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\begin{document}

\title{Increased fetal fraction is associated with the incidence of birth
weight discordance and selective fetal growth restriction:a
retrospective cohort study}



\author[1]{Renyi Hua}%
\author[1]{Yuanqing Xia}%
\author[1]{Shan Wang}%
\author[1]{Jinling Sun}%
\author[1]{Li  Gao}%
\author[1]{Yi Wu}%
\author[1]{Xinrong Zhao}%
\author[1]{Yanlin Wang}%
\affil[1]{International Peace Maternity and Child Health Hospital}%


\vspace{-1em}



  
  \date{May 25, 2024}


\begingroup
\let\center\flushleft
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\selectlanguage{english}
\begin{abstract}
\textbf{Objective} To test the hypothesis that the fetal fraction in
twin pregnancy would reflect birthweight discrepancy. \textbf{Design}
Retrospective cohort study. \textbf{Setting} International Peace
Maternity and Child Health Hospital in Shanghai, China
\textbf{Population} We included 237 twin pregnancies undergoing cfDNA
screening for aneuploidy and delivered at International Peace Maternity
and Child Health Hospital in Shanghai, China between January 2018 to
December 2021. Exclusion criteria including abnormal NIPT results,
fetal demise or structural abnormalities. \textbf{Methods} All women
with twin pregnancies were offered a scan to determine chorionicity as
well as first-trimester nuchal translucency (NT) at 11
\textsuperscript{+0}-13 \textsuperscript{+6} weeks. Dichorionic was
confirmed when ultrasound assessment clearly indicates two placentas.
The twin peak sign is used to distinguish chorionicity if only one
placenta is visualized. \textbf{Main Outcome Measures} Fetal fraction
and birth weight of the new borns were collected. Relationships between
fetal fraction and birth weight discordance or sFGR were analysed.
\textbf{Results} Fetal fraction was positively correlated with the
difference of birth weight (\selectlanguage{greek}β\selectlanguage{english}=0.004, 95\%CI:
0.001\textasciitilde{}0.006). Higher fetal fraction was significantly
associated with the increased risk of birthweight discordance of 20\%
(adjusted OR:1.073, 95\%CI: 1.009\textasciitilde{}1.142) and 25\%
(adjusted OR:1.092, 95\%CI: 1.006\textasciitilde{}1.185) and
sIUGR(adjusted OR:1.130, 95\%CI: 1.038\textasciitilde{}1.231). We
obtained the optimum cut-off point of fetal fraction [?] 11.790, [?] 14.800
and [?] 14.800 for birthweight discordance of 20\% and 25\% and sFGR,
respectively. \textbf{Conclusion} This study shown that fetal fraction
was positively correlated with the difference of birth weight. Fetal
fraction could be used as a biomarker in predating birth weight
discordance and sFGR, and help to make individualized clinical
monitoring plans for twin pregnancies.%
\end{abstract}%



\sloppy


\textbf{Original article}

\textbf{Increased fetal fraction is associated with the incidence of
birth weight discordance and selective fetal growth restriction:a
retrospective cohort study}

Renyi Hua\textsuperscript{\#1,2}, Yuanqing Xia\textsuperscript{\#1,2},
Shan Wang\textsuperscript{1,2}, Jinling Sun\textsuperscript{1,2}, Li
Gao\textsuperscript{1,2}, Yi Wu\textsuperscript{1,2}, Xinrong
Zhao\textsuperscript{1,2}, Yanlin Wang*\textsuperscript{1,2}

International Peace Maternity and Child Health Hospital of China Welfare
Institution (IPMCH), Shanghai, China

School of Medicine, Shanghai Jiao Tong University, Shanghai Key
Laboratory of Embryo Original Diseases

*Correspondent author: Yanlin Wang, International Peace Maternity and
Child Health Hospital of China Welfare Institution, 910 Heng Shan Road,
Shanghai, China, 200030; Tel: 0086-21-64070434

\textsuperscript{\#}Renyi Hua and Yuanqing Xia are joint first author

\textbf{Keywords} : twin pregnancy; Non-invasive prenatal testing; fetal
fraction; selective fetal growth restriction

\textbf{ABSTRACT}

\textbf{Objective}

To test the hypothesis that the fetal fraction in twin pregnancy would
reflect birthweight discrepancy.

\textbf{Design}

Retrospective cohort study.

