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\begin{document}

\title{Temporal persistence and long-term effect on noninvasive prenatal
testing by residual fetal cell-free DNA of demised co-twin after
selective fetal reduction in dichorionic diamniotic twin pregnancies}



\author[1]{Min Chen}%
\author[2]{Fengxia Su}%
\author[1]{Jia Wang}%
\author[3]{Li Zhou}%
\author[3]{Qiang Liu}%
\author[3]{Xiang Chai}%
\author[3]{Yu Yuan}%
\author[3]{Miao Cen}%
\author[2]{Yujing Wu}%
\author[4]{Yi  Wang}%
\author[4]{Yan Zhang}%
\author[2]{Fang Chen}%
\author[5]{Dunjin Chen}%
\author[2]{Ya Gao}%
\affil[1]{Department of Fetal Medicine and Prenatal Diagnosis, The Third Affiliated Hospital of Guangzhou Medical University}%
\affil[2]{BGI-Shenzhen}%
\affil[3]{Clinical Laboratory of BGI Health, BGI-Shenzhen}%
\affil[4]{MGI, BGI-Shenzhen,}%
\affil[5]{Department of Obstetrics, The Third Affiliated Hospital of Guangzhou Medical University}%


\vspace{-1em}



  
  \date{June 15, 2024}


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\selectlanguage{english}
\begin{abstract}
Objectives: To determine the temporal persistence of residual cell-free
DNA (cfDNA) of deceased co-twin in maternal circulation after selective
fetal reduction and evaluate its long-lasting effect on noninvasive
prenatal testing (NIPT) results. Design: Prospective observational study
Setting: The Third Affiliated Hospital of Guangzhou Medical University
Population: Dichorionic diamniotic twins (n=5) underwent selective fetal
reduction of a co-twin with trisomy. Methods: With consent, maternal
blood was collected immediately before reduction and periodically after
reduction until birth. CfDNA of each maternal blood sample was sequenced
for NIPT and analyzed for fetal trisomies and fetal fractions. Main
Outcome Measured: Detectable T-scores for trisomy identification and
three types of fetal fractions including the total fetal fraction, the
fetal fraction of the deceased co-twin, and the fetal fraction of the
surviving co-twin.%
\end{abstract}%



\sloppy


\textbf{Introduction}

The fetal demise of a co-twin (i.e., spontaneous reduction to a
singleton pregnancy) is common with an estimated prevalence of 6.2\% in
all twin pregnancies {[}1, 2{]}. This prevalence might be
underestimated, since the fetal demise occurred in the early trimester,
also known as the `vanishing twin', which may affect as high as 30\% of
early diagnosed pregnancies {[}3-6{]}. The reasons for fetal demise
could be complicated, yet chromosomal anomaly could be an important
factor, given the high rate of chromosome aneuploidy in early singleton
miscarriages {[}7-9{]}. It has been previously reported that the demised
fetus continues to give rise residual cell-free DNA (cfDNA) to maternal
circulation, which can cause false-positive results of fetal aneuploidy
and fetal gender when noninvasive prenatal testing (NIPT) is conducted
{[}10-12{]}. This poses technical uncertainties and counseling
difficulties when providing NIPT service in twin pregnancies, or
pregnancies with unaware fetus demise. So far, there is a lack of
clinical guidelines in terms of the interval after fetus demise to
provide NIPT, partly because the longitudinal change of residual cfDNA
of demise fetus has not been extensively studied. We aimed to use
dichorionic diamniotic (DCDA) twin pregnancies undergoing selective
fetal reduction of an aneuploidy co-twin as a model of natural fetal
demise to investigate the sequential changes of residual cfDNA of the
deceased fetus, as well as the persistent effect on NIPT.

