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\begin{document}

\title{Elevated nuchal translucency, is it time to discuss the cut off?. A
retrospective study}



\author[1]{Lucia Pasquini}%
\author[2]{Ilaria Ponziani}%
\author[2]{Elisa Spataro}%
\author[1]{Giulia Masini}%
\author[2]{Eleonora Biancareddu}%
\author[3]{Adalgisa Cordisco}%
\author[4]{Felice Petraglia}%
\affil[1]{Azienda Ospedaliero Universitaria Careggi}%
\affil[2]{University Hospital Careggi}%
\affil[3]{Piero Palagi Hospital}%
\affil[4]{University of Florence}%


\vspace{-1em}



  
  \date{June 18, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
Objectives To evaluate pregnancy and postnatal outcomes of fetuses with
NT between 95th and 99th percentile and whether they could benefit from
further investigations rather that routine scans. Design Retrospective
multicenter observational study. Setting Two Italian Fetal Medicine
centre Population All cases with NT between 95th and 99th percentile
(667) among women undergoing NT measurement between January 2015 and
December 2020. Methods Unfavourable outcome was considered as:
miscarriage or intrauterine fetal death (IUFD), chromosomal
abnormality/genetic syndrome, major malformation or neurodevelopmental
delay. Study population outcomes were compared with general population.
Main Outcome Measures Unfavourable pregnancy and postnatal outcomes.
Results The rate of unfavourable outcome was 25.44\%. We reported: 6
(0.90\%) second trimester miscarriage or IUFD, 90 (13.49\%) chromosomal
abnormalities/genetic syndromes, 57 (8.55\%) major malformations, 13
(1.95\%) cases of neurodevelopmental delay. The incidence of chromosomal
abnormalities/genetic syndromes and major malformations were
significantly higher (OR 6.99 (IC 95\% 4.33 - 11.28), p\textless{}0.001
and OR 17.77 (IC 95\%7.22 - 43.75), p\textless{}0.001 respectively)
compared to the general population. The incidence of neurodevelopmental
delay was not increased (OR of 0.64 CI 95\% 0.33 - 1.24 p=0.185).
Conclusions Fetuses with NT between 95th and 99th percentile have an
increased risk of pregnancy and postnatal adverse outcomes. According to
our data it is reasonable to consider a lower NT cut off
(NT\textgreater{}95th percentile) for offering further investigations
such as detailed ultrasound scan, fetal echocardiography and counselling
where the option of performing fetal karyotype and CGH array should be
discussed. Funding The authors have no funding to declare.%
\end{abstract}%



\sloppy


\textbf{Introduction}

Nuchal translucency (NT) is a subcutaneous fluid collection behind fetal
neck and it is physiologically present in the first trimester in all
fetuses\textsuperscript{1}. In the early '90, Nicolaides introduced NT
measurement as part of first trimester screening for trisomies 21, 13
and 18\textsuperscript{2} and over the years an increased NT has been
associated with several pathological conditions such as adverse
pregnancy outcome\textsuperscript{3-4}, chromosomal abnormalities other
than common trisomies, genetic syndromes\textsuperscript{5-9}, and
structural anomalies\textsuperscript{3;8}, in particular cardiac
malformations\textsuperscript{10-11}. Therefore, pregnancies with an
increased NT are considered at high risk and detailed fetal ultrasound,
fetal echocardiography, genetic counselling and chorionic villous
sampling (CVS)/amniocentesis are usually offered to these patients.

Reference ranges and percentiles for NT based on gestational age have
been established in a large study by Snijders et
al.\textsuperscript{12}. International guidelines, however, do not show
complete agreement regarding the cut off of NT value or percentile that
requires further investigations; the most commonly used are either 3.5
mm, 3 mm or the 99\textsuperscript{th}
percentile\textsuperscript{13-17}(Table 1). Usually, cases of NT between
the 95\textsuperscript{th}-99\textsuperscript{th} percentile are managed
as healthy pregnancies.

Recently, the accuracy of ultrasonographic technology has markedly
increased and new genetic tests such as CGH-array (Comparative Genomic
Hybridization) and exome sequencing, have been introduced in clinical
practice, allowing to be more accurate in the diagnosis of fetal
anomalies, chromosomal abnormalities and genetic syndromes.

The aim of our multicentric study was to evaluate all women with NT
between 95\textsuperscript{th} and 99\textsuperscript{th} percentile and
discuss a possible new cut off for fetal and genetic tools usually
provided to higher cut offs.

\textbf{Methods}

A multicenter retrospective observational study promoted by the Fetal
Medicine Unit of the Azienda Ospedaliero Universitaria Careggi
(Florence) with the collaboration of Piero Palagi Hospital (Florence).

All patients who underwent NT measurement in the first trimester between
January 2015 and December 2020 were included. Among them, all cases with
NT between 95\textsuperscript{th} and 99\textsuperscript{th} percentile
for gestational age were selected\textsuperscript{12}. Exclusion
criteria were: maternal age under 18 years, monochorionic placentation,
multiple pregnancies, incomplete data or absent informed consent for the
study.