\textbf{Setting}

International Peace Maternity and Child Health Hospital in Shanghai,
China

\textbf{Population}

We included 237 twin pregnancies undergoing cfDNA screening for
aneuploidy and delivered at International Peace Maternity and Child
Health Hospital in Shanghai, China between January 2018 to December
2021. Exclusion criteria including abnormal NIPT results, fetal demise
or structural abnormalities.

\textbf{Methods}

All women with twin pregnancies were offered a scan to determine
chorionicity as well as first-trimester nuchal translucency (NT) at
11\textsuperscript{+0}-13\textsuperscript{+6} weeks. Dichorionic was
confirmed when ultrasound assessment clearly indicates two placentas.
The twin peak sign is used to distinguish chorionicity if only one
placenta is visualized.

\textbf{Main Outcome Measures}

Fetal fraction and birth weight of the new borns were collected.
Relationships between fetal fraction and birth weight discordance or
sFGR were analysed.

\textbf{Results}

Fetal fraction was positively correlated with the difference of birth
weight (\selectlanguage{greek}β\selectlanguage{english}=0.004, 95\%CI: 0.001\textasciitilde{}0.006). Higher fetal
fraction was significantly associated with the increased risk of
birthweight discordance of 20\% (adjusted OR:1.073, 95\%CI:
1.009\textasciitilde{}1.142) and 25\% (adjusted OR:1.092, 95\%CI:
1.006\textasciitilde{}1.185) and sIUGR(adjusted OR:1.130, 95\%CI:
1.038\textasciitilde{}1.231). We obtained the optimum cut-off point of
fetal fraction [?] 11.790, [?] 14.800 and [?] 14.800 for birthweight
discordance of 20\% and 25\% and sFGR, respectively.

\textbf{Conclusion}

This study shown that fetal fraction was positively correlated with the
difference of birth weight. Fetal fraction could be used as a biomarker
in predating birth weight discordance and sFGR, and help to make
individualized clinical monitoring plans for twin pregnancies.

\textbf{Introduction}

In recent years, there has been a significant rise in twin pregnancies
due to the advancements in assisted reproductive technologies. However,
these pregnancies are more susceptible to complications such as fetal
growth restriction (FGR), premature birth, and perinatal loss. Twin
pregnancies can also experience selective fetal growth restriction
(sFGR), where one twin is compromised while the other grows normally.
This condition puts the affected twins at a higher risk of perinatal
mortality and morbidity\textsuperscript{{[}1{]}}. Additionally, compared
to non-sFGR pregnancies, those with sFGR tend to have earlier deliveries
and higher rates of neonatal unit admission\textsuperscript{{[}2{]}}.

It is estimated that 10-20\% of twin pregnancies are affected by sFGR.
Monochorionic twins have a higher incidence rate of 19.7\% compared to
dichorionic twins at 10.5\%\textsuperscript{{[}3{]}}. According to the
latest guidelines from the International Society of Ultrasound in
Obstetrics and Gynecology (ISUOG), sFGR is characterized by an estimated
fetal weight (EFW) below the 10th percentile in one twin and an
inter-twin EFW discordance of over 25\%. Additionally, a discordance
threshold of 20\% may be used to identify pregnancies with a higher
likelihood of negative outcomes\textsuperscript{{[}4{]}}. Detecting
pregnancies at risk of sFGR or birth weight discordance in the early
stages can enhance counseling, intervention timing, and potentially
enable preventive treatment trials. Various parameters identified during
early pregnancy have been linked to the development of sFGR later in
pregnancy. Although abnormal cord insertion is associated with sFGR in
twins, it is present in more than 55\% of all twin
pregnancies\textsuperscript{{[}5{]}}. A particularly isolated screening
test is urgently needed in clinical practice.

Placental pathology often correlates with the onset and prognosis of
sFGR. In cases of MC twins, sFGR is believed to be the result of an
unequal distribution of blood through placental anastomoses due to
uneven sharing of the placenta\textsuperscript{{[}6{]}}, whereas in DC
twins, from placental insufficiency in one of the
placentas\textsuperscript{{[}7{]}}. Throughout gestation, the villous
trophoblast in the placenta experiences ongoing turnover, which leads to
the release of apoptotic debris and cell-free DNA (cfDNA) into the
maternal bloodstream. Research suggests that levels of cfDNA may also
indicate placental issues. The amount of cfDNA specifically from the
fetus is known as fetal fraction, and it has been associated with
negative perinatal outcomes, including FGR in single
pregnancies\textsuperscript{{[}8, 9{]}}. Here, we testing the hypothesis
that the fetal fraction in twin pregnancy would reflect birthweight
discrepancy.

