Ultrasound in Obstet Gyne - 2024 - Bet - Adverse Pregnancy Outcome in Fetuses With Early Increased Nuchal Translucency

Bet B.B.
(Orcid ID: 0000-0003-2200-9607)

Lugthart Malou Anne (Orcid ID: 0000-0001-8385-2150)
Adverse pregnancy outcome in fetuses with early increased nuchal
translucency: prospective cohort study
B. B. Bet1,2, M. A. Lugthart1,2, I. H. Linskens2,3, M. C. van Maarle4, E. van Leeuwen1,2, E.
Pajkrt1,2
1
Department of Obstetrics and Gynecology, Amsterdam UMC location University of
Amsterdam, Amsterdam, The Netherlands

2
Amsterdam Reproduction and Development, Amsterdam, The Netherlands
3
Department of Obstetrics and Gynecology, Amsterdam UMC location Vrije Universiteit,
Amsterdam, The Netherlands

4
Department of Clinical Genetics, Amsterdam UMC location University of Amsterdam,
Amsterdam, The Netherlands
Corresponding author: Dr B. B. Bet, Amsterdam UMC location University of Amsterdam,
Meibergdreef 9, 1105 AZ, Amsterdam, The Netherlands (e-mail: b.bet@amsterdamumc.nl)
Short title: Early increased fetal nuchal translucency
Keywords: nuchal translucency, chromosomal anomalies, genetic disorders, structural
anomalies, adverse pregnancy outcome, non-invasive prenatal test, aneuploidy screening,
first-trimester anomaly scan, invasive prenatal testing
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CONTRIBUTION
What are the novel findings of this work?
This study found an adverse pregnancy outcome in 35.8% of fetuses with an increased nuchal
translucency (NT), defined as a NT≥2.5mm with a crown-rump length (CRL) <45mm. It is
confirmed in a prospective setting that fetuses with a persistently increased NT are associated
with a higher adverse outcome compared to fetuses with a normalized NT (65.2% versus
14.3%)
What are the clinical implications of this work?
Fetuses with an early increased NT are at high risk of adverse pregnancy outcome, therefore
we advise referral of all fetuses with an early increased NT to the fetal medicine unit for
detailed ultrasonography and counseling about diagnostic options. A follow-up NT can be of
prognostic value, but the risk of adverse outcome is still increased.

ABSTRACT
Objectives: An increased nuchal translucency (NT) ≥3.5mm is a well-established marker for
congenital anomalies and adverse pregnancy outcome between 11 and 14 weeks of
gestation. Little is known about its performance as a screening tool before 11 weeks of
gestation. We aimed to investigate in a prospective setting whether fetuses with an increased
NT before 11 weeks of gestation are at risk for an adverse pregnancy outcome.
Methods: This is a prospective cohort study including pregnant women with a viable fetus
with a NT≥2.5mm and a crown-rump-length (CRL) <45mm. All women were referred to our
fetal medicine unit (FMU) and scheduled for a follow-up scan where the NT was remeasured
after one week when the CRL was >45mm. Two groups were evaluated: cases with a
normalized NT(<3.5mm) and cases with a persistently increased NT (≥3.5mm). We monitored
the cases until four weeks after delivery. The main outcome was a composite adverse
outcome of aneuploidies, other genetic disorders, structural anomalies and pregnancy loss.
We performed subgroup analyses of NT thickness at inclusion and normalized or persistently
increased NT at follow-up.
Results: We included 109 cases of which 35.8% (39/109) had an adverse pregnancy
outcome. Of these 64.1% (25/39) were aneuploidies which corresponds to 22.9% (25/109)
aneuploidies in total. The subgroups of NT thickness at inclusion of 2.5-3.4, 3.5-4.4 and
≥4.5mm showed abnormal outcomes in 22.0% (9/41), 40.0% (18/45), 52.2% (12/23)
respectively. In fetuses with a normalized NT and without ultrasound abnormalities at follow-
up scan, the incidence of adverse outcome was 8.5% (5/59), of which 5.1% (3/59) were
aneuploidies.
Conclusion: Fetuses with an early increased NT thickness are at considerable risk of an
adverse pregnancy outcome, even if the NT normalizes after 11 weeks. Not all congenital
anomalies can be diagnosed with routine first-trimester screening such as non-invasive
prenatal testing and/or a first-trimester anomaly scan. Therefore, expectant parents should
always be referred to a FMU for detailed ultrasonography. Invasive prenatal testing should be
offered if an increased nuchal translucency of ≥2.5mm is observed before 11 weeks of
gestation.
INTRODUCTION
Increased nuchal translucency (NT) is a well-established marker for adverse pregnancy
outcomes given its association with aneuploidies, structural anomalies and a wide range of
genetic disorders1-7. Moreover, it is also associated with an increased chance of pregnancy
loss8. The current distribution model to determine an increased NT is correlated to the fetal
crown-rump length (CRL) ranging from 11+2 weeks (CRL=45mm) until 13+6 weeks of
gestation (CRL=84mm), together with 1st, 5th, 95th and 99th centiles9, 10. The 99th cut-off value
and significance of an increased NT with CRL <45mm is uncertain, as few publications on this
topic are available11-15. As the non-invasive prenatal test (NIPT) can already be performed
reliably from 10 weeks of gestation onwards, dating scans are performed more often at 9-10
weeks of gestation. Thus, an early increased NT will be a more common finding, with limited
possibilities to evaluate fetal anatomy at that gestational age.
When an increased NT is observed before 11 weeks of gestation, ultrasonographers in the
Netherlands are currently advised to repeat the ultrasound examination and NT measurement
in the correct timeframe (11+2 – 13+6). In case of NT normalization, women can opt for routine
first-trimester screening consisting of the NIPT and/or a first-trimester anomaly scan (FTAS)16.
When a persistent increased NT is observed, women are referred to a fetal medicine unit
(FMU) for detailed ultrasonography and counseled about diagnostic options. However, it
remains unclear if and when an increased NT will normalize in and if these fetuses will develop
normally as it is only studied in retrospective studies before11, 15. In our previous study, we
found an adverse pregnancy outcome in 24% of the cases with a normalized NT compared to
78% in the group with a persistently increased NT11. However, since these results were
possibly subjected to selection bias, we decided to perform a prospective cohort study.
We aimed to evaluate the clinical relevance of an early increased NT in terms of a composite
adverse pregnancy outcome, consisting of aneuploidies, other genetic disorders, structural
anomalies and pregnancy loss. We analyzed the association between NT thickness at