\textbf{Methods}

\textbf{Study population and study design}

This was a prospective observational study in women of DCDA twin
pregnancy opted for selective reduction of a co-twin in the Department
of Fetal Medicine and Prenatal Diagnosis, the Third Affiliated Hospital
of Guangzhou Medical University, China. Each woman has been previously
confirmed to carry dizygotic twin pregnancy by ultrasound, and opted for
fetal reduction due to an aneuploidy co-twin diagnosed by chorionic
villus sampling (CVS) or amniocentesis. Before reduction, each woman was
given a detailed explanation of the study, and written informed consent
of participating in this study was obtained. All women were above 18
years old, and their gestational ages were eight weeks or above at the
time of joining the study. Chromosomal abnormalities have not been found
in women and her husband, neither the couples have received a blood
transfusion, organ transplantation, cell therapy, or immunotherapy
before pregnancy. Fetal reductions were conducted by transabdominal
intracardiac injection of potassium chloride as the standard of practice
{[}13{]}. Immediately before and twice a month after reduction till
birth, the maternal blood of each woman was collected for NIPT for fetal
T21, T18, T13 to evaluate the residual cfDNA effect of the reduced
fetus. The couple's age, gestational age at each time of blood sampling,
ultrasound scanning results, prenatal diagnosis results, and other
relevant clinical information were recorded. This study was approved by
the Ethics Committee of the Third Affiliated Hospital of Guangzhou
Medical University (no.2019096) and the Institute Review Board of
BGI-Shenzhen (BGI-IRB 19081).

\textbf{Plasma cfDNA extraction and sequencing}

For each blood collection, five milliliters of peripheral blood was
obtained from the participating woman in an ethylene diamine tetraacetic
acid-anticoagulated (EDTA) tube. Plasma was separated within 8 hours
following a two-step centrifugation protocol {[}14{]}. Briefly, blood
was centrifugated at 1600 g for 10 minutes at 4\selectlanguage{ngerman}°C, and then the
supernatant was transferred to a new Eppendorf tube for second
centrifugation at 16000 g for 10 minutes at 4°C. The supernatant was
taken into a new tube and stored at -80°C for future use. All subsequent
procedures, including cfDNA isolation, library construction, and
sequencing, were performed at an ISO/IEC 17025 certified laboratory of
BGI-Shenzhen, China. CfDNA was extracted from 200\selectlanguage{greek}μ\selectlanguage{english}L plasma using a Micro
DNA Kit (Tiangen) following the manufacturer's instructions. The
extracted cfDNA was end-repaired and then ligated with adaptors for
multiplex sequencing. The ligated products were subjected to 12 cycles
of amplification using the Kapa HIFI hotstart ready master mix (Kapa
Biosystems), followed by quantitation using the dsDNA HS Assay kit 2.0
(Invitrogen) by Qubit2.0 and Agilent High Sensitivity DNA Kit with a
2100 Bioanalyzer (Agilent Technologies). PCR products were then
normalized and processed for circularization using MGIEasyTM DNA Library
Prep Kit (MGI, China) following the manufacturer's instructions
{[}15{]}. Briefly, PCR products were heat-denatured at 95 \selectlanguage{ngerman}°C for
3\selectlanguage{english} minutes to make single strand DNA circles (ssDNA circles), which were
then mixed with reagents of MGIEasyTM DNA Library Prep Kit and incubated
at 37\selectlanguage{english} degC for 30\selectlanguage{english} minutes to complete the circularization. The resulting
ssDNA circle was then used to generate DNA nanoballs (DNBs) by rolling
circle amplification (RCA) {[}16{]}. After RCA and the formation of
DNBs, the final product was measured by Qubit (Thermo Fisher) using the
ssDNA HS Assay kit (Invitrogen), and loaded on a DNBSEQ-500 platform
(MGI, China) for sequencing {[}17{]} following the manufacturer's
instructions. Sequencing was conducted with a strategy of single-end 35
base pairs. The data that support the findings of this study have been
deposited into CNSA (CNGB Nucleotide Sequence Archive\selectlanguage{english})of CNGBdb with
accession number CNP0000992~\selectlanguage{english}(\emph{https://db.cngb.org/cnsa/}~) upon
the request of accession code.

