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Clinical experience with noninvasive prenatal testing in Germany: Analysis of over 500 high-risk cases for trisomy 21, 18, 13 and monosomy X

Authors:
  • amedes Institut für Labormedizin und Klinische Genetik Nordrhein
  • amedes Institut für Labormedizin und Klinische Genetik Nordrhein

Abstract and Figures

Analysis of 545 NIPT high-risk cases with high risk for trisomy 21 (T21), 18 (T18), and 13 (T13), as well as monosomy X (MX) from routine NIPT testing in a single prenatal center in Germany. Analysis was performed using the VeriSeq NIPT Solution v2 (Illumina Inc., USA). The assessment of true vs false positive results were based on clinical outcome data. The average fetal fraction of 9.7% was within the expected range in T21 and MX but lower in T18 and T13. For all high-risk groups sensitivity and specificity was far above 99%. The positive predictive value (PPV) was highest at trisomy 21 with 94.1%, followed by trisomy 18 with 80.9%. For trisomy 13 and Monosomy X, the PPV was clearly lower at 60.5% and 65.6%, respectively. PPV was dependent on different indications and maternal age. We could show that statistical tools of the method like the log likelihood ratio (LLR) score and T-Statistics value are important to distinguish between (clinical) false positive and true positive NIPT results in trisomies. The relationship between results and quality scores is less significant for MX cases. The study shows that the Illumina VeriSeq v2 procedure is a highly reliable NIPT method with a low no call rate in the hands of experienced diagnostic laboratories.
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Research Article
Obstetrics and Gynecology Reports
Obstet Gynecol Rep, 2021 doi: 10.15761/OGR.1000157 Volume 5: 1-7
ISSN: 2515-2955
Clinical experience with noninvasive prenatal testing in
Germany: Analysis of over 500 high-risk cases for trisomy
21, 18, 13 and monosomy X
Bernd Eiben1*, Heike Borth1, Nargül Kutur1, Christina Courtis1, Anna Teubert2, Sarah Knippenberg2, Thomas Winkler1 and Ralf Glaubitz2
¹amedes genetics, Institut für Labormedizin und Klinische Genetik Rhein/Ruhr, Essen, Germany
2amedes genetics, Hannover, Georgstr. Germany
Abstract
Analysis of 545 NIPT high-risk cases with high risk for trisomy 21 (T21), 18 (T18), and 13 (T13), as well as monosomy X (MX) from routine NIPT testing in a
single prenatal center in Germany. Analysis was performed using the VeriSeq NIPT Solution v2 (Illumina Inc., USA). e assessment of true vs false positive results
were based on clinical outcome data.
e average fetal fraction of 9.7% was within the expected range in T21 and MX but lower in T18 and T13. For all high-risk groups sensitivity and specicity was
far above 99%. e positive predictive value (PPV) was highest at trisomy 21 with 94.1%, followed by trisomy 18 with 80.9%. For trisomy 13 and Monosomy X, the
PPV was clearly lower at 60.5% and 65.6%, respectively. PPV was dependent on dierent indications and maternal age.
We could show that statistical tools of the method like the log likelihood ratio (LLR) score and T-Statistics value are important to distinguish between (clinical) false
positive and true positive NIPT results in trisomies. e relationship between results and quality scores is less signicant for MX cases.
e study shows that the Illumina VeriSeq v2 procedure is a highly reliable NIPT method with a low no call rate in the hands of experienced diagnostic laboratories.
*Correspondence to: Bernd Eiben, amedes genetics, Institut für Labormedizin
und Klinische Genetik Rhein/Ruhr, Willy Brandt-Platz 4, D-45127 Essen,
Germany, E-mail: eiben@eurogen.de
Key words: fetal chromosomal aneuploidies, trisomy 21, trisomy 18, trisomy
13, monosomy X, fetal fraction, non-invasive prenatal testing (NIPT), positive
predictive value (ppv), VeriSeq NIPT Solution
Received: February 25, 2021; Accepted: March 08, 2021; Published: March 11, 2021
Introduction
Noninvasive Prenatal Testing (NIPT) is a molecular genetic test
that can determine whether a pregnancy is at high-risk for the common
aneuploidies (trisomy 21, trisomy 18, trisomy 13, and sex chromosome
aneuploidies) by analyzing circulating cell-free fetal DNA (cfDNA)
present in maternal plasma. e rst use of cfDNA for analysis of fetal
genetic status, fetal sex determination based on the presence or absence
of the Y chromosome, was reported in 1997 by Lo, et al. [1]. At that
time, sequencing methods suitable for routine evaluation of millions of
DNA fragments were not readily available nor was the corresponding
analysis and evaluation soware. us, cfDNA was not incorporated
into routine clinical use at that time. Since that time there have been
signicant advantages in next-generation sequencing technology as well
as the bioinformatics needed for analysis of sequencing results. ese
changes and commercialization of the cfDNA technology enabled the
implementation of NIPT in a clinical setting close to a decade ago. is
method initiated a revolution in prenatal testing: a move away from
invasive options, such as amniocentesis and chorionic villus sampling
(CVS), towards noninvasive prenatal screening.
In the 10 years since NIPT became available, it has become well
accepted in developed regions [2]. Numerous studies have been
published on the performance and use of NIPT. Most of these studies
are designed as multicenter studies and include comparatively few
abnormal trisomic pregnancies, with most samples originating from
unaected pregnancies. Here, we describe the clinical performance
of NIPT within our single prenatal unit, which is the primary NIPT
laboratory in Germany. e present work includes one of the largest
NIPT evaluations of trisomies 21, 18, and 13 as well as monosomy X
from a single prenatal unit and is intended to provide guidance in the
evaluation of such results for genetic counselors, gynecologists, and
diagnosticians in the laboratory.
