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\begin{document}

\title{Nephrin in maternal urine applied to a point-of-care kit to predict
preeclampsia: a prospective study.}



\author[1]{Kyong-No Lee}%
\author[2]{Subeen Hong}%
\author[3]{Tae Eun  Kim}%
\author[1]{Eun Ji  Oh}%
\author[4]{Ki-seok  Kim}%
\author[4]{Ju-Hyung  Kang}%
\author[4]{Min-Young  Yeo}%
\author[1]{Hyeon Ji  Kim}%
\author[3]{Jee Yoon Park}%
\affil[1]{Seoul National University Bundang Hospital Department of Obstetrics and Gynecology}%
\affil[2]{Catholic University of Korea School of Medicine}%
\affil[3]{Seoul National University Bundang Hospital}%
\affil[4]{Aptamer Sciences Inc}%


\vspace{-1em}



  
  \date{May 27, 2024}


\begingroup
\let\center\flushleft
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\selectlanguage{english}
\begin{abstract}
Objective: Nephrin is a protein in the glomerular podocyte slit
diaphragm; therefore, its presence in urine implies damage to podocytes.
This study aimed to determine the efficacy of nephrin as a biomarker in
maternal urine to predict preeclampsia (PE). Design and setting: This
prospective study included pregnant women admitted for delivery at Seoul
National University Bundang Hospital: March 2019 -- May 2020.
Population: Patients who had been diagnosed with PE were included and
patients without a history of underlying diseases were recruited for the
control group. Important clinical data has been collected. Methods:
Urine samples were obtained, and nephrin signaling was detected through
test strips using a lateral flow assay. Main Outcome Measures: The
results of the point-of-care test were compared between the 2 groups:
patients with PE, and without (control group) using the exact
concentration of nephrin by enzyme-linked immunosorbent assay (ELISA).
Results: Clinical characteristics -- maternal age, rate of nulliparity,
proportion of twin pregnancies, height, weight, cesarean section rate --
were comparable between the PE and control groups. Nephrin signals were
classified into four groups. In the PE group, signals 0, 1, 2, and 3
were found in 18.4\% (9/49), 44.9\% (22/49), 24.5\% (12/49), and 12.2\%
(6/49) of participants, respectively. This was significantly different
from the control group, in which 84.3\% (43/51) were found to have
signal 0 (P\textless{}0.001). Conclusions: Nephrin signaling in maternal
urine could be a noninvasive and useful test for predetecting severity
of PE.%
\end{abstract}%



\sloppy


\textbf{Nephrin in maternal urine applied to a point-of-care kit to
predict preeclampsia:a prospective study.}

Kyong-No Lee\textsuperscript{a}, Subeen Hong\textsuperscript{c}, Tae Eun
Kim\textsuperscript{a}, Eun Ji Oh\textsuperscript{a}, Ki-seok
Kim\textsuperscript{b}, Ju-Hyung Kang\textsuperscript{b}, Min-Young
Yeo\textsuperscript{b}, Hyeon Ji Kim\textsuperscript{a}, Jee Yoon
Park\textsuperscript{a}

\textsuperscript{a}Department of Obstetrics and Gynecology, Seoul
National University College of Medicine, Seoul National University
Bundang Hospital, Seongnam, Gyeonggi, Republic of Korea

\textsuperscript{b}Aptamer Sciences Inc., Seongnam, Gyeonggi, Republic
of Korea

\textsuperscript{c}Department of Obstetrics and Gynecology, College of
Medicine, The Catholic University of Korea, Seoul, Republic of Korea

\textbf{Correspondence to:}

Jee Yoon Park, MD

Department of Obstetrics and Gynecology Seoul National University
Bundang Hospital

82 Gumi-ro, 173 beon-gil, Bundang-gu, Seongnam-si, Gyeonggi-do, 13620,
Republic of Korea

Fax: +82-31-787-4054

Tel: +82-31-787-7266

E-mail: \emph{jyparkmd08@snubh.org}

\textbf{Running Title:} Predicting severity of preeclampsia with nephrin



\textbf{Abbreviations:}

\begin{itemize}
\tightlist
\item
  Area under the curve (AUC)
\item
  Enzyme-linked immunosorbent assay (ELISA)
\item
  Placental growth factor (PlGF)
\item
  Preeclampsia (PE)
\item
  Soluble endoglin (sEng)
\item
  Soluble fms-like tyrosine kinase 1 (sFlt-1)
\end{itemize}

\textbf{Abstract}

\textbf{Objective:} Nephrin is a protein in the glomerular podocyte slit
diaphragm; therefore, its presence in urine implies damage to podocytes.
This study aimed to determine the efficacy of nephrin as a biomarker in
maternal urine to predict preeclampsia (PE).

\textbf{Design and setting} : This prospective study included pregnant
women admitted for delivery at Seoul National University Bundang
Hospital: March 2019 -- May 2020.

\textbf{Population:} Patients who had been diagnosed with PE were
included and patients without a history of underlying diseases were
recruited for the control group. Important clinical data has been
collected.

\textbf{Methods:} Urine samples were obtained, and nephrin signaling was
detected through test strips using a lateral flow assay.

\textbf{Main Outcome Measures:} The results of the point-of-care test
were compared between the 2 groups: patients with PE, and without
(control group) using the exact concentration of nephrin by
enzyme-linked immunosorbent assay (ELISA).