\textbf{Methods}

\textbf{Study design and data collection}

We conducted a retrospective cohort study of all twin pregnancies
undergoing cfDNA screening for aneuploidy and delivered at International
Peace Maternity and Child Health Hospital in Shanghai, China between
January 2018 to December 2021. All women with twin pregnancies were
offered a scan to determine chorionicity as well as first-trimester
nuchal translucency (NT) at
11\textsuperscript{+0}-13\textsuperscript{+6} weeks. Dichorionic was
confirmed when ultrasound assessment clearly indicates two placentas.
The twin peak sign is used to distinguish chorionicity if only one
placenta is visualized. Following this, a thorough genetic consultation
was offered to all future parents of the twin cohort. The
characteristics of the tests and potential advantages and disadvantages
of the different modalities of prenatal genetic testing will be
discussed. Parents will also be informed that NIPT included information
on the test being validated for the detection of only T21, T18 and T13,
and the evidence of using NIPT in twin pregnancy is less than it is in
singletons,thus offering limited information compared with invasive
diagnostic testing. We used two platforms for cfDNA-BGI-Health
(Shenzhen, China) and Berry Genomics. The screening was performed
specific to the clinical site.

We abstracted maternal demographic characteristics, fetal fraction,
obstetric outcomes, and neonatal outcomes from the medical
record,including maternal age, race, parity, education levels,
pre-pregnancy body mass index (BMI), method of conception, smoking
status, gestational age delivery, and neonatal birthweight. Birth
discordant was defined as an inter-twin EFW discordance greater than
20\%. sFGR was defined as a condition in which the EFW of one twin is
less than the 10th centile and an inter-twin EFW discordance greater
than 25\%. Cases with chromosome abnormalities were excluded from this
study.

The exclusion criteria were the presence of twin-to-twin transfusion
syndrome (TTTS), monoamniotic twins, twin anemia--polycythemia sequence
(TAPS), and congenital, structural or genetic malformations in the
fetus. To assure the placentas would be intact, only MC twins gestations
delivered through cesarean section were included to ensure an intact
placenta.

The Quintero criteria were used to diagnose TTTS, while TAPS was
diagnosed based on a postnatal inter-twin hemoglobin difference of over
8 g/dL. Monochorionic twin pregnancies were identified in the first or
early second trimester through ultrasonographic criteria, including the
presence of a single placenta, a thin dividing membrane, and the absence
of a twin peak (lambda) sign. Obstetricians confirmed monochorionicity
through postpartum examination of the placenta, which showed a single
placenta with inter-twin anastomoses\textsuperscript{{[}4{]}}.

Selective fetal growth restriction (sFGR) was characterized by two
criteria: (I) a birth weight discordance of more than 25\% and (II) the
presence of an FGR twin with a birth weight below the 10th percentile.
The calculation for birth weight discordance involved subtracting the
weight of the FGR twin from the weight of the
appropriate-for-gestational-age (AGA) twin, dividing that difference by
the weight of the larger twin, and multiplying the result by
100\%\textsuperscript{{[}4, 10{]}}.

This study was approved by the Ethics Review Board of the International
Peace Maternity and Child Health Hospital Affiliated to the Shanghai
Jiao Tong University School of Medicine (No.GKLW2019-20).

\textbf{Statistical Analysis}

Categorical variables were described by percentage, and continuous
variables were described by mean and standard deviation. The
Kolmogorov-Smirnov test was used to test the normality of the continuous
variables. The group differences were examined using Chi-squared test,
Student t test, or Mann Whitney U test where appropriate.

The linear regression models were used to test the association of fetal
fraction with birthweight difference. Logistic regression was used to
obtain the odds ratio (OR) and 95\% confidence interval (CI), and
examine the effects of fetal fraction on birth weight discordance of
20\% and 25\% and sFGR.

The Optimal Cutpoints package was used to perform a receiver operator
characteristics curve (ROC) analysis for fetal fraction and birth
outcomes that have significance in above regression analysis, and
determine the optimum cut-off points. Then fetal fraction was converted
into according to the optimal cut-off points, and the association of
fetal fraction (categorical variable) on birth outcomes was assessed
using logistic regression.