inclusion and adverse outcome, and compared fetuses with a normalized NT or persistently
increased NT at follow-up.
METHODS
Study design and population
We conducted a prospective cohort study between May 2021 and September 2022 in
pregnant women with a gestational age between 9+0 and 11+2 weeks in the greater
Amsterdam region, the Netherlands. In the Netherlands, all pregnant women are offered a
dating scan and counseling on prenatal screening in the first trimester. These consultations
are primarily conducted at midwifery practices for low-risk pregnancies, while high-risk
pregnancies are handled by the obstetric departments of (academic) hospitals. Routinely
measurement of NT is not standard practice during the dating scan conducted between 9-10
weeks of gestation. However, occasionally an enlarged NT is visually observed and
subsequently measured. Consequently, we requested midwifery practices and affiliated
hospitals to refer eligible patients to our FMU.
Pregnant women were eligible if they had a singleton or a twin pregnancy with viable fetus(es)
and an isolated early increased NT. This was defined as a NT ≥2.5mm (the 99th percentile for
this gestational age9, 15, and in line with our previous study11) and a CRL between 25-44.9 mm.
We excluded pregnant women with a maternal age below sixteen years, with insufficient
knowledge of the Dutch or English language to comprehend the patient information, or with
an increased risk of a child with a monogenetic disease or chromosomal anomaly based upon
medical history. Cases with severe congenital anomalies present at inclusion, such as a body
stalk anomaly, were also excluded.
Study procedure
Women pregnant with a fetus with a NT ≥2.5mm and a CRL between 25-44.9mm were eligible
and were informed on study participation, first shortly by their primary healthcare provider and
in detail by the researcher by telephone. After oral consent, women were referred to the FMU
of the Amsterdam UMC, the ultrasound images of inclusion were retrieved and NT
measurements were verified by the authors. At the appointment at the FMU, we performed a
subsequent NT measurement at a minimum of seven days after the inclusion ultrasound, and
always between 11 and 14 weeks of gestation (maximum 14 days). A detailed ultrasound
examination was performed to detect congenital anomalies according to ISUOG guidelines17.
Prior to this follow-up scan the study information was repeated and informed consent was
signed. We examined normalization of NT according to the standards of the Fetal Medicine
Foundation (FMF)9. Therefore, a NT <3.5mm (<p99) was scored as normalized and a NT
≥3.5mm (≥p99) was scored as persistently increased. Women pregnant with a fetus with a
normalized NT and no other anomalies on the detailed ultrasound scan were referred back to
their primary care provider. They could opt for NIPT, a routine first-trimester anomaly scan
(FTAS) and/or second-trimester anomaly scan (STAS) locally. Parents were also counseled
about invasive diagnostic testing such as chorionic villus sampling and amniocentesis, and it
was performed if desired. In cases with a persistently increased NT or structural anomalies,
parents were offered invasive genetic testing and an advanced anomaly scan was performed
by a perinatologist at 20 weeks of gestation. However, they could also opt for NIPT and
sonographic follow-up after being informed about the limitations of these non-invasive tests.
In case of a normal anatomy scan, patients were referred back to their primary healthcare
provider. Alternatively, in case of abnormal findings, obstetric care was continued in the FMU.
Follow-up data was collected four weeks after birth or after termination of pregnancy. For
children born in our FMU, we used the electronic patient file to collect the data. For children
born elsewhere, we approached the midwife or affiliated hospital to obtain all necessary data.
All participants received a short digital questionnaire four weeks after birth, sent by e-mail, to
document unexpected postnatal findings (Appendix S1). If participants did not return the
questionnaire, subsequent information was obtained from their midwifery practice. The
flowchart of the study procedure is available in the supporting information as Figure S1.
Sample size calculation
The sample size calculation of this study focused on the comparison of chromosomal
anomalies between fetuses with persistently increased NT and normalized NT. We