\textbf{Results} : Clinical characteristics -- maternal age, rate of
nulliparity, proportion of twin pregnancies, height, weight, cesarean
section rate -- were comparable between the PE and control groups.
Nephrin signals were classified into four groups. In the PE group,
signals 0, 1, 2, and 3 were found in 18.4\% (9/49), 44.9\% (22/49),
24.5\% (12/49), and 12.2\% (6/49) of participants, respectively. This
was significantly different from the control group, in which 84.3\%
(43/51) were found to have signal 0 (\emph{P} \textless{}0.001).

\textbf{Conclusions} : Nephrin signaling in maternal urine could be a
noninvasive and useful test for predetecting severity of PE.

Funding: Seoul National University Bundang Hospital Research Fund
{[}grant number 06-2019-198{]}.

\textbf{Keywords:} preeclampsia, proteinuria, nephrin, biomarker,
preterm birth

\textbf{Tweetable abstract for social media:}

This study aimed to determine the efficacy of nephrin as a biomarker in
maternal urine to predict preeclampsia (PE). Patients who had been
diagnosed with PE were included and patients without a history of
underlying diseases were recruited for the control group. Urine samples
were obtained, and nephrin signaling was detected. Nephrin signals were
classified into four groups. In the PE group, signals 0, 1, 2, and 3
were found in 18.4\%, 44.9\%, 24.5\%, and 12.2\% of participants. In the
control group, in which 84.3\% were found to have signal 0 (\emph{P}
\textless{}0.001). The severity of PE was, however, implied according to
the intensity of signals in this study.

\textbf{Introduction}

Preeclampsia (PE) is identified in approximately 4--5\% of pregnancies
and is the second most common cause of pregnancy-related maternal death.
More than 90\% of maternal deaths occur in low-and lower-middle-income
countries\textsuperscript{1,2}. PE leads not only to maternal death and
short-term maternal morbidities but also to long-term cardiovascular
sequelae\textsuperscript{3}. By terminating pregnancy through early
diagnosis, more than half of all hypertension-related mortality can be
prevented\textsuperscript{4}. The diagnosis of PE is established when
women have high blood pressure and evidence of multi-organ involvement,
such as proteinuria, thrombocytopenia, liver involvement, or cerebral
symptoms\textsuperscript{5,6}. However, this method requires
considerable time and specialized facilities to obtain laboratory
findings and evidence of abnormal protein excretion in urine. In
countries where this method cannot be easily performed, the diagnosis
and treatment of PE may be delayed. To date, efforts have been made to
develop easy and simple methods for the diagnosis of PE. However, they
are still in the developmental stage and have not been fully validated.

Nephrin is a transmembrane protein of the slit diaphragm in podocytes
and consists of a renal filtration barrier\textsuperscript{7}. In kidney
injuries, damaged podocytes lead to alterations of the slit diaphragm
and foot process structure, resulting in nephrinuria. Thus, urine
nephrin is well known as an early biomarker for glomerular injury in
glomerulonephritis and diabetic nephropathy\textsuperscript{8-11}. In
addition, some studies have revealed that women with PE have
nephrinuria, which is considered a marker for possible renal damage and
a predictive indicator of severe PE\textsuperscript{12-15}. Using
aptamer-based technology, we attempted to develop a point-of-care test
quantifying nephrin excretion in urine, which could be used to rapidly
diagnose PE. The purpose of this study was to develop an assay to verify
the accuracy and performance of this test in the diagnosis of PE.

\textbf{Methods}

\emph{Development of the point-of-care test quantifying nephrin
excretion}

\emph{Modified systematic evolution of ligands by exponential enrichment
(SELEX)}

The advanced SELEX was used as described by Gold et al. Briefly, we
prepared a DNA library containing 40-nucleotide randomized region in
which dT is substituted with 5-(N-benzyl carboxyamide)-2'-deoxyuridine
(Bz-dU) or 5-(N-naphthylcarboxyamide)-2'-deoxyuridine (Nap-dU). The
randomized central region was flanked by two conserved regions of 17
nucleotides (5'-GAG TGACCGTCTGCCTG-40N-CAGCCACACCACCAGCC-3').
Twenty-five thermal cycles (93 for 30 s, 52 for 20 s, and 72 for 60
s) were conducted to amplify the library. The library was applied for
the SELEX process and the process was performed at 37 with following
steps. A mixture of 1 mmol of aptamer library dissolved in the buffer
(40 mM HEPES/pH 7.5, 120 mM NaCl, 5 mM KCl, 5 mM MgCl2, 0.002\% Tween
20) was heated at 95 for 3 min, and then slowly cooled to 37 at 0.1/s
for re-folding. To eliminate the non-specific binder aptamers, the
aptamer library solution was pre-incubated with His-tagged magnetic bead
(Invitrogen, Grand Island, NY), and supernatant was collected. The
aptamers in supernatant were incubated with 10 pmol of nephrin for 30
min and then the nephrin was captured through the His.