Material and methods
Study patient/Sample details
Our full cohort included more than 40,000 singleton and twin
pregnancies from a general German and Austrian pregnancy population
that elected to undergo NIPT between December 2017 and February
2021; a subset of these cases were reported previously [3]. Within the
full cohort, there were 545 cases reported by our NIPT assay at high
risk for trisomies 21, 18, and 13, as well as monosomy X, henceforth
referred to as the study cohort. Samples of at least 10 weeks of gestation
were included in the study; exclusion criteria were a known vanishing
twin or a higher-order multiple pregnancy. e amedes lab complies
with the provisions of the German Federal Data Protection Act. All data
were de-identied prior to enrollment in the study. Informed consent
was obtained from all patients participating in the study to use their
Eiben B (2021) Clinical experience with noninvasive prenatal testing in Germany: Analysis of over 500 high-risk cases for trisomy 21, 18, 13 and monosomy X
Volume 5: 2-7
Obstet Gynecol Rep, 2021 doi: 10.15761/OGR.1000157
t-test; a p value <0.05 was considered signicant. Binomial 95%
condence intervals (CI) were calculated for sensitivity and specicity
estimates.
In case of an inconclusive NIPT result, the analysis was repeated on
the initial sample and/or on a second blood sample. In these cases, the
result of the rst analysis was excluded from the statistical analysis, so
that only one result per patient was included in the statistical analysis.
In addition, in patients where a NIPT result was obtained from the
rst blood sample but a repeat analysis was requested (e.g., due to a
FF <2%), another blood sample was analyzed as a goodwill gesture.
In these cases, the result of the rst analysis was excluded from the
statistical evaluation, except in cases where the repeat analysis did not
yield a NIPT result; in the latter cases, the result of the rst analysis
was included in the statistics and the unsuccessful repeat analysis was
excluded.
Results
Demographics
Demographics of all patients analyzed are shown in Table 1. e
545 high-risk NIPT cases analyzed in the present study were part of a
collection of 41,607 pregnancy cases, including 13,607 cases published
previously [3]. e 41,607 cases included 40,871 (98.2%) singleton
pregnancies and 736 (1.8%) twin pregnancies (Table 1). e mean
maternal age was 33.5 years and the mean gestational age was 12.4
weeks. e primary indications for NIPT were patient anxiety (51%)
and advanced maternal age (40%).
Assay performance and clinical outcomes
Within 41,607 cases, 41,070 (98.7%) had an NIPT result with
the rst blood sample. e rst pass failure rate was improved from
2.0% (821/ 41,607) to 1.3% (537/ 41,607) by repeated analysis of the
initial sample. For the 537 pregnant women who did not get a result
with the rst blood draw, a repeated blood draw was oered, of which
459 (85.5%) patients accepted. Of these, 383 (83.4%) received an NIPT
data for appropriate quality control and improvement of the NIPT
assays.
Consistent with our previous study [3], indications for NIPT
included advanced maternal age, a positive screening test result
(ultrasound, serum markers), other medical reasons, or patient anxiety.
Other medical reasons included abnormal ultrasound, a history of
pregnancy complications including miscarriage or a previously aected
pregnancy (e.g., trisomy 21, 18, 13, monosomy X), a genetic aberration
in the family (e.g., trisomy 21), known diseases such as diabetes,
epilepsy, and carcinoma, medications such as chemotherapy, or
consanguinity. In the absence of information on the indication, patient
anxiety or advanced maternal age (if the patient was ≥ 35 years) were
used as indications. If multiple indications were provided, cases were
assigned to a sole indication with the following priority: (1) positive
screening test result, (2) advanced maternal age, (3) other medical
reasons, (4) patient anxiety. e correctness of the assignment of cases
to the dierent indication groups was dependent on the accuracy of the
information received from the attending gynecologists/pediatricians, as
was all other patient history information and feedback on the clinical
pregnancy outcome.
Clinical results for the 545 high-risk study cohort cases were
veried by invasive prenatal diagnostic procedures (cytogenetic
analysis aer CVS and/or amniocentesis), cytogenetic analysis of
products of conception (POC) or placenta, postmortem examinations
such as autopsy or macroscopic assessment of the abortion, postnatal
cytogenetic analyses, as well as ultrasound and newborn examination.
NIPT results that were positive for fetal aneuploidy were considered
conrmed if they were validated by either invasive prenatal diagnostics
or an abnormality observed on ultrasound that was consistent with the
high-risk NIPT. Low-risk NIPT results were considered conrmed if
the attending physician reported the birth of a healthy newborn lacking
physical features or phenotypes associated with trisomy 21, 18, 13, or
monosomy X.
VeriSeq NIPT solution v2 assay
e Illumina NIPT assay, VeriSeq NIPT Solution v2 (Illumina Inc.,
San Diego, CA, USA), was used for this study. is assay uses a paired-
end sequencing technique and reports fetal sex, risk for trisomy 21
(T21), 18 (T18), and 13 (T13), as well as sex chromosome aneuploidies.
We used a customized result reporting to meet amedes requirements:
reporting of sex chromosomes was explicitly limited to monosomy X
(MX) and XX/XY. Other sex chromosome aberrations are not analyzed
for medical and ethical reasons. is NIPT test is oered by the amedes
lab group in Germany and Austria under the name “fetalis”.
Data analysis was as previously described [3]. Briey, a fetal fraction
(FF) estimate was reported for each sample. Following data analysis, a
log likelihood ratio (LLR) score was provided for each sample. is is
the probability of a sample being aected given the sample’s estimated
FF and observed coverage. e assay soware also uses a dynamic
threshold metric known as the individualized Fetal Aneuploidy
Condence Test (iFACT), which determines whether there is sucient
sequencing coverage for each individual sample given the FF estimate
for that sample; samples that did not meet this threshold were reported
as QC failures. A T-Statistics value is provided by the soware and was
used to help dierentiate between low-risk and high-risk samples [3].
Statistics
Statistical data analysis was performed using Microso Excel 2016.