hypothesized that the incidence of chromosomal anomalies would be 27.4% or higher in the
group of fetuses with a persistently increased NT18, and 0.5% or lower in the group with a
normalized NT19. This would require a sample size of at least 34 patients in both groups to
show a significant difference. We used a Fisher's exact test using 97.5% one-sided
significance level and 90% power.
Study parameters and endpoints
The main outcome was a composite adverse pregnancy outcome that consisted of
aneuploidies, other genetic disorders, structural anomalies and pregnancy loss. We made
subgroup analyses based on NT thickness at the inclusion ultrasound and normalized or
persistently increased NT at follow-up scan. We evaluated which cases would have been
missed by routine first-trimester screening, consisting of NIPT and FTAS. We analyzed the
subgroup that would not have been routinely referred to the FMU in the current Dutch system,
namely the cases with NT at inclusion of 2.5-3.4mm. Lastly, pregnancy outcomes were
explored.
We divided the genetic outcomes in two categories: aneuploidies and other genetic disorders.
Other genetic disorders were defined as chromosomal aberrations (deletions, duplications,
inversions or translocations) and (mono)genetic diseases. We used these categories as the
NIPT is primarily designed for testing aneuploidy, the disorders as defined in “other genetic
disorders” could be missed with routine screening. We presented structural anomalies as
outcome only in cases without a chromosomal or genetic diagnosis. The aneuploidies, other
genetic disorders and structural anomalies were together categorized as congenital
anomalies. Next, pregnancy loss was determined as miscarriage or fetal death without a
congenital anomaly. A normal outcome was a live birth with normal physical examination until
four weeks after birth.
During the study period, NIPT was performed in the context of the Dutch TRIDENT study that
primarily tests for trisomy 21, 18 and 13, but also chromosomal aberrations on the other
autosomes (size resolution of 10–20Mb) could be tested if parents desired it. In these study,
sex chromosomes are not analyzed20.
Quantitative Fluorescence-Polymerase Chain Reaction (QF-PCR) was performed for
chromosome 21,18, 13, X and Y in all cases with invasive testing, followed by chromosome
microarray analysis when the QF-PCR result was normal. Whole Exome Sequencing (WES)
was reserved for cases with normal QF-PCR and microarray and with persistently increased
NT at follow-up scans or in case of other structural anomalies. Karyotyping was not performed
in all cases, only in the presence of a chromosome anomaly to determine heredity.
Data analysis
We described continuous data as mean with standard deviation (SD) or median with
interquartile rage (IQR) when appropriate. Categorical data were expressed as number with
percentages. The primary outcome of composite adverse pregnancy outcome was described
as number with percentage. Subgroup analysis of normalized NT and persistently increased
NT were performed using a Chi-square test to assess associations and the difference was
expressed as an odds ratio (OR). Furthermore, differences in baseline characteristics between
groups were analyzed using Chi-square test, Fisher’s exact test, or independent sample T-
test when appropriate. Trend analysis of NT measurement and composite adverse outcome
was assessed by scatterplots. We considered P values <0.05 as statistically significant.
Statistical analyses were done using IBM Corp SPSS Statistics version 25.0 (IBM, Armonk,
NY, USA) and RStudio version 1.2.1335.
Ethics
This study was registered in the Dutch National Trial Registry as NTR9494. The Medical Ethics
Committee of the Amsterdam UMC, location University of Amsterdam, approved the study
protocol in April 2021 (NL74879.018.21). All patients included gave written informed consent
before inclusion.
RESULTS
During the study period, 125 cases were referred for possible inclusion. Subsequently six
cases were excluded, because of severe structural anomalies at inclusion ultrasound (n=1),
non-viable pregnancy at follow-up scan (n=2), carriership of a genetic disease in (one of) the
parents (n=2) and no exact NT measurement at inclusion ultrasound (referred on
“eyeballing”)(n=1). Additionally, ten cases were excluded because women declined to
participate in this study. In total, 109 cases were included and the baseline characteristics of
the mother, pregnancy and fetus are presented in Table 1. Examples of an early increased
NT and NT-measurement at follow-up scan are shown in Figure 1.
After follow-up scan at the FMU, 101 of the 109 included cases (92.7%) opted for further
genetic testing. NIPT was solely performed in 49 cases (45.0%), invasive prenatal testing in
44 cases (40.4%) and postnatal testing in eight cases (7.3%). The invasive genetic tests
consisted of karyotype and/or QF-PCR (n=23), QF-PCR and microarray (n=19), QF-PCR,
microarray and WES (n=10).
In the remaining eight cases (7.3%), the parents opted out of genetic testing, all with a normal
outcome (live birth and normal follow-up after birth). Follow-up was completed for all
participants.
Composite adverse outcome
An adverse pregnancy outcome occurred in 39 of 109 cases (35.8%, 95%-CI 27.4-45.1%). Of
these, 36 (92.3%) were diagnosed with congenital anomalies and three cases resulted in
pregnancy loss. An aneuploidy was detected in 25 cases (22.9%) and in seven cases (6.4%)
another genetic disorder was found. Additionally, in cases without an aneuploidy or other
genetic disorder, a structural anomaly was diagnosed in four cases (3.7%). Structural
anomalies were also seen in 16 (64.0%) of the cases with an aneuploidy and in 6 (85.7%) of
the cases with another genetic disorder. Table 2 displays the relation between an early
increased NT thickness at inclusion and adverse pregnancy outcomes.