\emph{Binding affinity}

The aptamer-protein equilibrium dissociation constants (Kd) were
determined by the nitrocellulose-filter binding method. Before the
binding assay, aptamers were dephosphorylated using alkaline phosphatase
and their 5'-ends were radiolabeled using T4 polynucleotide kinase (New
England Biolabs) and {[}\textsuperscript{32}P{]}-ATP (Amersham Pharmacia
Biotech, Piscataway, NJ). The direct binding assays were conducted by
incubation of 10pM of \textsuperscript{32}P-labeled aptamers with
recombinant nephrin at a concentration ranging from 1 mM to 10 fM in the
selection buffer at 37. The fraction of aptamers bound to nephrin was
quantified with a PhosPhorImager (Fuji FLA-5100 Image Analyzer, Tokyo,
Japan). The data was corrected by subtraction of nonspecific binding
signal generated by binding of radiolabeled aptamers to the
nitrocellulose filter from the obtained signal.

\emph{Binding competition assay}

To select the aptamer pairs binding different regions of nephrin,
binding competition assays were performed using
{[}\textsuperscript{32}P{]}-labeled aptamer (hot aptamers) and unlabeled
aptamers (cold aptamers). We tested all aptamer pair candidates to
identify the best pair that did not compete with each other for binding
nephrin. Hot aptamers (2,000 cpm) and 25 pmole cold aptamers were
dissolved in S buffer (30 \selectlanguage{greek}μ\selectlanguage{english}L) and the solution was heated at 95 for 3
min and then slowly cooled to 25 at a rate of 0.1/s. After
transferring to a 96 well plate containing 30 \selectlanguage{greek}μ\selectlanguage{english}L of 10 nM nephrin, the
aptamer solution was incubated at 25 for 15 min. Then, 5.5 \selectlanguage{greek}μ\selectlanguage{english}L of Zorbax
resin solution was added to the reaction mixture followed agitation in a
Thermomixer for 1 min at 1,300 rpm. The mixture was applied to a PVDF
filter plate, and the hot aptamers bound to nephrin were quantified
using a phosphor-imager (Fuji FLA-5100 Imamge Analyzer).

\emph{Preparation of capture and detection aptamers for LFA}

The aptamers were modified to be used as capture or detection aptamers.
For capture aptamers, 5' biotin-labelled aptamers were conjugated with
neutravidin to efficiently spot them on the nitrocellulose membrane. The
biotin-labelled aptamers were dissolved in 5 \selectlanguage{greek}μ\selectlanguage{english}L of 1xPBS buffer at the
concentration of 80 \selectlanguage{greek}μ\selectlanguage{english}M. The solution was heated at 95 for 5 min and
cooled at 37 for 15 min, then mixed with 80 pmol of neutravidin (5 \selectlanguage{greek}μ\selectlanguage{english}L)
and incubated at 37 for 1 hour. The detection aptamers were prepared by
conjugating with streptavidin-gold nano particle (SA-GNP). The
biotin-labelled aptamers were dissolved in 100 \selectlanguage{greek}μ\selectlanguage{english}L of PBS buffer at a
concentration of 2 \selectlanguage{greek}μ\selectlanguage{english}M and the solution underwent heating and cooling as
mentioned above. The solution was mixed with 100 \selectlanguage{greek}μ\selectlanguage{english}L of SA-GNP (10 O.D
units/mL) and then 250 \selectlanguage{greek}μ\selectlanguage{english}L of 1xPBS was added. The mixture was incubated
for 1 hour at room temperature with shaking, them the mixture was
centrifuge at 4, 8,500 rpm. After removing the supernatant, the pellet
was resuspended with 100 uL of 1xPBS buffer. We applied 6 uL of the
detection aptamer solution for each strip.

\emph{Preparation of test strips}

The test strip consisted of a sample pad, a nitrocellulose membrane, and
an absorbent pad. A 0.8 mm sample pad was immersed in a sample pad
solution (10 mM sodium phosphate (pH 7.0), 10\% Tween 20) and dried at
room temperature in a desiccator. To assemble the test strip, a
nitrocellulose membrane was attached to the middle of the backing card,
and then a sample pad and an absorbent pad were attached to the bottom
and the top of the backing card, respectively. We spotted 0.5 \selectlanguage{greek}μ\selectlanguage{english}L of
capture aptamer solution and the control aptamer solution onto a
nitrocellulose membrane strip and dried for 2 days in room temperature.
The schematic illustration of the LFA system using a pair of nephrin
specific aptamers is described in figure 1.

\emph{Lateral flow assay}

We prepared 100 \selectlanguage{greek}μ\selectlanguage{english}L of each sample in test tubes and mixed it with 6 \selectlanguage{greek}μ\selectlanguage{english}L
of detection aptamer solution and 1 \selectlanguage{greek}μ\selectlanguage{english}L of DxSO\textsubscript{4}. Then
the samples were soaked via the sample pad on a test strip. We read the
signal after 15 mins of incubation and imaged it with a scanner. To
determine the limit of detection of LFA for nephrin, we tested with
commercially available nephrin. Various concentrations of nephrin (0,
0.5, 1, 2, 5 \selectlanguage{greek}μ\selectlanguage{english}g/mL) were prepared by spiking in the LF running buffer or
urine. Figure 2 demonstrates the competition of aptamers for binding to
nephrin protein. Positive signals were observed from nephrin protein
diluted from 5 \selectlanguage{greek}μ\selectlanguage{english}g/mL to 0.5 \selectlanguage{greek}μ\selectlanguage{english}g/mL. The limit of detection (LOD) of the
aptamer-based LFA was 0.5 \selectlanguage{greek}μ\selectlanguage{english}g/mL or lower.