Where applicable, statistical signicance was assessed using a Students
Study cohort
Cases, n 41,607
Singleton pregnancies, n (%) 40,871 (98.2)
Twin pregnancies, n (%) 736 (1.8)
Maternal age (year)
Mean ± SEM 33.45 ± 0.02
Range 16.25 – 55.87
Gestational age (week)
Mean ± SEM 12.38 ± 0.01
Range 10.00 – 36.57
BMI
Mean ± SEM 25.10 ± 0.03
Range 14.60 – 62.44
Indication for screening
Advanced maternal age, n (%) 16,675 (40.1)
Positive screening test result*, n (%) 1,890 (4.5)
Other medical reasons†, n (%) 1,918 (4.6)
Patient anxiety, n (%) 21,124 (50.8)
Table 1. Patient demographics for the full cohort
SEM: standard error of the mean; BMI: body mass index; *Positive screening test
result includes ultrasound or serum marker screening; †Other medical reasons include
e.g. abnormal ultrasound, known diseases of the patient (e.g., diabetes, epilepsy, and
carcinoma), medication (e.g., chemotherapy), or a high-risk family medical history such as
a previous miscarriage, a genetic aberration in a previous pregnancy (e.g., trisomy 21, 18,
13, monosomy X), a genetic aberration in the family (e.g., trisomy 21), or consanguinity.
Eiben B (2021) Clinical experience with noninvasive prenatal testing in Germany: Analysis of over 500 high-risk cases for trisomy 21, 18, 13 and monosomy X
Volume 5: 3-7
Obstet Gynecol Rep, 2021 doi: 10.15761/OGR.1000157
result with the second blood sample. Overall, a total of 41,453 (99.6%)
patients obtained an NIPT result. e nal failure rate was 0.4% (154/
41,607). e mean BMI was signicantly higher in cases without an
NIPT result (32.79 ± 0.67) compared with the low-risk group (25.07 ±
0.03; p<0.001).
Of the 41,453 patients who received an NIPT result, 40,908 (98.3%)
had a low-risk result and 545 (1.3%) patients were reported as at high-
risk for a fetal chromosome aneuploidy (Table 2). Clinical outcomes
were available for 7,012 (16.9%) cases; outcomes were based on invasive
diagnostic techniques, ultrasound, or newborn physical exam. Based on
clinical follow up for low-risk NIPT results (6,559/ 40,908), the negative
predictive value (NPV) was > 99.9% (6,558/ 6,559). In one case, a false
negative trisomy 21 result was observed, the fetal fraction of this false-
negative case was 3%.
e 545 cases with a high-risk NIPT result were actively followed up
for clinical outcome information. In 82.9% cases (452/ 545), outcome
information was obtained. ere were 93 cases classied as having
unknown clinical outcomes: In 62 cases, clinical outcome information
could not be obtained, or the pregnancy was terminated or ended in
miscarriage, but no information on the fetal genetic constellation
was available; 27 patients were recent cases and follow up diagnostic
testing has not yet been performed; four patients decided to continue
their pregnancies and no further information is available. Based on the
high-risk NIPT results cases with known outcomes, this NIPT has high
overall sensitivity (99.7%), specicity (99.1%), and positive predictive
value (PPV; 86.5%) (Table 2).
Of the 545 high-risk NIPT results, trisomy 21 was the most
commonly reported aneuploidy (61.5%), followed by trisomy 18
(20.6%), trisomy 13 (9.7%), and monosomy X (8.1%); there was one case
reported as a double aneuploidy (Table 3). Sensitivity and specicity
were far above 99% for each aneuploidy. e PPV was highest for
trisomy 21 at 94.1%, followed by trisomy 18 at 80.9%. e PPVs were
lower for trisomy 13 (60.5%) and monosomy X (65.6%) (Table 3).
Analysis of the high-risk NIPT results with respect to indication
was to investigate dierences between high- and low-risk populations.
is analysis showed that specicity and sensitivity levels were >99.0%
regardless of indication (Table 4). For each aneuploidy category, the PPV
in the advanced maternal age, high-risk screening, and other medical
reasons indication groups were higher than for the anxiety indication.
In trisomy 18, trisomy 13, and monosomy X high-risk NIPT results, the
PPV in the high-risk screening and other medical reasons indication
groups were above the PPV of the advanced maternal age group. For
trisomy 21 cases, the highest PPVs were observed in the high-risk
screening (98.6%) and advanced maternal age (95.0%) groups.
To investigate the role of maternal age in high-risk NIPT results,
the relationship between dierent maternal age groups and sensitivity,
specicity, and PPV was evaluated (Table 5). e sensitivity was ≥
98.6% and specicity was well above 99.0% in each age group. For cases
at high risk for trisomy 21, 18, and 13, the PPV was higher in cases
with an advanced maternal age (≥ 35 years) compared with younger
women (<35 years). is was particularly true for cases reported as
trisomy 21, where PPVs increased from 78.9% (25 to 29 years) and
93.0% (30 to 34 years) to 96.7% (35 to 39 years) and 94.3% (>40 years).
In the monosomy X risk group, an association with maternal age was
not evident.
Analysis of the incidence of the high-risk results for trisomies 21,
18, and 13 as well as monosomy X showed no signicant association
with gestational age at the time of sample collection for NIPT (Table 6).
Fetal fraction
e average FF was 9.7% in low-risk pregnancies. e average FF
was slightly lower in cases reported as trisomy 21 (9.4%), markedly
lower for cases reported as trisomy 18 (7.2%) and trisomy 13 (6.0%),
and unchanged in cases reported as monosomy X (10.1%) (Figure 1).