\textbf{Fetal fraction calculation}

Three different methods to calculate fetal fractions were used in this
study for different purposes, including an artificial neural network
model named the FF-QuantSC {[}18{]}, a Y-chromosome method {[}19{]}, and
the relative coverage of the trisomy chromosome. FF-QuantSC was used to
calculate the overall fetal fraction of both co-twins. In pregnancies
with one male co-twin, the Y-chromosome method was used to calculate the
fetal fraction of the male fetus. In pregnancies with two male fetuses,
the Y-chromosome method calculated the fetal fraction equal to an
overall fetal fraction, and results were compared with the FF-QuantSC
method. The relative coverage of the trisomy chromosome was specifically
used to calculate the fetal fraction of the trisomy co-twin who was
terminated by the selective reduction.

FF-QuantSC exploits an artificial neural network to calculate the fetal
fraction of both male and female co-twins {[}18{]}. Briefly, a fully
connected neural network model with a single hidden layer and 128
neurons was trained by the Vapnik-Chervonenkis dimension {[}20{]} and
trained with over 100 thousand of male pregnancies. Sequencing reads
were partitioned into continuous genomic windows of 60kb in length for
feature selection. Standardization by within-sample z-score
transformation was then used to generate a final feature matrix. After
training, the FF-QuantSC model was testified in six testing groups of
about 240 thousand pregnancies, including 36 thousand twin pregnancies
and 80 thousand female pregnancies {[}18{]}.

Y-chromosome method calculated fetal fractions using unique sequence
reads from the Y chromosome (eq.1).

Fetal fraction = 2*\(\frac{\overset{\overline{}}{\text{UR}_{Y}}}{\overset{\overline{}}{\text{UR}}}\)(1)

where \(\overset{\overline{}}{\text{UR}_{Y}}\) represents the unique reads on chromosome Y,
and \(\overset{\overline{}}{\text{UR}}\)represents the unique reads on autosome
chromosomes.

In pregnancies with a trisomy co-twin, fetal fraction was calculated
using the sequencing reads of the trisomic chromosome, typically
chromosome 13, 18, or 21 (eq.2).

\(\varepsilon_{i}=2\times\frac{\text{UR}_{i}-\overset{\overline{}}{\text{UR}}}{\overset{\overline{}}{\text{UR}}}\)\emph{i} =chromosome 13, 18, or 21 (2)

Where \(\varepsilon_{i,Y}\) represents the fetal fraction estimate by
chromosome \(i\); \emph{UR\textsubscript{i}} represents the
unique reads of chromosome \(i\); \(\overset{\overline{}}{\text{UR}}\)
represents the mean of unique reads on all autosomal chromosomes.

\textbf{Chromosome trisomy risk calculation}

Sequencing data were analyzed using a binary hypothesis T-score as
previously described {[}19{]}. Briefly, a binary hypothesis T-test and
logarithmic likelihood ratio L-score between the two t-tests were used
to classify whether the fetuses had T21, T18 or T13. T-score higher than
3 was used as the cut-off to identify fetal trisomy, and L-score higher
than 1 was used to determine the confidence.

\textbf{Results}

\textbf{Patients}

From January 2017 to January 2018, five women carrying DCDA twins with
one aneuploidy fetus were enrolled in this study (Table 1). The median
maternal age was 38.8 years and 80\% (4/5) of pregnant women had a
maternal age above 35 years. All women were conceived by in-vitro
fertilization and embryo transfer (IVF-ET), and each woman had received
the transplant of two embryos. During routine prenatal healthcare, all
women had at least one risk factor of fetal aneuploidy, including
advanced maternal age, increased fetal nuchal translucency (NT)
thickness, and structural abnormalities in ultrasound examination (Table
1). Fetal aneuploidies were confirmed by CVS or amniocentesis, showing
four women with a co-twin of trisomy 18 (T18) and one woman with a
co-twin of trisomy 21 (T21). The median of gestational age receiving
reduction procedure was 15 weeks. After reduction, each woman
voluntarily donated her blood samples every two to five weeks until
delivery. Depending on their willingness, patients 1, 2, and 5 gave
blood for seven times, five times, and six times after reduction,
respectively. Patient 3 and 4 both gave blood for nine times after
reduction.

\textbf{Fetal fractions}

The temporal dynamic changes of fetal fractions by three different
methods in all five pregnancies are shown in Figure 1, and detailed
information at each timepoints listed in Table S1.

