARS-CoV-2 nucleic acid test. Placental pathology showed
chronic ischemia performance.

\textbf{Strengths and Limitations}

Our study has several strengths. First, it was a retrospective cohort
study allowing us to evaluate the outcomes of COVID-19 from early
pregnancy to follow-up. Second, we have evaluated several parameters
during early pregnancy including clinical, laboratory, imaging and
placental pathology.

Meanwhile, our study has some limitations. Our study only enrolled five
cases with COVID-19. Therefore, our results may not be generalizable for
other populations. The number of cases is limited and further studies
are needed. Second, foetal and maternal outcomes were only observed
until two-months of follow-up, which provides limited information.
Further follow-up is important to assess the long-term effects of
maternal COVID-19 to the infant.

\textbf{Interpretation}

According to the data of 118 cases of COVID-19 pregnancy in
Wuhan\textsuperscript{2}, 64\% of the cases occurred during late
pregnancy, and the rate of severe disease in all patients with COVID-19
pregnancy was 8\%, which was lower than the average level of the general
population (15.7\%). There were no deaths reported. It has been
suggested that pregnancy might confer potential immunity. This was
consistent with our study wherein even severe COVID-19 improved after
proper treatment (Case A).

Among the three confirmed patients (Case A, B, C), only two (Case A and
C) showed elevated liver enzymes possibly because of COVID-19-related
hypoxia augmenting the inflammatory factors. Additionally, we found a
pro-inflammatory state in COVID-19 (elevated CRP and IL-6 levels). The
liver was also affected as seen from the abnormal liver function tests.
Finally, after the administration of antiviral therapy (umifenovir and
interferon atomisation) and liver supplements\selectlanguage{english},liver function profile
improved.

None of the patients showed significant foetal malformation or foetal or
placental virus infection. Even Case C, which was prematurely
terminated, had crown-lump length (CRL) at 12 weeks at par with age.

Placental pathology suggest chronic placental injury, hence has the
possibility of decreasing maternal blood perfusion into the villi, thus,
hindering maternal-foetal exchange\textsuperscript{2}. This may be
secondary to the hypoxic and inflammatory condition during COVID-19
affecting the placenta. Massive perivillous fibrin deposition and large
agglomeration of necrotic villi suggested placental infarction was also
found in Case B. The preterm premature rupture of membrane in Case B may
be not only due to transverse presentation but also associated with the
chronic ischemia and increased cytokines. Fortunately, only significant
placental infarction was associated with SGA
foetuses\textsuperscript{13}. In the present study, although placental
damaged was noted, the outcomes of were relatively ideal. The placental
potency may be enough to overcome the villi impairment. However, if a
mother had some complications associated with placental dysfunction
(preeclampsia, fetal growth retardation, et al), COVID-19 may severely
affect the placenta and foetus and result to adverse pregnancy outcome.
These studies indicated that pregnancy with COVID-19 during the first
and second trimester may result to full recovery allowing the
continuation of pregnancy to term with good maternal and foetal
outcomes.

All the placenta tests were negative for SARS-CoV-2 nucleic acid. In our
previous study, same negative results were found in the three cases who
had COVID-19 just before delivery\textsuperscript{9}. The recent study
cannot provide definite evidence to support intrauterine infection with
COVID-19.

Vertical transmission includes intrauterine infection during pregnancy,
intrapartum infection, and transmission through close contact and
breastfeeding after delivery. ACE2 is a functional receptor for
SARS-CoV-2 infection\textsuperscript{14}. Theoretically, ACE2 provides
the opportunity for SARS-CoV-2 infection. SARS-CoV-2 attaches to the
cells using S protein and enter cells through binding to ACE2. It was
suggested that the distribution and expression of ACE2 may be critical
for the target organs affected by SARS-CoV-2\textsuperscript{15,16}. The
available evidence suggests that ACE2 is widely expressed in female
reproductive system and the maternal-foetal interface, which includes
the stromal cells and perivascular cells of decidua, cytotrophoblasts
and syncytiotrophoblasts\textsuperscript{14,17}. Our results showed that
both the placenta and decidua expressed ACE2 during early pregnancy with
significantly higher levels than those during late pregnancy. However,
the histologic and real-time PCR of the placenta had the negative
results. Therefore, organs with high ACE2 expression may not always be
the target of SARS-CoV-2.

During vertical transmission, high viral loads of SARS-CoV-2 is
necessary. The placental villi may have more opportunities for
SARS-CoV-2 to enter the maternal blood allowing the virus to infect the
trophoblast cells. SARS-CoV-2 may then pass through the placental
villous lobules to enter the foetal capillaries and infect the foetus.
According to some reports, the lungs and the intestine may be major
viral target organs of SARS-CoV-2\textsuperscript{18}. Studies have
shown a high expression of ACE2 and TMPRSS2 in the lungs of
newborns\textsuperscript{19}. This places neonates at high risk for
SARS-CoV-2 infection.