We also evaluated performance in three FF subgroups: <4%, 4%-8%,
and > 8% (Table 7). For cases reported as trisomy 21, the FF was above
4% in around 95% of cases and above 8% in about half the cases; only a
5.7% had an FF less than 4%. e specicity and sensitivity for trisomy
21 was >99% when the FF was at least 4%. e PPV increased with
increasing FF from 71% to 99%. For trisomy 18 and trisomy 13, most
cases had a FF ≤ 8%. Importantly, sensitivity and specicity levels were
very high regardless of FF, >99.9% and ≥ 98.3%, respectively. Similar to
trisomy 21, the PPV for trisomy 18 increased with increasing FF. e
same association between PPV and FF was not observed for trisomy
Total cases, n 41,607
Low-risk
Cases, n (%) 40,908 (98.3)
Cases with follow-up (N), n (%) 6,559 (16.0)
TN, n (%) 6,558 (99.98)
FN, n (%) 1 (0.02)
NPV, % (TN/TP+FN) >99.9 (6,558/ 6,559)
High-risk
Cases, n (%) 545 (1.3)
Cases with follow-up (N), n (%) 452 (82.9)
Sensitivity, % (95% CI)
(TP/TP+FN)
99.7 (98.6 – 100.0)
(391/392)
Specicity, % (95% CI)
(TN+TP/TN+TP+FP)
99.1 (98.9 – 99.3)
(6,949/ 7,010)
PPV, % (TP/N) 86.5 (391/ 452)
Theoretical lower PPV, %
(TP/TP+FP+unk)
76.1
(391/ 514)
Theoretical upper PPV, %
(TP+unk/TP+FP+unk)
88.1
(453/ 514)
Table 2. Cases of clinical follow up, negative predictive value (NPV) for low-risk NIPT
results and sensitivity, specicity, and positive predictive value (PPV) for total high-risk
NIPT cases
n: Number; TP: True positive; FP: False positive; TN: True negative; FN: False negative;
N: Cases with obtained fetal outcome/with follow up; unk: Cases without obtained follow up.
Figure 1. Chromosomal risk status and fetal fraction
Values shown are mean ± standard error of the mean (SEM); t-test reference point is low-
risk NIPT result; * = p < 0.5, *** = p<0.001.
Eiben B (2021) Clinical experience with noninvasive prenatal testing in Germany: Analysis of over 500 high-risk cases for trisomy 21, 18, 13 and monosomy X
Volume 5: 4-7
Obstet Gynecol Rep, 2021 doi: 10.15761/OGR.1000157
High-risk NIPT result Trisomy 21 Trisomy 18 Trisomy 13 Trisomy 13 + 21 Monosomy X
Cases, n (%) 335 (61.5) 112 (20.6) 53 (9.7) 1 (0.2) 44 (8.1)
Cases with follow-up (N), n (%) 287 (85.7) 94 (83.9) 38 (71.7) 1 (-) 32 (72.7)
Sensitivity, % (95% CI)
(TP/TP+FN)
99.6 (98.0 – 100.0)
(270/ 271)
>99,9 (95.3 – 100.0)
(76/ 76)
>99,9 (85.2 – 100.0)
(23/ 23)
-
(1/ 1)
>99.9 (83.9 – 100.0)
(21/21)
Specicity, % (95% CI)
(TN+TP/TN+TP+FP)
99.8 (99.6 – 99.9)
(6,828/ 6,845)
99.7 (99.6 – 99.8)
(6,634/ 6,652)
99.8 (99.6 – 99.9)
(6,581/ 6,596)
-
(6,559/ 6,559)
99.8 (99.7 – 99.9)
(6,579/ 6,590)
PPV, % (TP/N) 94.1 (270/ 287) 80.9 (76/ 94) 60.5 (23/ 38) - (1/ 1) 65.6 (21/ 32)
Theoretical lower PPV, %
(TP/TP+FP+unk)
85.2
(270/ 317)
70.4
(76/ 108)
44.2
(23/ 52)
-
(1/ 1)
58.3
(21/ 36)
Theoretical upper PPV, %
(TP+unk/TP+FP+unk)
94.6
(300/ 317)
83.3
(90/ 108)
71.2
(37/ 52)
-
(1/ 1)
69.4
(25/ 36)
Table 3. Sensitivities, specicities, and positive predictive values (PPV) for high-risk NIPT cases
n: Number; TP: True positive; FP: False positive; TN: True negative; FN: False negative; N: Cases with obtained fetal outcome/with follow up; unk: Cases without obtained follow up.
Maternal age, years < 25 25 – 29 30 – 34 35 – 39 > 40
Cases, n (%) 1,057 (2.5) 6,889 (16.6) 16,099 (38.7) 14,020 (33.7) 3.541 (8.5)
Total high-risk cases, n (%) 8 (0.0) 41 (0.1) 125 (0.3) 220 (0.5) 151 (0.4)
Trisomy 21, 18, 13, 13 + 21
Cases, n (%) 6 (1.2) 36 (7.2) 103 (20.6) 207 (41.3) 149 (29.7)
Cases with follow-up (N), n (%) 5 (83.3) 29 (80.6) 83 (80.6) 172 (83.1) 131 (87.9)
Sensitivity, % (TP/TP+FN) >99.9 (2/ 2) >99.9 (22/ 22) 98.6 (71/ 72) >99.9 (160/ 160) >99.9 (115/ 115)
Specicity, % (TN+TP/TN+TP+FP) 98.4 (180/ 183) 99.4 (1,190/ 1,197) 99.5 (2,653/ 2,665) 99.5 (2,315/ 2,327) 97.4 (590/ 606)
PPV, % (TP/N) 40.0 (2/ 5) 75.9 (22/ 29) 85.5 (71/ 83) 93.0 (160/ 172) 87.8 (115/ 131)
Monosomy X
Cases, n (%) 2 (0.6) 5 (1.5) 22 (6.6) 13 (3.9) 2 (0.6)
Cases with follow-up (N), n (%) 1 (50.0) 3 (60.0) 17 (77.3) 9 (69.2) 2 (100.0)
Sensitivity, % (TP/TP+FN) - (1/ 1) >99.9 (2/ 2) >99.9 (11/ 11) >99.9 (5/ 5) >99.9 (2/ 2)
Specicity, % (TN+TP/TN+TP+FP) - (179/ 179) 99.9 (1,170/ 1,171) 99.8 (2,593/ 2,599) 99.8 (2,160/ 2,164) >99.9 (477/ 477)
PPV, % (TP/N) - (1/ 1) 66.7 (2/ 3) 64.7 (11/ 17) 55.6 (5/ 9) >99.9 (2/ 2)
Table 5. Sensitivities, specicities and PPV values for high-risk cases stratied by maternal age
n: Number; TP: True positive; FP: False positive; TN: True negative; FN: False negative; N: Cases with obtained fetal outcome/with follow up; unk: Cases without obtained follow up.