\emph{Study design and population}

This study was conducted with women admitted to the Seoul National
University Bundang Hospital between March 2019 and March 2020. Pregnant
female volunteers were recruited for this study. The case group included
women diagnosed with PE; and the control group consisted of women who
did not present with PE or kidney disease up until delivery. After
obtaining informed consent, we collected urine samples (15 ml) at
admission from women diagnosed with PE and at the time of
hospitalization for delivery from the normal control group. Spot urine
samples were stored at 4 \selectlanguage{ngerman}°C immediately after collection and tested
within 48 h. The calculation of the number of target participants was
based on a specificity of 92\%, according to the literature on the
number of samples required for the performance evaluation of the
diagnostic kit. The formula for the calculation is as follows:

\begin{equation}
N=P\ \times\left(1-P\right)\times\left(\frac{\mathcal{Z}_{\frac{a}{2}}}{d}\right)^{2}\nonumber \\
 \end{equation}

When the margin of error (d) is calculated as 0.08, at the significance
level \selectlanguage{greek}α\selectlanguage{english}=0.05, the minimum number of participants required to show a
specificity of 92\% or higher according to the above formula is 44.
Considering a 15\% dropout rate, 51 patients were required per group.
The results of the point-of-care test were compared (between the 2
groups) using the exact concentration of nephrin by enzyme-linked
immunosorbent assay (ELISA). The ELISA assay was performed twice to
measure the concentration of nephrin protein using the Human Nephrin
DuoSet\selectlanguage{ngerman}® ELISA Kit (R\&D Systems, Minneapolis, MN, USA) according to the
manufacturer's instructions. This study was approved by the
Institutional Review Board of the Seoul National University Bundang
Hospital (IRB No. B-1904/537-304).

\emph{Statistical analysis}

We used the Student's t-test for the analysis of continuous variables,
and the chi-square test or Fisher's exact test for the analysis of
categorical variables. To determine the signal intensity distribution of
the aptamer-based assay according to the severity of PE, a
linear-by-linear association was used. The Wilson interval method was
used to evaluate the following: diagnostic performance, sensitivity and
specificity, positive and negative predictive values, and positive and
negative likelihood ratios with 95\% confidence intervals. A\emph{P}
-value of \textless{}.05 was considered statistically significant. IBM®
SPSS® Statistics version 25.0 software (IBM® Inc., Armonk, NY) was used
for the analyses.

This study was supported by the Seoul National University Bundang
Hospital Research Fund {[}grant number 06-2019-198{]}.

\textbf{Results}

Table 1 presents the baseline characteristics of the study population.
There was no difference between the PE group and the control group in
age, parity, the proportion of twin pregnancies, preexisting
hypertension or diabetes mellitus, or the proportion of gestational
hypertension or diabetes mellitus. The gestational age at
hospitalization was lower in the PE group than in the control group
(32.8 weeks versus 37.0 weeks of gestation, respectively). The most
common sign in the PE group was high blood pressure, followed by
proteinuria, presence of PE-related symptoms, and elevation of liver
enzymes. Increased levels of creatinine, thrombocytopenia, and pulmonary
edema were observed in less than 10\% of the patients. In the control
group, no signs or symptoms except for high blood pressure and
proteinuria were observed.

Table 2 shows the signal intensity distribution of the aptamer-based
assay according to the severity of PE. Of the 50 women with PE, 37
(74\%) showed severe symptoms after admission, and 13 (26\%) were
diagnosed with PE without severe symptoms. Of the 52 women with signal 0
in the assay, 43 (83\%) were in the control group, and 9 (17\%) were
diagnosed with PE (regardless of the signal 0 in the assay). On the
other hand, 80\% of women with signal 2 and 71\% of those with signal 3,
respectively, showed PE with severe features. Only 14\% of women with
signals 2 or 3 were in the control group. The linear-by-linear
association between the signal intensity and severity of PE was shown to
be significant. The quantified concentration of nephrin measured from
clinical samples by ELISA and the receiver operating characteristic
curve are shown in Figure 3. The area under the curve (AUC) regarding
the diagnosis of PE was 0.688, and the \emph{P} -value was
\textless{}0.005.

Table 3 shows the diagnostic performance of the assay in PE and PE with
severe features when the intensity of the assay was \selectlanguage{english}[?]1 and \selectlanguage{english}[?]2,
respectively. When signal 1 (or higher) was positive, the detection rate
for PE was 82\% and the detection rate for PE with severe features was
89\%, which was significantly higher than that of signal 2 (or higher)
when positive. When signal 1 (or higher) was positive, the negative
predictive value for PE with severe features was 92.3\%. However, when
signal 2 (or higher) was positive, the false-positive rate
(1-specificity) was 6\%, which was significantly lower than that of
signal 1 (or higher).

\textbf{Discussion}

Since PE is a very serious complication during pregnancy, researchers
have been searching for various biological markers to enable early
detection. To date, clinical signs such as high blood pressure and
cerebral symptoms are considered to be the most important symptoms of
PE; many studies have reported the predictive value of soluble fms-like
tyrosine kinase 1 (sFlt-1) and placental growth factor
(PlGF)\textsuperscript{16-19}. However, few guidelines have selected the
sFlt-1/PlGF ratio as a diagnostic tool for PE, and a major disadvantage
of the sFlt-1/PlGF ratio is that it is measured in maternal blood, which
means the test is available only in medical centers. Here, we identified
the possibility of detecting a marker for PE in maternal urine. This has
significant implications for the early detection of PE because urine
tests are easier to perform and typically less costly than other tests
using samples--such as serum.