NT was measured according to the Fetal Medicine Foundation
guidelines\textsuperscript{18}. All ultrasound examinations were
performed by a certified FMF sonographer. Cases with positive combined
test or NT[?]3mm were offered genetic counselling, CVS or amniocentesis,
monthly detailed ultrasound and fetal echocardiography as per local
protocol. Information regarding maternal demographic characteristics,
genetic tests, ultrasound scans and other prenatal investigations (such
as fetal magnetic resonance imaging in selected cases), pregnancy
outcome and long-term postnatal outcome were obtained by reviewing
hospital records and patients' interviews. Patients were asked to
provide copy of medical records in case of postnatal diagnosis of
neonatal disease.

We considered as unfavourable outcome: miscarriage or intrauterine fetal
death (IUFD), the presence of chromosomal abnormality/genetic syndrome,
major malformation or neurodevelopmental delay. Major malformations were
defined as those requiring chronic medical treatment or major surgery
and/or associated with mental or physical disability. Minor
malformations were not included in the unfavourable outcome. In all
cases of neurodevelopmental delay the diagnosis was made by an expert
clinician.

Postnatal outcomes were compared with general population's data from the
EUROCAT (European Network of Population Based Registries for the
Epidemiological Surveillance of Congenital
Anomalies)\textsuperscript{19} register regarding chromosomal
abnormalities/genetic syndromes and major malformations. With regards to
neurodevelopmental delay, a recent review by Sotiriadis et
al.\textsuperscript{20} was used for comparison.

The study was approved by the local Ethic Committee (CEAVC n=17892) and
it was conducted in accordance with the standards of the Helsinki
Declaration.

Statistical analysis was performed using ``Statistical Package for
Social Sciences'' (SPSS Inc, version 21, Chicago, Il USA), and Microsoft
Office Excel package (Microsoft Corp., Redmond, WA, USA). Chi-square or
Fisher exact test were used to compare categorical variables and T Test
for continuous variables. A p-value of 0.05 was considered as
statistically significant. Odds ratio (OR) and 95\% confidence intervals
(IC 95\%) were also calculated.

\textbf{Results}

During the study period 667 fetuses met the inclusion criteria (on a
total of 12.250 NT tests). Mean maternal age was 35.64 \selectlanguage{ngerman}± 3.44 years, 617
(92.92\%) were spontaneous pregnancies while 47 (7.08\%) pregnancies
were conceived by assisted reproductive technology. All fetuses were
assessed by ultrasound follow up scans, while CVS or amniocentesis were
performed in 288 (43.37\%). For children with normal paediatric
examination at birth, mean age at the time of follow up was 2.65 years
(range 4 months-6 years). There was a live birth of a healthy fetus in
500 (74.96\%) cases. The outcome was unfavourable in 167 (25.44\%)
cases: there were 90 (13.49\%) cases of chromosomal
abnormalities/genetic syndromes, of which 45 (50\%) were trisomy 21, 57
(8.55\%) cases of major malformations of which 23 (3.45\%) were cardiac,
13 (1.95\%) cases of neurodevelopmental delay. In 6 cases a second
trimester miscarriage or IUFD occurred in fetuses with no detected
abnormalities (0.90\%). There was 1 (0.15\%) case of metabolic disease
(severe growth hormone (GH) deficiency). Details regarding postnatal
outcome including the time of diagnosis (prenatal or postnatal) are
reported in Table 2.

Details regarding each case of chromosomal abnormalities and genetic
syndromes are reported in Table S1. Major malformations and other
unfavourable outcome are listed in Table S2, cases of neurodevelopmental
delays are listed in Table S3.

The incidence of chromosomal abnormalities/genetic syndromes and major
malformations were significantly higher in our cohort (OR 6.99 (IC 95\%
4.33 - 11.28), p\textless{}0.001 and OR 17.77 (IC 95\%7.22 - 43.75),
p\textless{}0.001 respectively) compared to the general population.
Conversely, the incidence of neurodevelopmental delay was not increased
(OR of 0.64 CI 95\% 0.33 - 1.24 p=0.185) (Table 3 - Figure 1).

\textbf{Discussion}

Main Findings

Our present study showed that a NT between the 95\textsuperscript{th}and
the 99\textsuperscript{th} percentile is associated with unfavourable
outcome in 25.44\% of cases; in particular the risk of chromosomal
abnormalities/genetic syndrome and major malformations were 6.99 and
17.77 times higher, respectively, than the background risk of the
general population.

Interpretation

These data agree with previous studies reporting an increased risk of
adverse pregnancy outcome for mildly increased NT (NT
95\textsuperscript{th}-99\textsuperscript{th}percentile)\textsuperscript{4;21-22}.
Bardi et al. reported that 21.3\% of fetuses with an NT between the
95\textsuperscript{th} and the 99\textsuperscript{th} percentile had
congenital abnormalities, either genetic or structural.

The reported prevalence of healthy live-born children varied between
83\%\textsuperscript{4} and 78.2\%\textsuperscript{22}, similar to our
study (74.96\% of fetuses were born alive and healthy). A long term
follow up is crucial in terms of reporting adverse postnatal outcome, in
fact, in our cohort a high number of unfavourable diagnoses such as
genetic syndromes and neurodevelopmental delay were made between 6
months and 2 years of age.