Indication Adv. maternal age Screening Other med. reasons Anxiety
Cases, n (%) 16,675 (40.1) 1,890 (4.5) 1,918 (4.6) 21,124 (50.8)
Total high-risk cases, n (%) 287 (1.7) 125 (6.6) 23 (1.2) 110 (0.5)
Trisomy 21
Cases, n (%) 185 (55.2) 83 (24.8) 13 (3.9) 54 (16.1)
Cases with follow-up (N), n (%) 161 (87.0) 72 (86.7) 9 (69.2) 45 (83.3)
Sensitivity, % (TP/TP+FN) >99.9 (153/ 153) >99.9 (71/ 71) >99.9 (8/ 8) 97.4 (38/ 39)
Specicity, % (TN+TP/TN+TP+FP) 99.7 (2,698/ 2,706) 99.6 (256/ 257) 99.6 (282/ 283) 99.8 (3,592/ 3,599)
PPV, % (TP/N) 95.0 (153/ 161) 98.6 (71/ 72) 88.9 (8/ 9) 84.4 (38/ 45)
Trisomy 18
Cases, n (%) 62 (55.4) 25 (22.3) 4 (3.6) 21 (18.8)
Cases with follow-up (N), n (%) 57 (91.9) 20 (80.0) 3 (75.0) 14 (66.7)
Sensitivity, % (TP/TP+FN) >99.9 (49/ 49) >99.9 (18/ 18) >99.9 (3/ 3) >99.9 (6/ 6)
Specicity, % (TN+TP/TN+TP+FP) 99.7 (2,594/ 2,602) 99.0 (203/ 205) >99.9 (277/ 377) 99.8 (3,560/ 3,568)
PPV, % (TP/N) 86.0 (49/ 57) 90.0 (18/ 20) >99.9 (3/ 3) 42.9 (6/ 14)
Trisomy 13
Cases, n (%) 26 (49.1) 8 (15.1) 2 (3.8) 17 (32.1)
Cases with follow-up (N), n (%) 17 (65.4) 6 (75.0) 2 (100.0) 13 (76.5)
Sensitivity, % (TP/TP+FN) >99.9 (10/ 10) >99.9 (4/ 4) >99.9 (2/ 2) >99.9 (7/ 7)
Specicity, % (TN+TP/TN+TP+FP) 99.7 (2,555/ 2,562) 99.0 (189/ 191) >99.9 (276/ 276) 99.8 (3,561/ 3,567)
PPV, % (TP/N) 58.8 (10/ 17) 66.7 (4/ 6) >99.9 (2/ 2) 53.8 (7/ 13)
Monosomy X
Cases, n (%) 13 (29.5) 9 (20.5) 4 (9.1) 18 (40.9)
Cases with follow-up (N), n (%) 9 (69.2) 7 (77.8) 3 (75.0) 13 (72.2)
Sensitivity, % (TP/TP+FN) >99.9 (5/ 5) >99.9 (7/ 7) >99.9 (3/3) >99.9 (6/ 6)
Specicity, % (TN+TP/TN+TP+FP) 99.8 (2,550/ 2,554) >99.9 (192/ 192) >99.9 (277/277) 99.8 (3,560/ 3,567)
PPV, % (TP/N) 55.6 (5/ 9) >99.9 (7/ 7) >99.9 (3/3) 46.2 (6/ 13)
Table 4. Sensitivities, specicities and PPV values for high-risk cases stratied by indication
n: Number; TP: True positive; FP: False positive; TN: True negative; FN: False negative; N: Cases with obtained fetal outcome/with follow up; unk: Cases without obtained follow up.
Eiben B (2021) Clinical experience with noninvasive prenatal testing in Germany: Analysis of over 500 high-risk cases for trisomy 21, 18, 13 and monosomy X
Volume 5: 5-7
Obstet Gynecol Rep, 2021 doi: 10.15761/OGR.1000157
Gestational age, week+day 10+0 – 10+6 11+0 – 13+6 14+0 – 19+6 >20+0
Cases, n (%) 7,556 (18.2) 29,212 (70.2) 4,392 (10.6) 447 (1.1)
Total high-risk cases, n (%) 104 (1.4) 371 (4.9) 48 (0.6) 22 (0.3)
Trisomy 21, 18, 13, 13 + 21
Cases, n (%) 93 (18.6) 345 (68.9) 44 (8.8) 19 (3.8)
Cases with follow-up (N), n (%) 74 (79.6) 291 (84.3) 40 (90.9) 15 (78.9)
Sensitivity, % (TP/TP+FN) >99.9 (68/ 68) 99.6 (257/ 258) >99.9 (30/ 30) >99.9 (15/ 15)
Specicity, % (TN+TP/TN+TP+FP) 99.5 (1,248/ 1,254) 99.3 (4,922/ 4,956) 98.6 (691/ 701) >99.9 (67/ 67)
PPV, % (TP/N) 91.9 (68/ 74) 88.3 (257/ 291) 75.0 (30/ 40) >99.9 (15/ 15)
Monosomy X
Cases, n (%) 11 (25.0) 26 (59.1) 4 (9.1) 3 (6.8)
Cases with follow-up (N), n (%) 9 (81.8) 17 (65.4) 4 (100.0) 2 (66.7)
Sensitivity, % (TP/TP+FN) >99.9 (6/ 6) >99.9 (12/ 12) >99.9 (2/ 2) - (1/ 1)
Specicity, % (TN+TP/TN+TP+FP) 99.7 (1,186/ 1,189) 99.9 (4,677/ 4,682) 99.7 (663/ 665) - (53/ 54)
PPV, % (TP/N) 66.7 (6/ 9) 70.6 (12/ 17) 50.0 (2/ 4) - (1/ 2)
Table 6. Sensitivities, specicities and PPV values for high-risk cases stratied by gestational age
n: Number; TP: True positive; FP: False positive; TN: True negative; FN: False negative; N: Cases with obtained fetal outcome/with follow up; unk: Cases without obtained follow up.