In patient 1, both fetuses were female and fetal fractiosn could not be
calculated using Y-chromosome signals. However, the fetal fraction of
the reduced T18 fetus was calculated using trisomy signals, showing an
immediate decline from 3.27\% to 2.01\% after reduction and a mild
increase to 3.41\% at eight weeks after reduction. After that, the
demised T18 fetus gradually stopped the release of cfDNA at 16 weeks
after reduction as the fetal fraction dropped to close to 0. In
contrast, after a transient decline, the overall fetal fraction
maintained stable for several weeks, and then rise when the terminated
fetus reduced the release of cfDNA at eight weeks after reduction.

In patient 2, who had a male euploidy and a male T18 fetus, the fetal
fraction of the terminated T18 fetus increased from 1.80\% to the
maximum of 11.71\% at nine weeks after reduction and then rapidly
declined to close to 0 at 11 weeks after reduction. Since both fetuses
were male, the male fetal fractions calculated by the Y-chromosome
method and the overall fetal fractions calculated by FF-QuantSC were
almost overlapped, which showed peak levels at nine weeks after
reduction following the quick decline afterward. At 12 weeks after
reduction when the terminated fetus stopped the release of cfDNA, the
fetal fraction of the euploid fetus quickly rebound, and kept rising
until birth. It was noted that at 15 and 20 weeks after reduction, the
overall fetal fractions were slightly higher than the fetal fractions of
male fetuses.

Both patient 3 and 4 had discordant sex twins with a female trisomy and
a male euploid fetus. The fetal fractions of the terminated female fetus
showed the increase of fetal fraction to peak levels at seven to nine
weeks after reduction. After that, the terminated fetus in patient 3
gradually reduced the release of cfDNA and stopped discharge at 22 weeks
after reduction, while the fetal fraction of the terminated fetus in
patient 4 dropped to close 0 at 15 weeks after reduction. In both
patients, once the fetal fractions of the terminated female fetuses
began to decline, the fetal fractions of the euploid male fetuses
rapidly increased, especially when the fetal fraction of the terminated
female fetus was down to nearly 0. When the terminated female fetuses in
both patient 3 and 4 still gave rise substantial amount of cfDNA to
maternal circulation, the overall fetal fractions were higher than the
fetal fractions of each co-twin. However, after the fetal fractions of
the terminated female fetuses reduced to a relatively low level, the
total fetal fractions were closely correlated with the fetal fractions
of the euploid male fetuses.

In patient 5, since a male T18 fetus was terminated in the female-male
twins, the fetal fractions calculated with trisomy signals and Y
chromosome were almost identical, both showing a transient decrease
shortly after reduction and later rise to maximum levels at eight weeks
after reduction, and finally decline to nearly 0\% at 14 weeks after
reduction. Before seven weeks of post-reduction, the overall fetal
fraction maintained two to four folds higher than the fetal fraction of
the terminated fetus. Once the terminated fetus stopped the release of
cfDNA at 14 weeks after reduction, the overall fetal fraction began to
increase rapidly.

\textbf{NIPT results}

As a result of the persistence of a high fetal fraction of demised
trisomy co-twin, NIPT T-scores of the remaining euploid fetuses in all
five pregnancies were compromised for long periods after reduction
(Figure 2a). Detailed NIPT results of each time points are listed in
Table S1.

Patient 1 had her T-score of chromosome 18 reduced from 7.27 before
reduction to 4.55 at three weeks after reduction, and later rise to 8.20
at eight weeks after reduction. Then her T-score declined to 1.24 at 16
weeks after reduction and her NIPT results remained T18 low-risk
afterward.