The multivariate analyzes between fetal fraction and birth outcomes were
carried out on using multiple logistic regression and multiple linear
regression model. Model adjusted for maternal age, weight, primipara,
history of abortion, chorionicity, pregnancy via ART and physical
conditions (gestational hypertension, gestational diabetes).

All analyses were performed with the Statistical Package for the Social
Sciences (version 24; SPSS Inc, Chicago, IL) and the Optimal Cutpoints
and verification packages for R statistical software (version 3.5.1;
http://www.R-project.org). Significance was defined as a 2-tail
probability value of \textless{}0.05.

\textbf{Gestational Age Notation}

The authors follow the World Health Organization's evaluation of the
gestational age. Under this notation, the first day of the last
menstrual period (LMP) is day 0 of week 0. Therefore, days 0 to 6 are
completed week 0, days 7 to 13 are completed week 1, etc.

\textbf{Results}

\textbf{Study population}

A total of 237 twin pregnancies who had normal NIPT results and
delivered [?]28 weeks were included in study period. 5 cases of
intrauterine death of one fetus, 8 structural abnormality in one or both
foetuses were excluded from this study. The proportions of DC and MC
twins were 77.6\% (174) and 22.3\% (50) respectively. Compared to MC
group, DC group had higher maternal age (\emph{p} =0.006), longer
gestational weeks at delivery (\emph{p} \textless{}0.001), larger birth
weight of the foetuses (\emph{p} =0.001) and higher percentage of
pregnancies conceived by Assisted Reproductive Technology (\emph{p}
\textless{}0.001). The incidence of preterm delivery was higher in MC
group (\emph{p} =0.004). There was no difference in other pregnancy
complications such as gestational hypertension, gestational diabetes or
thyroid diseases. (Table 1).

\textbf{The association of Fetal fraction with birthweight discordance
and sIUGR}

As shown in Table 2, the adjusted model demonstrated that fetal fraction
was positively correlated with the difference of birth weight (\selectlanguage{greek}β\selectlanguage{english}=0.004,
95\%CI: 0.001\textasciitilde{}0.006). That is, for every one percent
increase in fetal fraction, the difference of birth weight between twins
increases by 0.4 percent.

The results of logistic analysis were shown in Table 3, indicating that
higher fetal fraction was significantly associated with the increased
risk of birthweight discordance of 20\% (adjusted OR:1.073, 95\%CI:
1.009\textasciitilde{}1.142) and 25\% (adjusted OR:1.092, 95\%CI:
1.006\textasciitilde{}1.185) and sIUGR (adjusted OR:1.130, 95\%CI:
1.038\textasciitilde{}1.231).

The optimum cut-off points for fetal fraction in birthweight discordance
and sIUGR were obtained with a ROC analysis (Table 4). Using ROC
analysis, we obtained the optimum cut-off point of fetal fraction [?]
11.790, [?] 14.800 and [?] 14.800 for birthweight discordance of 20\% and
25\% and sIUGR, respectively. For birthweight discordance of 20\%, the
AUC was 0.591, the sensitivity was 52.9\% and the specificity was
66.3\%(\emph{p} =0.017). The AUC of sIUGR was 0.620, with 37.5\%
sensitivity and 86.5\% specificity(\emph{p} =0.024).

Compared with women with fetal fraction of \textless{} 11.790, there
were a 1.091-fold higher risk of birth weight discordance of 20\%
(adjusted OR: 2.091, 95\%CI: 1.218\textasciitilde{}3.591) among these
with fetal fraction of [?]11.790, and significantly increased risks of
birth weight discordance of 25\% (adjusted OR: 3.045, 95\%CI:
1.297\textasciitilde{}7.149) and sIUGR (adjusted OR: 3.526, 95\%CI:
1.443\textasciitilde{}8.618) among these with fetal fraction of [?]14.800.