In the subgroup of 41 cases with a NT measurement of 2.5-3.4mm at inclusion, an adverse
pregnancy outcome was found in nine cases (22.0%, 95%-CI 12.0-36.7%)(Table 2). The
highest incidence of an adverse outcome was found in the subgroup with a NT ≥4.5mm at
inclusion, respectively twelve of 23 cases (52.2%, 95%-CI 33.0-70.8%)(Table 2). The rate of
adverse outcome increased with increasing NT but this was not associated with a higher
incidence of genetic anomalies, more cases had structural anomalies or were pregnancy
losses. Table 3 displays the variety of congenital anomalies, and by which prenatal test they
were found.
Normalized vs persistently increased NT
At follow-up NT measurement, the majority of cases, that is 63 out of 109 (57.8%), were
categorized as normalized (NT <3.5mm). An adverse pregnancy outcome was found in nine
cases (14.3%, 95%-CI 7.7-25.0%) within this group (Table 4). Four of them showed multiple
ultrasound anomalies and all had an abnormal pregnancy outcome. In fetuses with a
normalized NT and without ultrasound abnormalities at the follow-up scan, an adverse
outcome occurred in five of 59 cases (8.5%). Aneuploidies were diagnosed in three cases
(5.1%) within this group, consisting of two cases with Trisomy 21 and one case with Klinefelter
syndrome (Table 4). The other two anomalies were a chromosome 13 microdeletion and a
sacrococcygeal teratoma.
The persistently increased NT group consisted of 46 of 109 cases (42.2%) in which an
adverse pregnancy outcome was found in 30 cases (65.2%, 95%-CI 50.8-77.3%). In this
group, aneuploidies, other genetic disorders and structural anomalies were diagnosed in 20
(43.5%), five (10.9%) and two cases (4.3%), respectively. Additionally, there were three
pregnancy losses (6.5%).
Adverse pregnancy outcomes were found in 14.3% and 65.2% in fetuses with a normalized
and persistently increased NT, respectively, with an OR of 11.3 (95%-CI 4.4-28.5, p<.001). A
trend of an increasing NT related to an adverse pregnancy outcome and a decreasing NT
related to a normal outcome was seen and is presented in Figure 2.
In addition, in both groups an adverse pregnancy outcome was found in 8.5% (95%-CI 3.7-
18.4%) and 48.1% (95%-CI 30.7-66.0%) in fetuses without ultrasound abnormalities on follow-
up scan, respectively, with an OR of 10.0 (95%-CI 3.0-32.9, p<.001)(Table 4).
Looking at the characteristics of the normalized and persistently increased NT groups, there
was a difference in mean NT at inclusion of 3.6 ±0.8mm and 4.2 ±1.1mm (p<.001), and a
difference in mean NT at follow-up scan of 1.9±0.7 mm and 6.0 ±1.9mm (p<.001). Additionally,
the maternal age in the normalized NT group was significantly lower than in the persistently
increased group, 32.9 vs. 34.8 years (p=0.042). There was no difference between the groups
in CRL or gestational age at inclusion, BMI or nulliparity.
Lastly, Table 5 presents the relation between normalized or persistently increased NT and
outcome per subgroup of NT at inclusion. There was a statistically significant difference in
incidence of adverse outcome in the subgroup 3.5-4.4mm and ≥4.5mm between normalized
and persistently increased NT, of 13.0% vs 68.2% and 12.5% vs 73.3% respectively (Table
5).
Outcomes potentially missed by routine first-trimester screening
Two cases had a congenital anomaly that would have remained undetected by routine first-
trimester screening, namely the diagnosis of a Klinefelter syndrome and a sacrococcygeal
teratoma. In the Klinefelter case, the NT at inclusion was 3.2mm and at follow-up scan 0.9mm,
the FTAS was normal and the (Dutch) NIPT could not detect this anomaly as sex
chromosomes are not analyzed. The case with a sacrococcygeal teratoma showed no
ultrasound abnormalities until 19 weeks of gestation. All other cases had either a persistently
increased NT at follow-up scan, an abnormal NIPT result or showed ultrasound abnormalities
in the first trimester.