Several studies have suggested podocyturia as a novel marker for the
diagnosis of PE since significant excretion of podocytes in urine was
found among women diagnosed with PE; these studies also demonstrated
that acute but transient podocyturia in PE tends to be closely related
to continued heavy proteinuria\textsuperscript{20,21}. Nevertheless,
diagnosing podocyturia has only been done in laboratory settings
involving the incubation and staining of urinary cells with
podocyte-related proteins such as podocin and
nephrin\textsuperscript{12,22-25}. To solve this time-consuming problem,
many studies were conducted to discover the urinary podocyte-specific
proteins associated with PE, and several of them showed that the urinary
concentration of nephrin was significantly higher in patients with PE
than in those with normal pregnancies\textsuperscript{13,14,26}.

\textbf{Main findings:} In this study, we analyzed the diagnostic value
of nephrin for PE. In addition to measuring the urine concentration of
nephrin, we developed a point-of-care kit using nephrin-specific
aptamers. The signals expressed by the kit were related to the presence
and severity of PE. In the PE group: signals 1, 2, and 3 were found in
45\%, 25\%, and 12\% of participants, respectively. In the control
group: 84\% was found to have signal 0, and the difference between the
PE group and the control group was significant because signal 0 was
found in only 18\% of the PE group (\emph{P} \textless{}0.001). The
sensitivity and specificity of positive signals (1, 2, and 3) to predict
PE were 82\% and 84\%, respectively. This study suggests the possibility
of nephrin as a noninvasive and useful test to diagnose PE. Although the
predictive values still need to be further studied and refined, the
urine test kit using nephrin can be used to predict the development of
PE in the general population.

\textbf{Interpretation:} The critical interval (of increasing or
decreasing various biomarkers studied previously for the detection of
PE) is relatively large. For example, the time interval in which soluble
endoglin (sEng) increases is known to be from 5 weeks to 3 months after
the onset of PE. For the sFlt-1/PlGF ratio, the positive predictive
value reaches its highest point at 4 weeks of PE development. Compared
to those markers, the level of nephrin in urine abruptly increases
within 9 days of the onset of a clinical manifestation of
PE\textsuperscript{27,28}. This short interval of nephrin detection in
urine related to the development of PE could be useful to predict the
disease process early in pregnancy and to apply effective interventions
to manage the disease\textsuperscript{29-32}.

For the last few decades, nephrin as a structural component of the
podocyte slit diaphragm has been studied; however, the potential roles
in extra-renal tissues and in acquired kidney diseases are not yet well
known. Several groups studied the function of nephrin in various kidney
diseases either by analyzing the production of nephrin mRNA and/or
protein or by sequencing the nephrin gene for mutations in the samples
of affected patients\textsuperscript{10,33-36}. In a previous study, Jim
et al. investigated whether the detection of nephrin in urine could be
used as an early biomarker of diabetic nephropathy\textsuperscript{10}.
Another study investigated the association between nephrinuria and
various traits causing renal dysfunctions in type 2
diabetes\textsuperscript{37}.

\textbf{Strengths and limitations:} A limitation of the present study is
the relatively small sample size; however, the data collected
prospectively were sufficient in showing one of the possibilities of
nephrin: as a valuable predictive tool for PE, and to suggest the use of
nephrin for an easy point-of-care test. Since urine is non-invasive and
an easily obtainable sample, which can be acquired readily and
repeatedly, using urinary markers to detect PE warrants the affirmative
market feasibility in medical practice. Research on urine markers is
especially promising because proteinuria and kidney malfunction are the
main features of PE. Since the samples were collected at the time of
diagnosis for PE, the association between nephrin and the development of
PE might not represent the predictive value of nephrin.

\textbf{Conclusion:}

The severity of PE was, however, implied according to the intensity of
signals in this study. Future studies with large sample sizes are
essential to compensate for the inconsistency of urine markers and to
purify the specific markers, preventing any contamination caused by
common urinary tract infections.

\textbf{Acknowledgements:} none

\textbf{Disclosure of interests} : none

\textbf{Contribution to authorship} :

Kyong-No Lee: Drafting of the manuscript

Subeen Hong: Drafting and editing of the draft

Tae Eun Kim: Editing of the draft

Eun Ji Oh: Editing of the draft

Ki-seok Kim, Ju-Hyung Kang, Min-Young Yeo: Planning, Analyzing data

Hyeon Ji Kim: Drafting and editing of the draft

Jee Yoon Park: Planning, analyzing data, drafting, and editing the
draft.

\textbf{Details of Ethics approval:} This study was approved by the
Institutional Review Board of the Seoul National University Bundang
Hospital (IRB No. B-1904/537-304).

\textbf{Funding} : This study was supported by the Seoul National
University Bundang Hospital Research Fund {[}grant number
06-2019-198{]}.

\textbf{Reference}

1. Anderson UD, Olsson MG, Kristensen KH, Akerstrom B, Hansson SR.
Review: Biochemical markers to predict preeclampsia. Placenta. 2012;33
Suppl:S42-7.