\textbf{Discussion}

In this study, we assessed the association between fetal fraction and
birth weight discordant and sIUGR. It is showed that fetal fraction was
positively correlated with the difference of birth weight. For every one
percent increase in fetal fraction, the difference of birth weight
between twins increases by 0.4 percent. Higher fetal fraction was
significantly associated with the increased risk of birthweight
discordance of 20\% and 25\% and sIUGR. Using ROC analysis, we obtained
the optimum cut-off point of fetal fraction [?] 11.790, [?] 14.800 and [?]
14.800 for birthweight discordance of 20\% and 25\% and sIUGR,
respectively. For birthweight discordance of 20\%, the AUC was 0.591,
the sensitivity was 52.9\% and the specificity was 66.3\%(\emph{p}
=0.017). The AUC of sIUGR was 0.620, with 37.5\% sensitivity and 86.5\%
specificity(\emph{p} =0.024). Compared with women with fetal fraction of
\textless{} 11.790, there were a 1.091-fold higher risk of birth weight
discordance of 20\% among these with fetal fraction of [?]11.790, and
significantly increased risks of birth weight discordance of 25\% and
sFGR among these with fetal fraction of [?]14.800.

Cell-free fetal DNA actually originates from
placenta\textsuperscript{{[}11{]}}.~ Throughout a typical pregnancy,
cffDNA experiences a gradual increase, comprising roughly 13\% of all
cell-free DNA found in maternal plasma towards the end of gestation,
before swiftly decreasing to undetectable levels after
childbirth\textsuperscript{{[}12, 13{]}}. The size of the placenta and
the rate of trophoblast apoptosis are factors that affect the release of
cffDNA\textsuperscript{{[}9{]}}. According to reports, cffDNA triggers
the secretion of type 1 interferons such as IFN-\selectlanguage{greek}β \selectlanguage{english}and IFN-\selectlanguage{greek}α, \selectlanguage{english}along with
other pro-inflammatory agents\textsuperscript{{[}14{]}}. The
introduction of CpG resulted in gestational hypertension and heightened
vasoconstriction in rats. This suggests that cffDNA has the ability to
initiate inflammatory cascades as a pro-inflammatory trigger.

Research has demonstrated that oxidative stress results in trophoblast
apoptosis and elevated cffDNA release\textsuperscript{{[}15{]}}. The
liberation of cffDNA can be affected by doxorubicin and high-mobility
group box protein-1, which are inflammatory agents. Conversely,
lipopolysaccharide does not affect the release of early or term
cffDNA\textsuperscript{{[}16{]}}, suggesting that cffDNA is involved in
sterile inflammation as opposed to infectious processes. Sterile
inflammation may lead to placental dysfunction and consequently cause
pregnancy complications.

In order to predict negative outcomes earlier and non-invasively, many
studies have linked pregnancy complications with cffDNA plasma
concentrations. Pregnancies affected by pre-eclampsia, preterm delivery,
and FGR were found to have higher cffDNA concentrations during the
second trimester. Additionally, there was a noticeable increase in
cffDNA levels three weeks prior to the onset of
symptoms\textsuperscript{{[}17{]}}. In twins with birth weight
discordant, we were able to see an increase in cffDNA which was
represented by fetal frection. Increased levels of cffDNA are also a
result of intrauterine surgeries, such as laser ablation, used to treat
twin-twin transfusion syndrome\textsuperscript{{[}18{]}}. It is possible
that the rise in cffDNA levels observed in twins with discordant birth
weight and selective fetal growth restriction (sFGR) is a result of the
repair process. In cases where there is discordant growth in DC
pregnancies, the incidence of preeclampsia can be as high as 37.5\%,
which is significantly greater than the 21.9\% rate observed in MC sFGR
pregnancies\textsuperscript{{[}19, 20{]}}. It is known that placental
insufficiency affects only one fetus and results in selective growth
restriction in DC pregnancies\textsuperscript{{[}10{]}}. After corrected
for both preeclampsia and gestational diabetes, increased cffDNA is
still associated with the incidence of birth weight discordance and
sFGR, which showed that there may be other pathological mechanisms
involved. Placentae that are affected by uneven sharing have fetal
circulations that are more tightly connected with larger and more
frequent arterio-arterial (AA) anastomoses. This can be helpful for
growth-restricted fetuses, as it allows their co-twin to compensate to
some extent for the inadequacy of their own placenta. However, this
close connection also poses risks to appropriately grown fetuses, who
are vulnerable to sudden changes in the blood pressure of their smaller
co-twin. Although unequal placental sharing is the primary cause of sFGR
in MC pregnancies, the interdependent feto-placental circulations play a
crucial role in the prognosis and perinatal outcome. In cases of sFGR,
especially those with abnormal umbilical artery Doppler, glucose
transporters (GLUTs) were found to be elevated due to hypo-perfusion. We
suspect that increased cffDNA in MC twins with sFGR is a result of
apoptosis in such hypoxic environments. Additionally, hypermetabolism
may also be a factor in elevated cffDNA levels since the placenta
attempts to increase glucose transport to the fetal circulation as an
adaptive response\textsuperscript{{[}21{]}}. We suspect that increased
cffDNA in MC twins with sFGR is a consequence of apoptosis in such
hypoxic environment. Further, hypermetabolism may also be a reason for
elevated cffDNA since the placenta attempts to increase glucose
transport to the fetal circulation as an adaptive response.