Cases with NT consistently <3.5mm
The NT was normalized in 32 of the 41 cases (78.0%) in the subgroup with NT 2.5-3.4mm,
and thus never measured above 3.5mm (Table 5). Five of these 32 cases (15.6%, 95%-CI
6.9-31.8%) had an adverse pregnancy outcome, consisting of Trisomy 21 (n=2, NT at follow-
up of 2.1mm and 2.2mm, both without other structural anomalies), chromosome 13
microdeletion (n=1, NT at follow-up of 3.3mm, cerebellar vermis hypoplasia seen at 14 weeks
of gestation), Klinefelter syndrome (n=1), and a sacrococcygeal teratoma (n=1).
Pregnancy outcomes
Of the total cohort, 74 children (67.9%) were live born. Their median gestational age at delivery
was 39 weeks and 6 days (IQR 38+2–40+5), the mean birthweight was 3312gr ±673 and
51.4% had a female gender.
Termination of pregnancy (TOP) occurred in 24 cases (22.0%), miscarriage in six cases
(5.5%) and fetal death in five cases (4.6%). The mean gestational age at TOP was 14 weeks
and 5 days. In eight of these 24 cases (33.3%), the parents opted for NIPT and the mean
gestational age at TOP was 15 weeks and 4 days. Compared to the 16 cases in which NIPT
was not performed, the mean gestational age at TOP was 14 weeks and 2 days (p=0.267).
DISCUSSION
Principal findings
Fetuses with an early increased NT are at high risk of adverse pregnancy outcome. In our
cohort of 109 fetuses, we found aneuploidies, other genetic disorders and structural anomalies
in 22.9%, 6.4% and 1.8%. Additionally, three pregnancy losses occurred without other
diagnosis and therefore the composite adverse pregnancy outcome was 35.8%. Fetuses with
a normalized NT were significantly different from fetuses with a persistently increased NT at
follow-up scan, with an adverse outcome in 14.3% vs 65.2%. In fetuses with a normalized NT
and without ultrasound abnormalities at follow-up scan, the incidence of adverse outcome was
8.5%.
Interpretation
The limited retrospective studies on early increased NT have shown that there is an
association with adverse outcome, comparable to that of increased NT between 11-14 weeks’
gestation11, 13-15. Grande et al. showed that 64% of fetuses with trisomy 21 already had an
increased NT before 11 weeks of gestation, suggested that re-evaluation was not needed13.
Additionally, Scholl et al. analyzed fetuses with cystic hygroma before 11 weeks and reported
a high number of adverse outcome (43%), but did not evaluate the exact thickness of the NT14.
Ramkrishna et al. was the first to provide a follow-up NT in fetuses with increased NT
(>2.2mm) and CRL between 28-44 and distinguished between fetuses with neck edema or
generalized edema15. Normalized NT (<3.5mm) occurred in the majority of the cases (81.9%)
but was not measured in all cases. Our results also showed that NT normalizes after 11 weeks
of gestation in the majority of the cases (58%). This is higher than the subgroup analysis of
Lugthart et al. in which 44% had a normalized NT11, and can possibly be explained by the
retrospective design of that study and the risk of selection bias therein as only severe cases
were potentially referred. In addition, our study demonstrates a superior outcome within the
normalized NT subgroup, with a lower incidence of adverse outcome respectively 14.3% (9 of

63 cases) in this prospective cohort compared to 24.0% (6 of 25 cases) in our previous
retrospective cohort . Both studies, however, demonstrate a clinically relevant high rate of
11
congenital anomalies (chromosomal, genetic and/or structural anomalies) when compared to
the baseline prevalence of approximately 2.5% derived from EUROCAT data21.
The findings of this prospective study highlight the need to evaluate the NT during a dating
scan between 9-11 weeks of gestation, which serves as the initial step of a prenatal screening
program. In cases where an enlarged NT is visualised or suspected at this gestation, we
recommend to conduct a re-evaluation of NT after one week, prior to NIPT, to define the best
diagnostic approach for each individual patient and prevent any delay in diagnosis.
Furthermore, a FTAS at 13-14 weeks of gestation should be performed to assess fetal
(cardiac) anatomy. We observed a slight delay in TOP among cases that chose NIPT as initial
screening test instead of directly undergo invasive prenatal testing. Although not significant,
this finding emphasizes the importance of early invasive testing, if desired.
Although routine screening (NIPT, FTAS, STAS) would classify nearly all abnormal cases as
high-risk for abnormal outcome in our study, invasive genetic testing is superior for achieving
an accurate diagnosis. The genetic disorders in our cohort were diagnosed using microarray
or WES, highlighting their additional diagnostic value in fetuses with increased nuchal
translucency22-24. Performing NIPT only could lead to false reassurance of expectant parents
and potentially waste resources if invasive testing is performed independent of NIPT results.
Moreover, there was one case of false-negative NIPT in our cohort (trisomy 18).
Nevertheless, even if the NT is normalized during re-evaluation, it is important to inform
parents that the risk of chromosomal and structural anomalies remains higher than the
baseline risk (2.5%, EUROCAT)21. Consequently, we recommend referring all patients with a
NT ≥2.5mm and a gestational age before 11 weeks to a FMU, where both invasive and non-
invasive diagnostic tests can be offered after counselling. This enables parents to make
informed decisions regarding reproductive choices.