Patient 2 had her T-score of chromosome 18 dramatically rise from 4.55
at two weeks after reduction to 24.62 at nine weeks after reduction, and
then rapidly declined to 2.09 at 12 weeks after reduction for T18
low-risk afterward. Interestingly, her T-score of chromosome 13 reached
3.33 at 15 weeks after reduction, and then rise to 5.65 at 20 weeks
after reduction, indicating high risk of trisomy 13 (T13) (Figure 2b).
This was consistent with the results of her fetal fraction, showing that
at 15 and 20 weeks post-reduction, the overall fetal fraction calculated
by FF-QuantSC was slightly higher than the fetal fractions of both male
fetuses calculated by the Y-chromosome method. A later confirmation in
placenta tissues by sequencing placental tissues validated the presence
of placenta mosaicism of T13 (Figure 2c).

In patient 3, in the first two weeks after reduction, her T-score of
chromosome 21 only mildly increased from 10.04 to 13.94. After that, her
T-score reached a maximum of 36.60 at eight weeks after reduction, and
then quickly declined to 8.76 at 12 weeks after reduction. Since then,
her NIPT results maintained high risk for T21 for a very long time until
22 weeks after reduction.

In patient 4, her T-score of chromosome 18 also showed an initial mild
but later faster increase, and reached a maximum of 17.27 at seven weeks
after reduction. After that, the T-score gradually declined to 1.85 at
15 weeks after reduction. Since then, the NIPT results changed to T18
low risk.

Patient 5 had her T-score of chromosome 18 reduced from 12.39 before
reduction to 4.84 at two weeks after reduction, and rise to 16.04\% at
seven weeks after reduction. The T-score then continuously declined, and
the NIPT results changed to T18 low-risk at 14 weeks after reduction.

\textbf{Prognosis}

All five women gave live birth to a healthy baby at 37 to 39 gestational
weeks without neonatal abnormalities.

\textbf{Discussion}

\textbf{Main findings}

Using selective reduction of a co-twin with aneuploidy as the simulation
of twin demise, we demonstrated that the fetal fraction of the
terminated fetus underwent dynamic changes and persistent effect
throughout pregnancy. After fetal reduction, the fetal fraction of the
terminated fetus took 7 to 9 weeks to reach a peak level, and then
gradually dropped down to nearly 0\% at 15 to 16 weeks after reduction.
In one case (patient 3), the residual cfDNA of the terminated fetus even
gave rise to observable fetal fraction at 20 weeks after reduction,
equivalent to 37 gestational weeks in this woman. On the other hand, in
patient 3 and 4, we observed that the fetal fraction of the remaining
normal fetus appeared to maintain stable or mildly increase until the
fetal fraction of the trisomy co-twin declined.

The influence of the residual cfDNA of a terminated fetus was apparent,
as we found the persistent detection of fetal trisomy after the fetal
reduction, ranging from 12 to 22 weeks post-reduction with the median of
15.8 weeks. Importantly, in all pregnancies, T-scores demonstrated close
correlations to the fetal fractions of the terminated fetus, showing a
rapid increase from two to three weeks after reduction and reached the
maximum values at seven to nine weeks after reduction, following the
gradual decline to close to 0 in the late third trimester. In patient 3
who had a very long presence of residual cfDNA of the terminated T21
fetus, NIPT showed positive results until 20 weeks after reduction,
which was concordant to the fetal fraction results.

\textbf{Strengths and limitations}

The major strength of this study was the consecutive observational
approach and prospective collection of blood samples. Previously,
several studies have reported the disturbance of NIPT by residual cfDNA
of twin demise {[}11, 21, 22{]}. However, very few studies demonstrate
the longitudinal effect of residual cfDNA after twin demise. We
successfully established the consecutive profiles of residual cfDNA
changes after twin demise by densely testing blood samples until birth,
which provides the robust evidence to elucidate an important question:
how long the placenta of a deceased co-twin can release cfDNA into the
maternal circulation? Another strength of this study was a combination
of three different methods of calculating fetal fractions. This helps
compare the total fetal fraction measured by FF-QuantSC, the fetal
fraction of the demised co-twin measured by relative coverage of the
trisomy chromosome, and in two cases (patient 3 and 4) the fetal
fraction of the survived euploid fetuses measured by Y-chromosome.