The incidence of chromosomal abnormalities and genetic syndromes varied
in previous studies. Kagan et al. in 2006\textsuperscript{6} reported a
prevalence of 7.1\% for NT between 95\textsuperscript{th} and
99\textsuperscript{th} percentile, but the only genetic investigation
performed was however standard karyotype. In a study by Tekesin et
al.\textsuperscript{22} the incidence of abnormal fetal karyotype was
18.2\% in a selected high risk population with NT between
95\textsuperscript{th} and 99\textsuperscript{th} percentile. In our
study group, which mainly included a low risk population, fetuses with
these conditions accounted for 13.49\% of cases.

Furthermore, according to recent studies\textsuperscript{23-24}, the use
of CGH-arrays in fetuses with NT\textgreater{}3.5 mm can reveal
approximately 5\% copy number variants (CNVs) with pathogenetic or
uncertain significance. Despite the level of anxiety that variants of
unknown significance (VOUS) could bring, it is now mandatory that
couples who are offered karyotyping are at least informed on the
existence of this genetic test.

The most common malformations reported in our population were cardiac
defects (40.35\% of all malformations detected). This result is
consistent with the incidence reported in a meta-analysis by Sotiriadis
et al.\textsuperscript{10} where 45\% of fetuses with cardiac
malformation showed an NT
\textgreater{}95\textsuperscript{th}percentile, but only in 20\% of them
NT was above 99\textsuperscript{th}percentile. Therefore, the majority
of fetuses with cardiac defects showed an NT between
95\textsuperscript{th} and 99\textsuperscript{th}percentile. Similar
percentages (34.1\%-41.57\%) can be observed in more recent
studies\textsuperscript{21-22}. These data suggest that an
NT\textgreater{}95\textsuperscript{th} percentile could be the proper
cut off to offer fetal echocardiography.

With regards to neurodevelopmental delay , Hellmuth et al. in
2017\textsuperscript{25} found no increased risk in a cohort of children
with NT between 95th and 99th percentile at first trimester scan, with a
mean follow up of 4.4 years. The present study confirms the reported
results, showing that the incidence of those disorders is 1.95\% in our
population, not significantly different from the general population.

A critical point in the management of pregnancies with NT between
95\textsuperscript{th} and 99\textsuperscript{th} percentile is the
level of investigations that should be offered to couples. At the
moment, international guidelines do not include
NT\textgreater{}95\textsuperscript{th} percentile among the cases that
should benefit from further examinations than the routine ones. Souka et
al. in 2005\textsuperscript{3} proposed a specific management for
fetuses with NT between 95\textsuperscript{th} and
99\textsuperscript{th} percentile which included a detailed ultrasound
at 20 weeks with examination of fetal heart, preferably performed by a
fetal echocardiography specialist. This protocol was also recommended by
De Domenico et al.\textsuperscript{9}. Moreover, both authors stated
that fetal karyotype should be performed based on patients-specific risk
for chromosomal anomalies. Based on the high prevalence of chromosomal
anomalies and genetic syndromes in our cohort, we suggest that fetal
karyotyping should be offered also in case of NT between
95\textsuperscript{th} and 99\textsuperscript{th} percentile.

Strengths and Limitations

Strength of our study is the inclusion of a low risk population; this
could have reduced population selection bias. Even though the study
involved more than one Centre, a single protocol was shared and the
management of all the patients was homogeneous. In addition, the long
term follow-up of children was crucial because several postnatal
diagnosis were made after 6 months of age.

A possible limitation of the study is that not all patients underwent
fetal karyotyping and CGH-array. In particular, postnatal genetic
examination of liveborns who showed normal long term follow up was not
performed and this could lead to the underestimation of genetic
disorders.

\textbf{Conclusion}

In conclusion, fetus with NT between 95\textsuperscript{th} and
99\textsuperscript{th} percentile are at increased risk of pregnancy and
postnatal adverse outcomes; these data should guide clinicians in the
management of pregnancies wit this condition. Our data support the new
lower cut off of NT (NT\textgreater{}95th percentile) for offering
further investigations to couples. These should include detailed
ultrasound scan, fetal echocardiography and counselling where the option
of performing fetal karyotype and CGH array should be discussed.

\textbf{Conflict of Interest}

The authors report no relevant financial, personal, political,
intellectual or religious interest that could have influenced the
outcome of this work.

\textbf{Contribution to Authorship}

The study was designed by IP, ES, FP and LP. The data were acquired by
IP, EB and AC, and interpreted and analyzed by IP, ES and GM. Manuscript
was drafted by IP, ES and EB, and revised for important intellectual
content by GM, AC, FP and LP. All authors read and approved the final
version of the manuscript.

\textbf{Details of Ethical Approval}

The study was approved by the local Ethic Committee (CEAVC - Comitato
Etico Area Vasta Centro n=17892). All the procedures were conducted in
accordance with the standards of the Helsinki Declaration.

\textbf{Data Availability Statement}

The data that support the findings of this study are available from the
corresponding author upon reasonable request.

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