The results of this prospective study demands further research in the evaluation of the NT
before the traditional timeframe of 11-14 weeks. First, an important area of further research is
determining the normal range of NT measurement in fetuses with a CRL between 25-45mm.
There are some studies done, but currently no 99th percentiles are available and thus no clear
guidelines on normal ranges can be determined12, 13. This can lead to unnecessary anxiety in
pregnant women and the risk of over- or under-testing of the fetus. Therefore, it is also
essential to investigate the psychological impact of receiving a diagnosis of early increased
NT with unclear significance on expectant parents.
Moreover, the long-term outcomes of early increased NT should be evaluated as well. This
study evaluated all fetuses until four weeks after birth, however, it is possible that certain
adverse outcomes will be revealed after that period.
Lastly, it is important to investigate the value of NT evaluation at the dating scan in the context
of the total prenatal screening program25. As a first-trimester anomaly scan at 11-14 weeks’
gestation, including a NT measurement, is currently being researched in the Netherlands, it is
unclear how useful the evaluation of the early NT is.
Strengths and limitations
The prospective design of this study enabled us to collect reliable and comprehensive data,
minimized the risk of selection bias as we included patients from the entire Amsterdam region,
and allowed us to follow all cases until four weeks after birth.
However, some limitations should be mentioned as well. The main limitation is that we were
unable to provide information on the total population (denominator), as NT measurement is
not part of routine practice before 11 weeks of gestation. This resulted in the selective
measurement of NT in cases where it appeared larger than normal (nominator). While this
approach aligns with standard clinical practice involving a pre-NIPT scan, it deviates from the
scientific ideal approach of measuring NT in all cases.

We included all fetuses with increased NT from our region to minimize selection bias, however
not all sonographers performing dating scans are qualified to measure NT according to the
ISUOG guidelines17. Nevertheless, in almost all cases, we obtained the ultrasound images
and re-evaluated the NT according to the guidelines. In addition, not all cases included in this
study underwent the same (amount of) genetic testing, and there is a possibility that some
(mono)genetic disorders may have been missed. However, it would be unethical and
impractical to conduct a prospective study in which invasive genetic tests are mandatory.
Moreover, we did not compare the results to a control group of women without an early dating
scan. Lastly, our sample size calculation enabled us to compare outcomes of fetuses with
normalized and persistently increased NT, however, the sample size was insufficient to
establish normal values of NT at a CRL of 25-45mm.
Conclusions
Fetuses with an early increased NT are at high risk of adverse pregnancy outcome, therefore
we advise referral of all fetuses with a NT ≥2.5mm and CRL <45mm to the FMU for detailed
ultrasonography and counseling about further diagnostic options. A repeated NT
measurement can be of prognostic value, however, the risk of adverse outcome is still
increased. Therefore, expectant parents should be informed about their options and should
not be offered solely routine first-trimester screening as NIPT and/or FTAS.

in the study and the staff at the Fetal Medicine Unit of the Amsterdam UMC, who facilitated
We acknowledge the support of all the pregnant women and their partners who participated
the execution of the study.

Acknowledgments
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FIGURE LEGENDS
Figure 1 Examples of early increased NT (A1, B1, C1) and the follow-up NT measurements
(A2, B2, C2). Fetus A was diagnosed with Trisomy 18. The NT-measurements were 4.0mm
at inclusion and 8.5mm at follow-up (persistently increased NT). Fetus B was diagnosed with
a chromosome 13 microdeletion. The NT-measurements were 2.7mm at inclusion and 3.2mm
at follow-up (normalized NT). Fetus C was diagnosed with Trisomy 21. The NTmeasurements
were 2.7mm at inclusion and 2.1mm at follow-up (normalized NT).
Figure 2 Trend analysis of nuchal translucency and outcome. A. NT at inclusion (CRL <45mm)
and NT at follow-up scan of 70 cases with abnormal pregnancy outcome. B. NT at inclusion
and NT at follow-up scan of 39 cases with an adverse pregnancy outcome. NB: Three outliers
were removed from this graph, due to low CRL <27mm (n=2) and high NT >10mm (n=1).
TABLES
Table 1. Characteristics of the mother, pregnancy and fetus
(n=109)
Parameter N(%), mean ±SD, median
[IQR]
Maternal age (years) 33.7 ±5.0
Weight (kg) 65.0 [58.8 – 75.0]
BMI (kg/m2) 23.2 [20.7 – 26.3]
Smoking 1 (0.9)
Nulliparity 52 (47.7)
Singleton gestation 106 (97.2)
Inclusion ultrasound
Nuchal translucency (mm) 3.6 [3.2 – 4.3]
Crown-rump length (mm) 39.3 [35.1 – 42.4]
Gestational age (weeks, 10w5d ± 3d
days)
Follow-up ultrasound
Nuchal translucency (mm) 2.7 [1.7 – 5.4]
Crown-rump length (mm) 52.3 [49.0 – 55.6]
Gestational age (weeks, 11w5d ± 3d
days)
Abbreviations: BMI; body mass index, IQR; interquartile range, NIPT; non-invasive prenatal
test, SD; standard deviation.

Table 2. Degree of early NT enlargement and relationship with adverse pregnancy outcome,
N (%)
Adverse pregnancy outcome
Congenital anomalies
Live
NT at Other Structural
Total Pregnancy birth,
inclusion Aneuploidies genetic anomalies Total
cases loss † no
(mm) disorders *
defects
2.5 – 3.4 41 6 (14.6) 1 (2.4) 2 (4.9) - 9 32 (78.0)
(22.0)
3.5 – 4.4 45 13 (28.9) 4 (8.9) - 1 (2.2) 18 27 (60.0)
(40.0)
≥ 4.5 23 6 (26.1) 2 (8.7) 2 (8.7) 2 (8.7) 12 11 (47.8)
(52.2)
Total 109 25 (22.9) 7 (6.4) 4 (3.7) 3 (4.6) 39 70 (64.2)
(35.8)
* Structural anomaly without the diagnosis of a chromosomal or genetic disorder.