2. Venkatesha S, Toporsian M, Lam C, Hanai J, Mammoto T, Kim YM, et al.
Soluble endoglin contributes to the pathogenesis of preeclampsia. Nat
Med. 2006;12(6):642-9.

3. Paauw ND, Lely AT. Cardiovascular Sequels During and After
Preeclampsia. Adv Exp Med Biol. 2018;1065:455-70.

4. Rana S, Lemoine E, Granger JP, Karumanchi SA. Preeclampsia:
Pathophysiology, Challenges, and Perspectives. Circ Res.
2019;124(7):1094-112.

5. Ozkara A, Kaya AE, Ba\selectlanguage{polish}ş\selectlanguage{english}bu\selectlanguage{polish}ğ \selectlanguage{english}A, \selectlanguage{ngerman}Ökten SB, Do\selectlanguage{polish}ğ\selectlanguage{english}an O, \selectlanguage{ngerman}Ça\selectlanguage{polish}ğ\selectlanguage{english}lar M, et al.
Proteinuria in preeclampsia: is it important? Ginekol Pol.
2018;89(5):256-61.

6. Gestational Hypertension and Preeclampsia: ACOG Practice Bulletin,
Number 222. Obstet Gynecol. 2020;135(6):e237-e60.

7. Tryggvason K, Wartiovaara J. Molecular basis of glomerular
permselectivity. Curr Opin Nephrol Hypertens. 2001;10(4):543-9.

8. Ziyadeh FN, Wolf G. Pathogenesis of the podocytopathy and proteinuria
in diabetic glomerulopathy. Curr Diabetes Rev. 2008;4(1):39-45.

9. Alter ML, Kretschmer A, Von Websky K, Tsuprykov O, Reichetzeder C,
Simon A, et al. Early urinary and plasma biomarkers for experimental
diabetic nephropathy. Clin Lab. 2012;58(7-8):659-71.

10. Jim B, Ghanta M, Qipo A, Fan Y, Chuang PY, Cohen HW, et al.
Dysregulated nephrin in diabetic nephropathy of type 2 diabetes: a cross
sectional study. PLoS One. 2012;7(5):e36041.

11. Matsusaka T, Sandgren E, Shintani A, Kon V, Pastan I, Fogo AB, et
al. Podocyte injury damages other podocytes. J Am Soc Nephrol.
2011;22(7):1275-85.

12. Hauser PV, Collino F, Bussolati B, Camussi G. Nephrin and
endothelial injury. Curr Opin Nephrol Hypertens. 2009;18(1):3-8.

13. Son GH, Kwon JY, Lee S, Park J, Kim YJ, Yun B, et al. Comparison of
serum and urinary nephrin levels between normal pregnancies and severe
preeclampsia. Eur J Obstet Gynecol Reprod Biol. 2013;166(2):139-44.

14. Wang Y, Zhao S, Loyd S, Groome LJ. Increased urinary excretion of
nephrin, podocalyxin, and \selectlanguage{greek}β\selectlanguage{english}ig-h3 in women with preeclampsia. Am J
Physiol Renal Physiol. 2012;302(9):F1084-9.

15. Jung YJ, Cho HY, Cho S, Kim YH, Jeon JD, Kim YJ, et al. The Level of
Serum and Urinary Nephrin in Normal Pregnancy and Pregnancy with
Subsequent Preeclampsia. Yonsei Med J. 2017;58(2):401-6.

16. Garovic VD, Wagner SJ, Petrovic LM, Gray CE, Hall P, Sugimoto H, et
al. Glomerular expression of nephrin and synaptopodin, but not podocin,
is decreased in kidney sections from women with preeclampsia. Nephrol
Dial Transplant. 2007;22(4):1136-43.

17. Brown MA, Magee LA, Kenny LC, Karumanchi SA, McCarthy FP, Saito S,
et al. Hypertensive Disorders of Pregnancy: ISSHP Classification,
Diagnosis, and Management Recommendations for International Practice.
Hypertension. 2018;72(1):24-43.

18. Verlohren S, Herraiz I, Lapaire O, Schlembach D, Zeisler H, Calda P,
et al. New gestational phase-specific cutoff values for the use of the
soluble fms-like tyrosine kinase-1/placental growth factor ratio as a
diagnostic test for preeclampsia. Hypertension. 2014;63(2):346-52.

19. Verlohren S, Herraiz I, Lapaire O, Schlembach D, Moertl M, Zeisler
H, et al. The sFlt-1/PlGF ratio in different types of hypertensive
pregnancy disorders and its prognostic potential in preeclamptic
patients. Am J Obstet Gynecol. 2012;206(1):58.e1-8.

20. Aita K, Etoh M, Hamada H, Yokoyama C, Takahashi A, Suzuki T, et al.
Acute and transient podocyte loss and proteinuria in preeclampsia.
Nephron Clin Pract. 2009;112(2):c65-70.

21. Garovic VD, Craici IM, Wagner SJ, White WM, Brost BC, Rose CH, et
al. Mass spectrometry as a novel method for detection of podocyturia in
pre-eclampsia. Nephrol Dial Transplant. 2013;28(6):1555-61.