True positive False positive
LLR Score T-Statistics value LLR Score T-Statistics value
Trisomy 21 179.1 ± 9.7 16.6 ± 0.5 20.9 ± 9.8 4.9 ± 0.9
Trisomy 18 170.5 ± 19.3 16.2 ± 1.0 44.1 ± 18.0 8.0 ± 1.6
Trisomy 13 126.6 ± 21.7 14.6 ± 1.5 47.1 ± 17.2 8.6 ± 1.6
Monosomy X 50.8 ± 8.5 -24.2 ± 2.2 48.8 ± 18.9 -22.0 ± 4.4
Table 8. Relationship between NIPT results and LLR scores/T-Statistics values
Values shown as mean ± standard error of the mean (SEM).
Fetal fraction, % < 4% 4% – 8% > 8%
Cases, n (%) 1,015 (2.4) 17,522 (42.3) 22,916 (55.3)
Total high-risk cases, n (%) 47 (4.6) 266 (1.5) 232 (1.0)
Trisomy 21
Cases, n (%) 19 (5.7) 148 (44.2) 168 (50.1)
Cases with follow-up (N), n (%) 14 (73.7) 127 (85.8) 146 (86.9)
Sensitivity, % (TP/TP+FN) 90.9 (10/ 11) >99.9 (116/ 116) >99.9 (144/ 144)
Specicity, % (TN+TP/TN+TP+FP) 97.8 (180/ 184) 99.6 (3,094/ 3,105) 99.9 (3,554/ 3,556)
PPV, % (TP/N) 71.4 (10/ 14) 91.3 (116/ 127) 98.6 (144/ 146)
Trisomy 18
Cases, n (%) 15 (13.4) 64 (57.1) 33 (29.5)
Cases with follow-up (N), n (%) 10 (66.7) 57 (89.1) 27 (81.8)
Sensitivity, % (TP/TP+FN) >99.9 (7/ 7) >99.9 (45/ 45) >99.9 (24/ 24)
Specicity, % (TN+TP/TN+TP+FP) 98.3 (177/ 180) 99.6 (3,023/ 3,035) 99.9 (3,434/ 3,437)
PPV, % (TP/N) 70.0 (7/ 10) 78.9 (45/ 57) 88.9 (24/ 27)
Trisomy 13
Cases, n (%) 13 (24.5) 32 (60.4) 8 (15.1)
Cases with follow-up (N), n (%) 9 (69.2) 23 (71.9) 6 (75.0)
Sensitivity, % (TP/TP+FN) >99.9 (6/ 6) >99.9 (14/ 14) >99.9 (3/ 3)
Specicity, % (TN+TP/TN+TP+FP) 98.3 (176/ 179) 99.7 (2,992/ 3,001) 99.9 (3,413/ 3,416)
PPV, % (TP/N) 66.7 (6/ 9) 60.9 (14/ 23) 50.0 (3/ 6)
Monosomy X
Cases, n (%) 0 (0.0) 22 (50.0) 22 (50.0)
Cases with follow-up (N), n (%) - 17 (77.3) 15 (68.2)
Sensitivity, % (TP/TP+FN) - (0/ 0) >99.9 (12/ 12) >99.9 (9/ 9)
Specicity, % (TN+TP/TN+TP+FP) - (170/ 170) 99.8 (2,990/ 2,995) 99.8 (3,419/ 3,425)
PPV, % (TP/N) - (0/ 0) 70.6 (12/ 17) 60.0 (9/ 15)
Table 7. Sensitivities, specicities and PPV values for high-risk cases for cases with fetal fractions < 4%, 4%-8%, and > 8%
n: Number; TP: True positive; FP: False positive; TN: True negative; FN: False negative; N: Cases with obtained fetal outcome/with follow up; unk: Cases without obtained follow up.
Eiben B (2021) Clinical experience with noninvasive prenatal testing in Germany: Analysis of over 500 high-risk cases for trisomy 21, 18, 13 and monosomy X
Volume 5: 6-7
Obstet Gynecol Rep, 2021 doi: 10.15761/OGR.1000157
13. All monosomy X cases had at least 4% FF, and all specicity and
sensitivity levels were very high regardless of FF.
By analyzing the LLR score, which is a degree of probability that
a fetal aberration is present, it became obvious that all true positive
trisomy high-risk cases had approximately 3-8 fold higher LLR scores
and 1.8–3.4 fold higher T-Statistics values compared to false positive
cases (Table 8). No dierences were found between true positive and
false positive monosomy X cases.
Discussion and conclusion
NIPT for trisomy 21, 18, and 13 as well as fetal sex and sex
chromosome aberrations has been rapidly adopted into clinical practice
worldwide. NIPT in Germany, Austria, and Switzerland is regulated by
a number of professional societies, including the German Society of
Human Genetics [4,5], the German Society for Ultrasound in Medicine
(DEGUM), Austrian Society for Ultrasound in Medicine (ÖGUM),
Swiss Society for Ultrasound in Medicine (SGUM), and Fetal Medicine
Foundation (FMF) Germany [6,7]. Our laboratory follows these
guidelines and recommendations. Here we showed the low failure rate
of 0.4% and high performance of the NIPT assay used in our laboratory
across maternal ages, gestational ages, and FF values in a large clinical
population.
NIPT is increasingly being adopted by smaller laboratories in their
routine services. ere is a need for larger collections of aneuploid
cases within individual laboratories to provide sucient experience
to evaluate questionable ndings and to serve as a knowledge base to
enable a laboratory to advise the referring gynecologists and the patients
about the signicance of the NIPT ndings. e amedes genetic unit is
one of the largest labs in Germany. We have been performing genetic
counselling, amniocentesis and CVS for over 30 years, rst trimester
screening for 20 years, and we have 10 years experience with NIPT.