†
Pregnancy loss (miscarriage or fetal death) without another diagnosis, thus no aneuploidy,
no other genetic or structural anomalies.
Abbreviations: NT; nuchal translucency.

Table 3. Overview of abnormal findings grouped by type of test.
Subdivided for normalized and persistently increased NT groups, N
(%)
Total Normalized Pers.
(n=109) NT (n=63) increased
NT (n=46)
Abnormal NIPT 5 (4.6)
Trisomy 21 2 (1.8) 1 (1.6) 1 (2.2)
Trisomy 18 1 (0.9) - 1 (2.2)
Trisomy 13 2 (1.8) 1 (1.6) 1 (2.2)
Abnormal QF-PCR 20 (18.3)
Trisomy 21 (in total, n=7, 5 (4.6) 1 (1.6) 4 (8.7)
6.4%)
Trisomy 18 (in total, n=11, 10 (9.2) - 10 (21.7)
10.1%)
Trisomy 13 (in total, n=4, 2 (1.8) 1 (1.6) 1 (2.2)
3.7%)
Klinefelter syndrome (XXY) 1 (0.9) 1 (1.6) -
Monosomy X 2 (1.8) - 2 (4.3)
Abnormal Microarray 3 (2.8)
22q11 deletion syndrome 1 (0.9) - 1 (2.2)
13q31.1q34 microdeletion 1 (0.9) 1 (1.6) -
15q11.2 microdeletion † 1 (0.9) - 1 (2.2)
Abnormal WES 4 (3.7)
MYRF gene mutation 1 (0.9) - 1 (2.2)
MED13L gene mutation 1 (0.9) - 1 (2.2)

MED12L gene mutation 1 (0.9) 1 (1.6) -
CBL-gene mutation ‡ 1 (0.9) - 1 (2.2)
Abnormal FTAS 3 (2.8)
Fetal hydrops 2 (1.8) - 2 (4.3)
Bilateral radial aplasia § 1 (0.9) 1 (1.6) -
Abnormal STAS 1 (0.9)
Sacrococcygeal teratoma * 1 (0.9) 1 (1.6) -
Total 36 (33.0) 9 (14.3) 27 (58.7)
* Potentially missed by routine first-trimester screening consisting of NIPT and FTAS, as the
Dutch NIPT does not include sex chromosomes and no ultrasound abnormalities were seen
in the first trimester.
†
Additional finding, definite relation with increased NT could not be made.
‡
Noonan-like syndrome
§
No underlying genetic cause was found with QF-PCR, microarray or WES.
Abbreviations: FTAS; first-trimester anomaly scan, NIPT; non-invasive prenatal test, NT;
nuchal translucency, QF-PCR; Quantitative Fluorescence-Polymerase Chain Reaction,
STAS; second-trimester anomaly scan, WES; whole exome sequencing.

Table 4. Normalized or persistently increased NT at follow-up scan between 11 and 14 of
gestation, and the relation with adverse outcome, N (%)
Adverse pregnancy outcome
Congenital anomalies
Live
Other
Follow-up scan Total Structural Pregnancy birth,
Aneuploidies genetic Total
11-14 weeks cases anomalies Loss no
disorders
defects
Normalized 63 5 (7.9) 2 (3.2) 2 (3.2) - 9 (14.3) 54 (85.7)
No US 59 3 (5.1) * 1 (1.7) † 1 (1.7) ‡ - 5 (8.5) 54 (91.5)
anomalies
US anomalies 4 2 (50.0) § 1 (25.0) ¶ 1 (25.0) ** - 4 (100) 0 (0)
Persistently 46 20 (43.5) 5 (10.9) 2 (4.3) 3 (6.5) 30 16 (34.8)
increased (65.2)
No US 27 6 (22.2) †† 4 (14.8) ‡‡ - 3 (11.1) 13 14 (51.9)
anomalies (48.1)
US anomalies 19 14 (73.7) §§ 1 (5.3) ¶¶ 2 (10.5) *** - 17 2 (10.5)
(89.5)
* Trisomy 21 (n=2), Klinefelter syndrome (n=1)
†
13q31.1q34 deletion (n=1, cerebellar vermis hypoplasia was seen at 14 weeks of gestation)
‡
Sacrococcygeal teratoma (n=1)
§
Trisomy 13 (n=2)
¶
MED12L gene mutation (n=1)
** Bilateral radial aplasia (n=1)

††
Trisomy 21 (n=4), Trisomy 18 (n=1), Monosomy X (n=1)
‡‡
22q11 deletion syndrome (n=1), 15q11.2 microdeletion (n=1), MED13L gene mutation
(n=1), CBL-gene mutation (n=1)
§§
Trisomy 21 (n=1), Trisomy 18 (n=10), Trisomy 13 (n=2), Monosomy X (n=1)
¶¶
MYRF gene mutation (n=1)
*** Fetal hydrops (n=2)
Abbreviations: FU; follow-up, NT; nuchal translucency, US; ultrasound.