22. Kandasamy Y, Smith R, Lumbers ER, Rudd D. Nephrin - a biomarker of
early glomerular injury. Biomark Res. 2014;2:21.

23. Zhai T, Furuta I, Akaishi R, Ishikawa S, Morikawa M, Yamada T, et
al. Alteration of podocyte phenotype in the urine of women with
preeclampsia. Sci Rep. 2016;6:24258.

24. Zhao S, Gu X, Groome LJ, Wang Y. Decreased nephrin and GLEPP-1, but
increased VEGF, Flt-1, and nitrotyrosine, expressions in kidney tissue
sections from women with preeclampsia. Reprod Sci. 2009;16(10):970-9.

25. Collino F, Bussolati B, Gerbaudo E, Marozio L, Pelissetto S,
Benedetto C, et al. Preeclamptic sera induce nephrin shedding from
podocytes through endothelin-1 release by endothelial glomerular cells.
Am J Physiol Renal Physiol. 2008;294(5):F1185-94.

26. Son GH, Kim JH, Hwang JH, Kim YH, Park YW, Kwon JY. Urinary
excretion of nephrin in patients with severe preeclampsia. Urinary
nephrin in preeclampsia. Hypertens Pregnancy. 2011;30(4):408-13.

27. Levine RJ, Maynard SE, Qian C, Lim KH, England LJ, Yu KF, et al.
Circulating angiogenic factors and the risk of preeclampsia. N Engl J
Med. 2004;350(7):672-83.

28. Lynch AM, Murphy JR, Gibbs RS, Levine RJ, Giclas PC, Salmon JE, et
al. The interrelationship of complement-activation fragments and
angiogenesis-related factors in early pregnancy and their association
with pre-eclampsia. BJOG: An International Journal of Obstetrics \&
Gynecology. 2010;117(4):456-62.

29. Staff AC, Benton SJ, von Dadelszen P, Roberts JM, Taylor RN, Powers
RW, et al. Redefining preeclampsia using placenta-derived biomarkers.
Hypertension. 2013;61(5):932-42.

30. Roberts JM, Bell MJ. If we know so much about preeclampsia, why
haven't we cured the disease? J Reprod Immunol. 2013;99(1-2):1-9.

31. Myers JE, Kenny LC, McCowan LM, Chan EH, Dekker GA, Poston L, et al.
Angiogenic factors combined with clinical risk factors to predict
preterm pre-eclampsia in nulliparous women: a predictive test accuracy
study. BJOG: An International Journal of Obstetrics \& Gynecology.
2013;120(10):1215-23.

32. Tarca AL, Romero R, Benshalom-Tirosh N, Than NG, Gudicha DW, Done B,
et al. The prediction of early preeclampsia: Results from a longitudinal
proteomics study. PLoS One. 2019;14(6):e0217273.

33. Ruotsalainen V, Ljungberg P, Wartiovaara J, Lenkkeri U, Kestil\selectlanguage{ngerman}ä M,
Jalanko H, et al. Nephrin is specifically located at the slit diaphragm
of glomerular podocytes. Proceedings of the National Academy of
Sciences. 1999;96(14):7962.

34. Patrakka J, Tryggvason K. Nephrin--a unique structural and signaling
protein of the kidney filter. Trends Mol Med. 2007;13(9):396-403.

35. Verma R, Kovari I, Soofi A, Nihalani D, Patrie K, Holzman LB.
Nephrin ectodomain engagement results in Src kinase activation, nephrin
phosphorylation, Nck recruitment, and actin polymerization. J Clin
Invest. 2006;116(5):1346-59.

36. Ohmori T, De S, Tanigawa S, Miike K, Islam M, Soga M, et al.
Impaired NEPHRIN localization in kidney organoids derived from nephrotic
patient iPS cells. Sci Rep. 2021;11(1):3982.

37. Ng DP, Tai BC, Tan E, Leong H, Nurbaya S, Lim XL, et al. Nephrinuria
associates with multiple renal traits in type 2 diabetes. Nephrol Dial
Transplant. 2011;26(8):2508-14.

\textbf{Table 1.} The baseline characteristics of study population\selectlanguage{english}
\begin{longtable}[]{@{}llll@{}}
\toprule
\begin{minipage}[b]{0.24\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[b]{0.24\columnwidth}\raggedright\strut
Control (n=53)\strut
\end{minipage} & \begin{minipage}[b]{0.24\columnwidth}\raggedright\strut
Preeclampsia (n=50)\strut
\end{minipage} & \begin{minipage}[b]{0.24\columnwidth}\raggedright\strut
\emph{p} value\strut
\end{minipage}\tabularnewline
\midrule
\endhead
\begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
Age (years)\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
34.8\selectlanguage{ngerman}±4.4\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
35.2\selectlanguage{ngerman}±4.0\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
0.624\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
Nulliparity\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
39 (73.6\%)\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
36 (72.0\%)\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
0.857\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
Twin pregnancy\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
16 (30.2\%)\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
11 (22.0\%)\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
0.345\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
Preexsiting hypertension\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
1 (1.9\%)\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
3 (6.0\%)\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
0.353\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
Gestational hypertension\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
3 (5.7\%)\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
3 (6.0\%)\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
1.000\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
Preexsiting diabetes\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
0 (0.0\%)\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
1 (2.0\%)\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
0.485\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
Gesetatioanl diabetes\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
7 (13.2\%)\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
10 (20.0\%)\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
0.353\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
Gestational age at admission\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
37.0\selectlanguage{ngerman}±2.9\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
32.8\selectlanguage{ngerman}±3.3\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
\textless{}0.001\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
Presence of severe symptom\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
0 (0.0\%)\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
20 (40.0\%)\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
\textless{}0.001\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
High blood pressure (\textgreater{}160/90mmHg)\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
7 (13.2\%)\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
50 (100\%)\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
\textless{}0.001\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
Proteinuria\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
\ldots{} Dipstick \textgreater{} 1+\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
7 (13.2\%)\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
47 (94.0\%)\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
\textless{}0.001\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
\ldots{} Proteinuria in 24 hour \textgreater{} 300mg or
Protein/Creatinine ratio \textgreater{} 0.3\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
0 (0.0\%)\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
38 (76.0\%)\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
\textless{}0.001\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
Elevation of liver enzyme\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
0 (0.0\%)\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
12 (24.0\%)\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
\textless{}0.001\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
Elevation of Creatinine\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
0 (0.0\%)\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
5 (10.0\%)\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
0.024\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
Low platelet count \textless{} 10,000\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
0 (0.0\%)\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
2 (4.0\%)\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
0.233\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
Pulmonary edema\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
0 (0.0\%)\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
2 (4.0\%)\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
0.233\strut
\end{minipage}\tabularnewline
\bottomrule
\end{longtable}