The limitations of this study included a small sample size and the
restriction of selecting DCDA twins with a trisomy co-twin to simulate a
natural twin demise. Due to the small sample size, statistical analysis
could not be conducted. For the ease of analyzing the individual fetal
fraction of different co-twins, we only used dizygotic twins in our
study. It is not clear if the same observations can be replicated in
monozygotic twins, especially monochorionic twins who have a
significantly higher risk of fetal demise {[}23, 24{]}. Moreover, the
results obtained using the reduced trisomy co-twin might not fully
represent natural twin demise, which can be caused by many other reasons
with or without chromosomal abnormalities {[}7, 25{]}. Besides, in our
study, women opted for reduction at 14 to 17 gestational weeks, thus
missing the opportunities to observe residual cfDNA at early pregnancy
when most vanishing twins occur. Lastly, because of the difficulties
during clinical practice, the time points of sample collection in each
woman were not identical. In one case (patient 2), the blood sample
before the reduction was missing.

\textbf{Interpretations}

At the first two to three weeks after fetal reduction, there were
discordant observations of fetal fraction changes, as two cases showed a
bland increment of cfDNA release of demised co-twin while two other
cases showed a slight decline. After that, all cases demonstrated
consistent patterns of fast increase of a fetal fraction of demised
co-twin, and reached the top level at 7-9 weeks after reduction and the
cfDNA disappeared at 12-16 weeks after a demise or even longer. This
indicates that the fetal demise induced increased release of residual
cfDNA into maternal circulation for a substantial period. A recent study
by Bevilacqua et al. may observe the a similar phenomenon {[}26{]}, in
which the fetal aneuploidy risk of NIPT after the selective reduction of
a trisomy co-twin also displayed the `rise-and-drop' changes in two
cases.

Recently, Boyd described the histopathological changes of the placenta
in intrauterine demise {[}27{]}. After fetal demise, maternal perfusion
and fetal blood flow pressure disappear, which leads to the compromise
of villous capillary integrity and endothelial karyorrhexis, eventually
resulting in fibrosis avascular villi and solid fabrin deposition of
intervillous space. Meanwhile, villous edema and intravillous hemorrhage
accompanied by maternal inflammation could also occur to increase organ
autolysis {[}27{]}. It was believed that intravascular karyorrhexis and
multifocal villous obliteration might occur soon after fetal demise,
whereas extensive obliteration of stem villi and have the level of
villous fibrosis may take 14 days or more {[}27, 28{]}. Thus, we
speculated that the initial discordant change patterns of residual cfDNA
released from demised co-twin reflects the complicity of placental
autolysis immediately after the cease of fetal blood flow and maternal
perfusion. In contrast, the afterward rapid increase of residual cfDNA
release was the result of extensive villous fibrosis and stem villi
obliteration occurred at least 14 days after fetal demise. Also, it
appeared that the fetal fraction of the surviving euploid fetus
increased a lot faster when the release of residual cfDNA from the
demised fetus declined at 7-9 weeks after reduction. This may reflect
that the normal development and cfDNA emitting of the surviving fetus is
influenced by the demised fetus.

Gestational age at fetal demise might have important roles in
determining how long the residual cfDNA of the demised fetus can last.
Gromminger et al. reported a vanishing twin that cfDNA of the demised
co-twin only contributed to a small proportion of total fetal fraction,
presumably because of early pregnancy demise {[}22{]}. However, another
case reported by Niles et al. demonstrated different findings, in which
a co-twin demised at 6+1 weeks showed persistent false positive NIPT
results for at least 15 weeks after fetal demise {[}29{]}. Our study
added no evidence since the lack of samples with fetal demise at early
pregnancy. Hence, further studies are still needed to explain the effect
of gestational age on residual cfDNA clearance.