During our time performing NIPT, we have used each of the major
NIPT methods in our lab and have chosen to use the Illumina VeriSeq
NIPT Solution platform for the last 3 years. With this platform, we have
processed more than 40,000 samples, including 545 reported as high-
risk for a fetal chromosome aneuploidy. Follow up was obtained for
82.9% of the high-risk cases. erefore, we feel the high performance
values determined here are a reliable estimate of the true performance
of this assay in a clinical setting. e likelihood that the PPV in
particular could shi in one direction or the other is relatively low, but it
is possible that some subgroups are not a reliable estimate given the low
number of cases. Since the sensitivities and specicities were well above
99% for all high-risk aneuploidy groups studied, this data supports
the accuracy of NIPT in general as previously reported in a systematic
review [8]. e technical data presented here must not be taken out
of context of clinical ndings. Irreversible clinical decisions should not
be made based on NIPT results alone. High-risk NIPT results should
always be conrmed by amniocentesis or CVS.
e Illumina evaluation soware includes tools like the LLR
score and the T-Statistics value. In our daily work, these parameters
have proved to be very helpful in discriminating between true and
false positive cases for the common trisomies. We use this valuable
information in the consultation of each case.
We think our subgroup analyses for PPVs are of particular interest
and value. Consistent with the literature [9,10], the highest PPV was for
trisomy 21, with lower values for trisomy 18 and trisomy 13. is nding
can be explained by the dierence in prevalence between the trisomies:
the highest in trisomy 21 and the lowest in trisomy 13. Previous studies
[11,12] have shown that the PPV for monosomy X cases reported by
NIPT are in the low [13] to medium range [14], as was also observed in
this study. What is the reason for this? One study reported that 8.6% of
positive results for sex chromosome aneuploidy were due to maternal
mosaicism [15]. Such maternal sex chromosome abnormalities may
cause discordant results between NIPT result and the fetus [16]. But
an early detection of phenotype-genotype-sex discordance is important
to nd evidence of underlying genetic, chromosomal, or biochemical
disease, and also to enable time-critical postnatal treatment [17]. ey
emphasized the need for simultaneous detailed ultrasound examination
to detect and quickly clarify discrepant situations. Importantly, as the
NIPT method used here incorporates fragment length information,
this reduces the likelihood of maternal chromosome anomalies causing
a high-risk call by NIPT.
If we look at the impact of dierent indications on the PPVs we
can see that the higher the basic risk of an indication, the higher the
PPV. For example, the PPVs for the indication “increased maternal age
or “high-risk screening” are signicantly higher than for the indication
“anxiety”. is is likely explained by a lower background prevalence
of fetal aneuploidy in the lower risk (anxiety) indication group [18].
Women from low-risk indications and younger women should be
counseled about a lower PPV.
Fetal fraction is one of the most important quality control
parameters for NIPT and should be considered for proper counseling
and further clinical management [19]. e average FF is around 10%.
e FF depends on factors, such as the maternal BMI (FF decreases
with increasing BMI) [20,21], the type of trisomy (lower FF in trisomies
13 and 18) [22], and the presence of mosaicism [23]. Signicantly
lower FF was found also in this study in cases screened positive for
trisomy 18 and trisomy 13. Dierent NIPT approaches have dierent
minimum threshold values of FF for reporting, with some failing all
samples below a set threshold (between 2% and 4%) [19]. For this
reason, it is very important to choose an NIPT platform that includes
eective FF quality metrics to minimize failures linked to technical
reasons [24]. e method employed here uses a dynamic threshold
metric named iFACT, which determines whether there is sucient
sequencing coverage for each individual sample given the FF estimate
for that sample. is metric enables accurate reporting at low fetal
fractions and a low failure rate. is is evident from the data reported
here where fetal aneuploidies were accurately reported at FF below 4%.
is is particularly important for trisomy 13 and 18 as the average FF
is much lower for these aneuploidies. e overall no call rate for the
initial blood sample in this study was 1.3% and use of a second blood
sample reduced the failure rate to a low 0.4%. us, 99.6% of all 41,607
pregnant women received an NIPT result. To our knowledge this is the
lowest no call rate reported.
Our experience with over 40,000 clinical NIPT cases shows that
the present NGS-based NIPT method can reliably identify the most
frequent aneuploidies occurring prenatally. Fortunately, the majority of
NIPT results are normal, so that these results lead to a rapid relief of the
pregnant women.
Acknowledgements
Wolf Kupatt and Christoph Keck (amedes) have always supported
and advanced the NIPT project excellently. In addition, we want to
thank the many gynaecologists who gave us valuable information on
the fetal outcome.
Eiben B (2021) Clinical experience with noninvasive prenatal testing in Germany: Analysis of over 500 high-risk cases for trisomy 21, 18, 13 and monosomy X
Volume 5: 7-7
Obstet Gynecol Rep, 2021 doi: 10.15761/OGR.1000157
Funding
Assistance with data analysis and paper preparation was provided
by Illumina, Inc. but no nancial renumeration was provided directly
to the authors.
Competing interest
e authors declare that they have no conict of interest.
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Copyright: ©2021 Eiben B. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use,
distribution, and reproduction in any medium, provided the original author and source are credited.
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Background Maternal X chromosome abnormalities may cause discordant results between non-invasive prenatal screening tests and diagnostic evaluation of the fetus/newborn, leading to unnecessary invasive testing. Women with X chromosome abnormalities are at increased risk for reproductive, pregnancy or other health complications, which may be reduced or ameliorated by early diagnosis, monitoring and intervention. Objective To validate a single-nucleotide polymorphism-based non-invasive prenatal test to identify X chromosome abnormalities of maternal origin. Study Design All tests unable to evaluate fetal risk for aneuploidy due to uninformative algorithm results were eligible for inclusion. Two groups of cases were prospectively identified; Group A (n=106) where a maternal X chromosome abnormality was suspected and Group B (control group, n=107) where a fetal chromosome abnormality involving chromosome 13, 18, 21 or X was suspected, but did not meet criteria for reporting. Maternal DNA was isolated from the plasma-depleted cellular pellet and sent to a reference laboratory for blinded analysis using chromosomal microarray. A chromosome abnormality involving chromosomes 13, 18, 21 or X was reported by the reference laboratory if ≥5 Mb in size and present in ≥20% of the DNA. Results A maternal X chromosome abnormality was suspected in 1/1,305 tests (149/194,385; 0.08%). In Group A, a maternal X chromosome abnormality was confirmed in 100/106 cases (94.3% positive predictive value, one-sided 97.5% confidence interval 88.1–100.0%). Turner syndrome was the most commonly suspected maternal abnormality (58/106, 54.7%), with confirmation of mosaic or non-mosaic 45,X by microarray in 38/58 (65.5%) cases. Non-invasive prenatal screening tests suspected the presence of maternal 47,XXX with or without mosaicism in 40/106 (37.7%) cases, confirmed by microarray in 38/40 (95.0%). In Group B (n=107), no maternal microarray abnormalities were reported, providing a negative predictive value of 100% (one-sided 97.5% confidence interval: 96.6–100.0%). Conclusions When non-invasive prenatal testing suspected a maternal X chromosome abnormality, maternal microarray confirmed an X chromosome abnormality with 94.3% positive predictive value. Of the maternal X chromosome abnormalities detected by array, >50% were 45,X. When fetal chromosome abnormalities involving chromosomes 13, 18, 21 or X were suspected, no maternal chromosomes abnormalities were reported, yielding a negative predictive value of 100%. Women with maternal X abnormalities suspected with non-invasive prenatal testing may be at increased risk for reproductive and health complications; early evaluation and treatment may prevent long-term consequences or disability.