\textbf{Table 2.} Distribution of signal according to the severity of
preeclampsia\selectlanguage{english}
\begin{longtable}[]{@{}lllll@{}}
\toprule
\begin{minipage}[b]{0.19\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[b]{0.19\columnwidth}\raggedright\strut
Control (n=53)\strut
\end{minipage} & \begin{minipage}[b]{0.19\columnwidth}\raggedright\strut
Preeclampsia without severe feature (n=13)\strut
\end{minipage} & \begin{minipage}[b]{0.19\columnwidth}\raggedright\strut
Preeclampsia with severe feature (n=37)\strut
\end{minipage} & \begin{minipage}[b]{0.19\columnwidth}\raggedright\strut
\emph{p-}value\strut
\end{minipage}\tabularnewline
\midrule
\endhead
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
0 (n=52)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
43 (82.7\%)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
5 (9.6\%)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
4 (7.7\%)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\textless{}0.001\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
1 (n=29)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
7 (24.1\%)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
6 (20.7\%)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
16 (55.2\%)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
2 (n=15)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
2 (13.3\%)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
1 (6.7\%)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
12 (80.0\%)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
3 (n=7)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
1 (14.3\%)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
1 (14.3\%)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
5 (71.4\%)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\bottomrule
\end{longtable}

\textbf{Table 3.} Diagnostic performances of the point-of-care test
using nephrin\selectlanguage{english}
\begin{longtable}[]{@{}llllllll@{}}
\toprule
\begin{minipage}[b]{0.10\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[b]{0.10\columnwidth}\raggedright\strut
Sensitivity\strut
\end{minipage} & \begin{minipage}[b]{0.10\columnwidth}\raggedright\strut
Specificity\strut
\end{minipage} & \begin{minipage}[b]{0.10\columnwidth}\raggedright\strut
PPV\strut
\end{minipage} & \begin{minipage}[b]{0.10\columnwidth}\raggedright\strut
NPV\strut
\end{minipage} & \begin{minipage}[b]{0.10\columnwidth}\raggedright\strut
pLR\strut
\end{minipage} & \begin{minipage}[b]{0.10\columnwidth}\raggedright\strut
nLR\strut
\end{minipage} & \begin{minipage}[b]{0.10\columnwidth}\raggedright\strut
nLR\strut
\end{minipage}\tabularnewline
\midrule
\endhead
\begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
Signaling [?]1\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
Preeclampsia with severe feature\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
89.2* (74.6-97.0)\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
72.7* (60.4-83.0)\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
64.7 (50.1-77.6)\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
92.3 (81.5-97.9)\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
3.27\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
3.27\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
0.15\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
Preeclampsia (total)\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
82.0* (68.6-91.4)\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
81.1** (68.0-90.6)\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
80.4 (66.9-90.2)\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
82.7 (69.7-91.8)\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
4.35\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
4.35\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
0.22\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
Signaling [?]2\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
Preeclampsia with severe feature\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
46.0* (29.5-63.1)\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
92.4* (83.2-97.5)\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
77.3 (54.6-92.2)\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
75.3 (64.5-84.2)\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
6.06\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
6.06\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
0.58\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
Preeclampsia (total)\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
38.0* (24.7-52.8)\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
94.3** (84.3-98.8)\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
86.4 (65.1-97.1)\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
61.7 (50.3-72.3)\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
6.71\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
6.71\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
0.66\strut
\end{minipage}\tabularnewline
\bottomrule
\end{longtable}

PPV, positive predictive value; NPV, negative predictive value; pLR,
positive likelihood ratio; nLR, negative likelihood ratio

* P values \textless{}0.001 for the comparison between signaling [?]1 and
signaling [?]2

** P values \textless{}0.05 for the comparison between signaling [?]1 and
signaling [?]2\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/figure-1/figure-1}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/figure-2/figure-2}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/figure-3/figure-3}

\end{center}
\end{figure}

\selectlanguage{english}
\FloatBarrier
\end{document}