The fetal demise of a co-twin has been known as an important factor
causing false positive NIPT results {[}30, 31{]}. It has been estimated
that a substantial proportion of false positive NIPT results ranging
from 15\% to 33\% might be caused by the fetal demise of unaware twin
pregnancies {[}32-34{]}. A key concern for clinical practice of
providing NIPT service is how long the residual cfDNA from the demise
co-twin remains in maternal circulation, and whether NIPT should be
provided if the fetal demise of a co-twin is identified. Two previous
studies have provided preliminary evidence. Curnow et al. demonstrated
in five vanishing twins that the fetal cfDNA from vanished twins was
detectable at eight weeks after demise {[}21{]}. However, their study
was based on retrospective NIPT data of samples known with a fetus
deceased at seven to eight weeks, and thus provided no follow-up of the
rest of pregnancy. Bevilacqua et al. also used fetal reduction of a
trisomic co-twin as a model for natural demise, and reported the
prospective cfDNA testing results in seven cases {[}26{]}. However, due
to minimal sampling frequency, although they found transiently
detectable aneuploidy signals at three to eight weeks following
reduction, they could not conclude the predictable patterns of
aneuploidy risk and fetal fraction. In contrast, our study demonstrated
that after fetal reduction, there was a consistent pattern of the change
of fetal trisomy T-score and fetal fraction.

Both the fetal fraction of demised fetus and the trisomy T-score
increased to a peak level at seven to nine weeks after reduction,
possibly because of the increased rate of placental tissue autolysis
after fetus deceased. Although continuous decline after eight weeks
post-reduction, the detectable fetal fraction of demised fetus and
trisomy T-score still lasted from 12 to 16 weeks after reduction, and
even up to 20 weeks post-reduction. This finding is consistent with a
sporadic case showing NIPT false positive results of fetal sex and
aneuploidy at 15 weeks after a demised co-twin {[}29{]}. However, this
phenomenon has not been thoroughly proven. Based on our results,
residual cfDNA of fetal demise remained in maternal circulation had a
long-lasting effect on NIPT by inducing false positive results for a
long time of pregnancy. Therefore, the current data does not support
providing NIPT to pregnancies given the identification of fetal demise.

\textbf{Conclusions}

Continuous detection of a significant risk of fetal trisomy after fetal
reduction indicated that residual cfDNA remained in maternal plasma for
around 12 to 22 weeks after fetal demise. The persistent detection of
residual cfDNA of the demised co-twin has a long-lasting influence on
the results of NIPT. It should be cautious about providing NIPT in
pregnancies with a risk of fetal demise.

\textbf{Conflicts of Interest}

None of the authors have any conflicts of interest to declare. Completed
disclosure of interest forms is available to view online as supporting
information.

\textbf{Contribution to authorship}

MC and YG designed the study. FS and JW wrote the first version of the
manuscript. MC and JW recruited patents and collected samples and
clinical follow-up. FS, YW, YW, YZ, FC performed sample preparation and
sequencing experiments. LZ, QL, XC, YY, MC conducted bioinformatics
analysis. MC, FS, JW, and YG interpreted sequencing results and clinical
implications. MC, DC and YG overviewed the study, finalized the
manuscript, and provided funding.

\textbf{Details of ethics approval}

This study was approved on May 21, 2019 by the Ethics Committee of the
Third Affiliated Hospital of Guangzhou Medical University (approval
no.2019096) and the Institute Review Board of BGI-Shenzhen (approval
no.19081).

\textbf{Funding}

The study was supported by the National Key Research and Development
Program of China (2018YFC 1004104), the National Natural Science
Foundation of China (NSFC) (No. 81671470), and Shenzhen Municipal
Government of China (JCYJ20180703093402288 and JCYJ20170412153136375).

\textbf{Acknowledgments}

We thank all the participating women for their cooperation in this
study. We thank Dr. Tze Kin Lau for valuable suggestions in revising the
manuscript.

\textbf{Suppporting Information}

Additional supporting information may be found online in the Supporting
Information section at the end of the article.

\textbf{Table S1.} Detailed information of patients that had
NIPT-positive results.