Table 5. Analysis of NT measurement at inclusion ultrasound and relation
between normalized or persistently increased NT and outcome, N(%)
Subgroup Total Normal Adverse P-value
outcome outcome
NT 2.5-3.4mm at 41 32 (78.0) 9 (22.0)
inclusion
Normalized 32 27 (84.4) 5 (15.6)
Persistently 9 5 (55.6) 4 (44.4) 0.087
increased
NT 3.5-4.4mm at 45 27 (60.0) 18 (40.0)
inclusion
Normalized 23 20 (87.0) 3 (13.0)
Persistently 22 7 (31.8) 15 (68.2) <.001
increased
NT ≥4.5mm at 23 11 (47.8) 12 (52.2)
inclusion
Normalized 8 7 (87.5) 1 (12.5)
Persistently 15 4 (26.7) 11 (73.3) 0.009
increased
Abbreviations: NT; nuchal translucency.

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Bet B.B.

(Orcid ID: 0000-0003-2200-9607)

Adverse pregnancy outcome in fetuses with early increased nuchal

translucency: prospective cohort study

B. B. Bet1,2, M. A. Lugthart1,2, I. H. Linskens2,3, M. C. van Maarle4, E. van Leeuwen1,2, E.

Amsterdam, Amsterdam, The Netherlands

Amsterdam, The Netherlands

Amsterdam, The Netherlands

Corresponding author: Dr B. B. Bet, Amsterdam UMC location University of Amsterdam,

Meibergdreef 9, 1105 AZ, Amsterdam, The Netherlands (e-mail: b.bet@amsterdamumc.nl)

Short title: Early increased fetal nuchal translucency

Keywords: nuchal translucency, chromosomal anomalies, genetic disorders, structural

anomalies, adverse pregnancy outcome, non-invasive prenatal test, aneuploidy screening,

first-trimester anomaly scan, invasive prenatal testing

What are the novel findings of this work?

translucency (NT), defined as a NT≥2.5mm with a crown-rump length (CRL) <45mm. It is

What are the clinical implications of this work?

detailed ultrasonography and counseling about diagnostic options. A follow-up NT can be of

prognostic value, but the risk of adverse outcome is still increased.

Objectives: An increased nuchal translucency (NT) ≥3.5mm is a well-established marker for

congenital anomalies and adverse pregnancy outcome between 11 and 14 weeks of

gestation. We aimed to investigate in a prospective setting whether fetuses with an increased

NT before 11 weeks of gestation are at risk for an adverse pregnancy outcome.

normalized NT(<3.5mm) and cases with a persistently increased NT (≥3.5mm). We monitored

We performed subgroup analyses of NT thickness at inclusion and normalized or persistently

aneuploidies in total. The subgroups of NT thickness at inclusion of 2.5-3.4, 3.5-4.4 and

respectively. In fetuses with a normalized NT and without ultrasound abnormalities at follow-

Conclusion: Fetuses with an early increased NT thickness are at considerable risk of an

anomalies can be diagnosed with routine first-trimester screening such as non-invasive

offered if an increased nuchal translucency of ≥2.5mm is observed before 11 weeks of

Increased nuchal translucency (NT) is a well-established marker for adverse pregnancy

genetic disorders1-7. Moreover, it is also associated with an increased chance of pregnancy

possibilities to evaluate fetal anatomy at that gestational age.

When an increased NT is observed before 11 weeks of gestation, ultrasonographers in the

possibly subjected to selection bias, we decided to perform a prospective cohort study.

We aimed to evaluate the clinical relevance of an early increased NT in terms of a composite

adverse pregnancy outcome, consisting of aneuploidies, other genetic disorders, structural

anomalies and pregnancy loss. We analyzed the association between NT thickness at

Study design and population

pregnancies are handled by the obstetric departments of (academic) hospitals. Routinely

weeks of gestation. However, occasionally an enlarged NT is visually observed and

subsequently measured. Consequently, we requested midwifery practices and affiliated

hospitals to refer eligible patients to our FMU.

stalk anomaly, were also excluded.

always between 11 and 14 weeks of gestation (maximum 14 days). A detailed ultrasound

examination was performed to detect congenital anomalies according to ISUOG guidelines17.

signed. We examined normalization of NT according to the standards of the Fetal Medicine

Foundation (FMF)9. Therefore, a NT <3.5mm (<p99) was scored as normalized and a NT

was performed if desired. In cases with a persistently increased NT or structural anomalies,

Sample size calculation

anomalies between fetuses with persistently increased NT and normalized NT. We

significance level and 90% power.

Study parameters and endpoints

subgroup analyses based on NT thickness at the inclusion ultrasound and normalized or

Other genetic disorders were defined as chromosomal aberrations (deletions, duplications,

inversions or translocations) and (mono)genetic diseases. We used these categories as the

disorders” could be missed with routine screening. We presented structural anomalies as

genetic disorders and structural anomalies were together categorized as congenital

four weeks after birth.

sex chromosomes are not analyzed20.

Quantitative Fluorescence-Polymerase Chain Reaction (QF-PCR) was performed for

in all cases, only in the presence of a chromosome anomaly to determine heredity.

as number with percentage. Subgroup analysis of normalized NT and persistently increased

expressed as an odds ratio (OR). Furthermore, differences in baseline characteristics between