A Lamouroux et al\textsuperscript{20} reported three swabs positive for
SARS-CoV-2 in 11 placental or membrane swabs sent following delivery in
women with moderate to severe COVID-19. None of the infants tested
positive for SARS-CoV-2 on days 1 to 5 of life, and none demonstrated
COVID-19 symptoms. While there were no clinical signs of vertical
transmission, they postulated on possible intrapartum viral exposure.
For infants delivered vaginally, viral contamination may be from room
air, vaginal secretions, maternal blood, or amniotic fluid. In our
study, no virus or viral infection was found in the placenta. Wang et
al\textsuperscript{21} reported a case of positive SARS-CoV-2 throat
swab test in a newborn 36 hours after delivery. Airborne transmission
possibility after delivery cannot be excluded. The risk of neonatal
infection from postpartum exposure via respiratory secretions and close
contact should be avoided. The neonate throat swab tests and placenta
tests were negative in our cases. This reminds obstetricians that
although there is no clear evidence of intrauterine infection, careful
attention to protect the neonate from exposure to SARS-CoV-2 during and
after delivery should be observed.

In clinical studies, most of the neonates were negative of
SARS-CoV-2\textsuperscript{2,4}. The placenta may not be the main target
organ of SARS-CoV-2. There is no clear evidence of intrauterine
infection, which requires further study regarding the specific
mechanisms involved.

\textbf{CONCLUSION}

Pregnancy complicated with COVID-19 does not show significant effects on
pregnancy outcomes. No adverse foetal outcomes were observed during the
neonatal follow-up (until 3 months) in the infants born to mothers
infected with COVID-19 during early pregnancy. COVID-19 during pregnancy
may lead to a systemic inflammatory response and hypoxia, resulting in
elevated liver enzymes and placental insufficiency. The placenta has a
potential reserve capacity allowing pregnancy to continue to term.
Although the placenta and decidua express ACE2, the placenta does not
appear be a target organ for SARS-CoV-2. Possibly, the placental barrier
can effectively prevent SARS-CoV-2 from causing intrauterine infection.

\textbf{Acknowledgements: None}

\textbf{Disclosure of interests:} All authors declare no conflict of
interest.

\textbf{Contribution to Authorship:}

ZY and ZL conceived the study. HBX and NX performed placental study. MH
carried out the CT image data collection. SY collected the clinical
data. ZY wrote the first draft. NX and ZL reviewed the article.

\textbf{Details of Ethics Approval:}

The study was approved by the Medical Ethics Committee of the Union
Hospital, TongJi Medical College of HuaZhong University of Science and
Technology {[}2020, NO. 0144{]}. Written informed consent was obtained
from the patients before enrolment and when data were collected.

Funding

Our study has been funded by the National Natural Science Foundation of
China, Hubei Provincial Natural Science Foundation of China.

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Figure Legend

Fig 1. Chest CT image of the cases.

A1, B1, C1 were the CT scanning images of pregnant women complicated
with COVID-19. Multiple or scattered membranous glass density
shadows/dense porphyry shadows surrounding the parenchyma of both lungs
could be seen at the onset of Covid-19, which indicated the possibility
of viral pneumonia is high. A2,C2 were the CT image which showed lesions
in lung were improved after being treated for 7 days. A3, B2, C3 were
the CT image at the time before delivery, no obvious abnormality was
observed.

Fig 2. Pathological findings of placental tissue

Case A : Pregnant woman of 24weeks complicated with Covid-19, and
delivered at 40 weeks. Placental chronic ischemia was shown : presence
of multiple placental calcifications (black arrow) and dense small villi
with increased syncytial knots (blue arrow) . (1: HE x40, 2: x100)

Case B: Pregnant woman of 26 weeks complicated with COVID-19, and
delivered at 35 weeks. Placental chronic infarct was seen at the
maternal side (40 times magnification) . In the middle part of the
placenta, normal villi structure could be seen. (100 times
magnification).

Case C: Pregnant woman of 6 weeks complicated with COVID-19, and the
pregnancy was terminated at 12 weeks. Part of the villous was edema and
fibrosis (Arrow) (40 times).'' No definite thrombosis was found.

Case D: Pregnant women of 38 weeks who was serum IgG positive
asymptomatic case. Chronic placental ischemia infarction could be seen.
(1: HE x40, 2: x100).

Fig 3. Representative ACE2 Immunohistochemical staining in placenta.

ACE2 was positive in both villi and decidua, which was mainly expressed
in membrane and cytoplasm. It expressed stronger in 12 weeks (Case C) (x
100 times). ACE2 evaluation criteria: the area quartering method was 1,
2, 3 and 4 scores, and the staining intensity was 1 (weak), 2 (medium)
and 3 (strong), respectively. The total scores were area scores multiply
staining intensity scores.

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