\textbf{Reference}

1. Ong SS, Zamora J, Khan KS, Kilby MD: \textbf{Prognosis for the
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Table1 Characteristics and clinical details of pregnancies. ART,
artificial reproductive technology; DCDA, dichorionic diamniotic;
IVF-ET, in vitro fertilization and embryo transfer; NT, nuchal
translucency.\selectlanguage{english}
\begin{longtable}[]{@{}llllll@{}}
\toprule
\begin{minipage}[b]{0.14\columnwidth}\raggedright\strut
\textbf{Samples}\strut
\end{minipage} & \begin{minipage}[b]{0.14\columnwidth}\raggedright\strut
\textbf{Patient 1}\strut
\end{minipage} & \begin{minipage}[b]{0.14\columnwidth}\raggedright\strut
\textbf{Patient 2}\strut
\end{minipage} & \begin{minipage}[b]{0.14\columnwidth}\raggedright\strut
\textbf{Patient 3}\strut
\end{minipage} & \begin{minipage}[b]{0.14\columnwidth}\raggedright\strut
\textbf{Patient 4}\strut
\end{minipage} & \begin{minipage}[b]{0.14\columnwidth}\raggedright\strut
\textbf{Patient 5}\strut
\end{minipage}\tabularnewline
\midrule
\endhead
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\textbf{Maternal age (year)}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
42\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
40\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
31\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
44\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
37\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\textbf{Pregnancy history}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
G1P0\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
G3P0\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
G1P0\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
G2P1\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
G2P1\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\textbf{Chorionicity}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
DCDA\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
DCDA\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
DCDA\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
DCDA\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
DCDA\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\textbf{ART method}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
IVF-ET\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
IVF-ET\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
IVF-ET\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
IVF-ET\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
IVF-ET\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\textbf{Number of embryo transfer}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
2\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
2\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
2\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
2\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
2\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\textbf{Gestational age for ultrasound (week)}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
13\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
14\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
12+5\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
12+6\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
12+5\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
\textbf{Ultrasound examination}\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
Fetus 1: NT 1.5mm; Fetus 2: NT 4.3mm, nasal bone loss, tricuspid
regurgitation, single umbilical artery\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
Fetus 1: NT 2.0mm; Fetus 2: NT 3.5mm, nasal bone loss, omphalocele\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
Fetus 1: NT 1.8mm; Fetus 2: NT 2.0mm, nasal bone loss\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
Fetus 1: NT 2.7mm, nasal bone loss, tricuspid regurgitation, single
umbilical artery; Fetus 2: NT 2.5mm\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
Fetus 1: NT 3.3mm; Fetus 2: NT 2.6mm, nasal bone loss, omphalocele\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
\textbf{Prenatal diagnosis}\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
Fetus 1: 46, XX; Fetus 2: 47, XX, +18\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
Fetus 1: 46, XY; Fetus 2: 47, XY, +18\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
Fetus 1: 46, XY; Fetus 2: 47, XX, +21\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
Fetus 1: 47, XX, +18; Fetus 2: 46, XY\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
Fetus 1: 46, XX; Fetus 2: 47, XY, +18\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\textbf{Gestational age for reduction surgery (week)}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
14+3\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
14+0\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
17+4\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
15+5\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
14+5\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\textbf{Delivery}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
One female with no apparent abnormalities\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
One male with no apparent abnormalities\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
One male with no apparent abnormalities\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
One male with no apparent abnormalities\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
One female with no apparent abnormalities\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\textbf{Gestational age of birth (weeks)}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
38+6\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
36+5\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
39+2\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
37+1\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
38+5\strut
\end{minipage}\tabularnewline
\bottomrule
\end{longtable}

\textbf{Hosted file}

\verb`Figure1. Fetal fractions at multiple time points before and after fetal reduction in five twin pregnancies.docx`  available at \url{https://authorea.com/users/316246/articles/446393-temporal-persistence-and-long-term-effect-on-noninvasive-prenatal-testing-by-residual-fetal-cell-free-dna-of-demised-co-twin-after-selective-fetal-reduction-in-dichorionic-diamniotic-twin-pregnancies}

\textbf{Hosted file}

\verb`Figure2.NIPT results by T-scores at each timepoint in five twin pregnancies.docx`  available at \url{https://authorea.com/users/316246/articles/446393-temporal-persistence-and-long-term-effect-on-noninvasive-prenatal-testing-by-residual-fetal-cell-free-dna-of-demised-co-twin-after-selective-fetal-reduction-in-dichorionic-diamniotic-twin-pregnancies}

\selectlanguage{english}
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\end{document}