\textbf{Conclusion:}

High fetal fraction is associated with poor placental development and
dysfunction. Cutoff of both 11.790 and 14.800 can be use as potential
biomarker in predict birth weight discordance and sFGR.

\textbf{Author contributions}

Renyi Hua: conceptualisation, methodology, investigation, resources,
writing -- original draft, visualisation, project administration.
Yuanqing Xia: formal analysis, data curation.Shan Wang and Jinling Sun:
conceptualization, methodology, visualisation. Li Gao, Yi Wu and Xinrong
Zhao: conceptualisation, methodology, formal analysis, investigation,
resources, data curation, project administration. Yanlin Wang:
methodology, writing -- review \& editing, supervision.

\textbf{Funding support:}

This work was supported by China National Key Research and Development
Grant(2018YFC1004600)

\textbf{Conflict of interests:}

None declared.

\textbf{Ethics approval:}

This study was approved by the Ethics Review Board of the International
Peace Maternity and Child Health Hospital Affiliated to the Shanghai
Jiao Tong University School of Medicine (No.GKLW2019-20).

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{[}18{]}. Wataganara T, Gratacos E, Jani J, et al. Persistent elevation
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{[}19{]}. Sparks TN, Nakagawa S, Gonzalez JM. Hypertension in
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\textbf{Table 1. Demographics of DC and MC groups}\selectlanguage{english}
\begin{longtable}[]{@{}lllll@{}}
\toprule
& \textbf{Overall} & \textbf{DC} & \textbf{MC} & p\tabularnewline
& mean\selectlanguage{ngerman}±SD/n(\%) & mean\selectlanguage{ngerman}±SD/n(\%) & mean\selectlanguage{ngerman}±SD/n(\%) &\tabularnewline
\textbf{Maternal characteristic} & n=224 & n=174 & n=50 &\tabularnewline
Age(year), n(\%) & & & & \textbf{0.006}\tabularnewline
mean\selectlanguage{ngerman}±SD & 31.97 \selectlanguage{ngerman}±3.75 & 32.30 \selectlanguage{ngerman}±3.49 & 30.82 \selectlanguage{ngerman}±4.40 &\tabularnewline
\textless{}35 & 180 (80.4) & 137 (78.7) & 43 (86.0) &\tabularnewline
[?]35 & 44 (19.6) & 37 (21.3) & 7 (14.0) &\tabularnewline
Maternal weight, kg & 59.09 \selectlanguage{ngerman}±8.98 & 59.52 \selectlanguage{ngerman}±9.38 & 57.59 \selectlanguage{ngerman}±7.28 &
0.228\tabularnewline
First gestation, n (\%) & & & & 0.625\tabularnewline
Yes & 141 (62.9) & 111 (63.8) & 30 (60.0) &\tabularnewline
No & 83 (37.1) & 63 (36.2) & 20 (40.0) &\tabularnewline
Primipara, n (\%) & & & & 0.081\tabularnewline
Yes & 199 (88.8) & 158 (90.8) & 41 (82.0) &\tabularnewline
No & 25 (11.2) & 16 (9.2) & 9 (18.0) &\tabularnewline
History of abortion, n (\%) & & & & 0.404\tabularnewline
No & 155 (69.2) & 118 (67.8) & 37 (74.0) &\tabularnewline
Yes & 69 (30.8) & 56 (32.2) & 13 (26.0) &\tabularnewline
Pregnancy via ART, n (\%) & & & &
\textbf{\textless{}0.001}\tabularnewline
No & 72 (32.1) & 42 (24.1) & 30 (60.0) &\tabularnewline
Yes & 152 (67.9) & 132 (75.9) & 20 (40.0) &\tabularnewline
Fetal fraction & 10.73 \selectlanguage{ngerman}±4.15 & 10.57 \selectlanguage{ngerman}±4.25 & 11.30 \selectlanguage{ngerman}±3.78 &
0.079\tabularnewline
Gestational hypertension, n (\%) & & & & 0.976\tabularnewline
No & 184 (82.1) & 143 (82.2) & 41 (82.0) &\tabularnewline
Yes & 40 (17.9) & 31 (17.8) & 9 (18.0) &\tabularnewline
Gestational diabetes, n (\%) & & & & 0.634\tabularnewline
No & 180 (80.4) & 141 (81.0) & 39 (78.0) &\tabularnewline
Yes & 44 (19.6) & 33 (19.0) & 11 (22.0) &\tabularnewline
Thyroid disease, n (\%) & & & & 0.977\tabularnewline
None & 191 (85.3) & 147 (84.5) & 44 (88.0) &\tabularnewline
Subclinical hypothyroidism & 5 (2.2) & 4 (2.3) & 1 (2.0)
&\tabularnewline
Hypothyroidism & 16 (7.1) & 13 (7.5) & 3 (6.0) &\tabularnewline
Subclinical hyperthyroidism & 7 (3.1) & 6 (3.4) & 1 (2.0)
&\tabularnewline
Hyperthyroidism & 5 (2.2) & 4 (2.3) & 1 (2.0) &\tabularnewline
\textbf{Fetal characteristic} & n=448 & n=348 & n=100 &\tabularnewline
Gestational age at delivery, week & 35.87 \selectlanguage{ngerman}±1.99 & 36.08 \selectlanguage{ngerman}±1.85 & 35.16
\selectlanguage{ngerman}±2.27 & \textbf{\textless{}0.001}\tabularnewline
Preterm birth, n (\%) & & & & \textbf{0.004}\tabularnewline
No & 200 (44.6) & 168 (48.3) & 32 (32.0) &\tabularnewline
Yes & 248 (55.4) & 180 (51.7) & 68 (68.0) &\tabularnewline
Birth weight, g & 2413.55\selectlanguage{ngerman}±466.54 & 2456.14\selectlanguage{ngerman}±436.53 & 2265.35\selectlanguage{ngerman}±535.05 &
\textbf{0.001}\tabularnewline
Difference of birth weight & 0.12 \selectlanguage{ngerman}±0.11 & 0.11 \selectlanguage{ngerman}±0.11 & 0.12 \selectlanguage{ngerman}±0.12 &
0.331\tabularnewline
Birth-weight discordance of 20\%, n (\%) & Birth-weight discordance of
20\%, n (\%) & & & 0.795\tabularnewline
No & 380 (84.8) & 296 (85.1) & 84 (84.0) &\tabularnewline
Yes & 68 (15.2) & 52 (14.9) & 16 (16.0) &\tabularnewline
Birth-weight discordance of 25\%, n (\%) & Birth-weight discordance of
25\%, n (\%) & & & 0.988\tabularnewline
No & 412 (92.0) & 320 (92.0) & 92 (92.0) &\tabularnewline
Yes & 36 (8.0) & 28 (8.0) & 8 (8.0) &\tabularnewline
sFGR, n (\%) & & & & 0.706\tabularnewline
No & 416(92.9) & 324 (93.1) & 92 (92.0) &\tabularnewline
Yes & 32 (7.1) & 24 (6.9) & 8 (8.0) &\tabularnewline
\bottomrule
\end{longtable}

\textbf{Table 2 Linear regression analyses of fetal fraction and
difference of birth-weight}\selectlanguage{english}
\begin{longtable}[]{@{}lllll@{}}
\toprule
\textbf{Outcome} & \selectlanguage{greek}\textbf{β(95\%CI)}\selectlanguage{english} & p & \selectlanguage{greek}\textbf{β(95\%CI)}\selectlanguage{english}
\textsuperscript{a} & p\tabularnewline
Difference of birth-weight & 1.076(1.046\textasciitilde{}1.107) &
\textbf{0.000} & 1.004(1.001\textasciitilde{}1.006) &
\textbf{0.003}\tabularnewline
\bottomrule
\end{longtable}

\textsuperscript{a}Model adjusted for maternal age, weight, history of
abortion, primipara, pregnancy via ART, chorionicity, gestational
hypertension and gestational diabetes

\textbf{Table 3 Logistic regression analyses of fetal fraction and
birth-weight discordance and sFGR}\selectlanguage{english}
\begin{longtable}[]{@{}lllll@{}}
\toprule
\textbf{Outcome} & \textbf{OR(95\%CI)} & p & \textbf{aOR(95\%CI)}
\textsuperscript{a} & p\tabularnewline
Birth-weight discordance-20\% & 1.079(1.018\textasciitilde{}1.144) &
\textbf{0.011} & 1.073(1.009\textasciitilde{}1.142) &
\textbf{0.025}\tabularnewline
Birth-weight discordance-25\% & 1.082(1.005\textasciitilde{}1.166) &
\textbf{0.036} & 1.092(1.006\textasciitilde{}1.185) &
\textbf{0.035}\tabularnewline
sFGR, n (\%) & 1.117(1.035\textasciitilde{}1.205) & \textbf{0.004} &
1.13(1.038\textasciitilde{}1.231) & \textbf{0.005}\tabularnewline
\bottomrule
\end{longtable}

\textsuperscript{a}Model adjusted for maternal age, weight, history of
abortion, primipara, pregnancy via ART, chorionicity, gestational
hypertension and gestational diabetes

\textbf{Table 4 ROC analyses for fetal fraction and birth outcomes}\selectlanguage{english}
\begin{longtable}[]{@{}llll@{}}
\toprule
\textbf{Outcome} & \textbf{Cut-off value} & \textbf{AUC (95\%CI)} &
p\tabularnewline
birth-weight discordance of 20\% & 11.790 &
0.591(0.516\textasciitilde{}0.666) & \textbf{0.017}\tabularnewline
birth-weight discordance of 25\% & 14.800 &
0.569(0.466\textasciitilde{}0.673) & 0.083\tabularnewline
sFGR & 14.800 & 0.620(0.514\textasciitilde{}0.725) &
\textbf{0.024}\tabularnewline
\bottomrule
\end{longtable}

\textbf{Table 5 Logistic regression analyses of fetal fraction and
birth-weight discordance and sFGR}\selectlanguage{english}
\begin{longtable}[]{@{}llllll@{}}
\toprule
\textbf{Outcome} & \textbf{Fetal fraction} & \textbf{OR(95\%CI)} & p &
\textbf{aOR(95\%CI)} \textsuperscript{a} & p\tabularnewline
Birth-weight discordance of 20\% & Birth-weight discordance of 20\% &
Birth-weight discordance of 20\% & & &\tabularnewline
& {[}0, 11.79\%) & 1 & & 1 &\tabularnewline
& {[}11.79\%, 100\%{]} & 2.215(1.315\textasciitilde{}3.731) &
\textbf{0.003} & 2.091(1.218\textasciitilde{}3.591) &
\textbf{0.008}\tabularnewline
Birth-weight discordance of 25\% & Birth-weight discordance of 25\% &
Birth-weight discordance of 25\% & & &\tabularnewline
& {[}0, 11.79\%) & 1 & & 1 &\tabularnewline
& {[}11.79\%, 14.80\%) & 0.985(0.379\textasciitilde{}2.559) & 0.976 &
0.843(0.306\textasciitilde{}2.32) & 0.741\tabularnewline
& {[}14.80\%, 100\%{]} & 3.167(1.444\textasciitilde{}6.944) &
\textbf{0.004} & 3.045(1.297\textasciitilde{}7.149) &
\textbf{0.011}\tabularnewline
sFGR & & & & &\tabularnewline
& {[}0, 11.79\%) & 1 & & 1 &\tabularnewline
& {[}11.79\%, 14.80\%) & 1.286(0.48\textasciitilde{}3.445) & 0.617 &
1.211(0.43\textasciitilde{}3.407) & 0.717\tabularnewline
& {[}14.80\%, 100\%{]} & 4.133(1.815\textasciitilde{}9.411) &
\textbf{0.001} & 3.526(1.443\textasciitilde{}8.618) &
\textbf{0.006}\tabularnewline
\bottomrule
\end{longtable}

\textsuperscript{a}Model adjusted for maternal age, weight, history of
abortion, primipara, pregnancy via ART, chorionicity, gestational
hypertension and gestational diabetes\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image1/image1}

\end{center}
\end{figure}

\textbf{Figure legends} :

Figure 1: ROC Curves for fetal faction and birth outcomes.

\emph{A: ROC Curve for fetal faction and birth weight discordance of
20\%; B: ROC Curve for fetal faction and birth weight discordance of
25\%; C: ROC Curve for fetal faction and sFGR}

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