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Introduction: Noninvasive prenatal testing (NIPT), using cell-free fetal DNA, has increasingly been adopted as a screening tool for fetal aneuploidies. Several studies have discussed benefits and limitations of NIPT compared to both ultrasound and invasive procedures, but in spite of some shortcomings NIPT has become extensively used within the last five years. This study aims to describe the current use of NIPT in Europe, Australia and the USA. Material and methods: We conducted a survey to describe the current use of NIPT. Colleagues filled in a simple email-based questionnaire on NIPT in their own country, providing information on: 1) Access to NIPT, 2) NIPT's chromosomal coverage, 3) financial coverage of NIPT for the patient and 4) the proportion of women using NIPT in pregnancy. Some data are best clinical estimates, due to a lack of national data. Results: In Europe, 14 countries have adopted NIPT into a national policy/program. Two countries (Belgium and the Netherlands) offer NIPT for all pregnant women, whereas most other European countries have implemented NIPT as an offer for higher risk women after first trimester screening. In Australia, either Combined First Trimester Screening (cFTS) or NIPT are used as primary prenatal screening tests. In the USA, there are no national consensus policies on the use of NIPT, however, NIPT is widely implemented. In most European countries offering NIPT, the proportion of women using NIPT is well below 25%. In the Netherlands, Austria, Italy, Spain and most Australian and American States, 25-50% of women have NIPT performed and only in Belgium it is above 75%. In most countries NIPT reports on trisomy 13, 18 and 21, and often also on sex chromosome aneuploidies. Only in Belgium, the Netherlands, Lithuania, Greece, Cyprus and Italy is NIPT offered predominantly as a genome-wide test (including some microdeletions or a whole genome coverage). Conclusions: NIPT has been widely adopted throughout Europe, Australia and the USA, but only some countries/states have a national policy on the use of NIPT. The variation in NIPT utilization is considerable.
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Prenatal diagnosis of sex discordance is a relatively new phenomenon. Prior to cell‐free DNA testing, the diagnosis of a disorder of sexual differentiation was serendipitous, either through identification of ambiguous genitalia at the mid‐trimester morphology ultrasound or discovery of genotype‐phenotype discordance in cases where preimplantation genetic diagnosis or invasive prenatal testing had occurred. The widespread integration of cfDNA testing into modern antenatal screening has made sex chromosome assessment possible from 10 weeks of gestation, and discordant fetal sex is now more commonly diagnosed prenatally, with a prevalence of approximately 1 in 1500‐2000 pregnancies. Early detection of phenotype‐genotype sex discordance is important as it may indicate an underlying genetic, chromosomal or biochemical condition and it also allows for time‐critical postnatal treatment. The aim of this paper is to review cfDNA and ultrasound diagnosis of fetal sex, identify possible causes of phenotype‐genotype discordance and provide a systematic approach for clinicians when counselling and managing couples in this circumstance. This article is protected by copyright. All rights reserved.
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The fetal fraction (FF) is a function of both biological factors and bioinformatics algorithms used to interpret DNA sequencing results. It is an essential quality control component of noninvasive prenatal testing (NIPT) results. Clinicians need to understand the biological influences on FF to be able to provide optimal post-test counseling and clinical management. There are many different technologies available for the measurement of FF. Clinicians do not need to know the details behind the bioinformatics algorithms of FF measurements, but they do need to appreciate the significant variations between the different sequencing technologies used by different laboratories. There is no universal FF threshold that is applicable across all platforms and there have not been any differences demonstrated in NIPT performance by sequencing platform or method of FF calculation. Importantly, while FF should be routinely measured, there is not yet a consensus as to whether it should be routinely reported to the clinician. The clinician should know what to expect from a standard test report and whether reasons for failed NIPT results are revealed. Emerging solutions to the challenges of samples with low FF should reduce rates of failed NIPT in the future. In the meantime, having a “plan B” prepared for those patients for whom NIPT is unsuccessful is essential in today’s clinical practice.
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Objective: To follow up the noninvasive prenatal testing (NIPT) results of 31515 singleton pregnancies in Fujian province, southeastern China and assess its performance in low-, moderate- and high-risk pregnancies. Methods: Cell-free plasma DNA was subjected to low-coverage whole-genome sequencing. Standard Z-score analysis of the mapped sequencing reads was used to call fetal aneuploidies, including trisomies 21, 18 and 13 (T21, T18, T13) and sex chromosome aneuploidies (SCAs). NIPT positive results were confirmed by amniocentesis and karyotyping. Results: In the study population, the positive rate of chromosomal abnormalities detected by NIPT was 1.38%. A higher rate of chromosomal abnormalities was found in the high-risk group (1.57%) compared to moderate- (1.05%) and low-risk groups (1.18%) (P < 0.05). The sensitivity and specificity was 98.96% (95/96) and 99.97% (31401/31419) for detection of T21, 100% (25/25) and 99.97% (31479/31490) for T18, 100% (7/7) and 99.97% (31500/31508) for T13 and 100% (61/61) and 99.74% (31372/31454) for SCAs. Positive predictive values (PPVs) were high for T21 (84.07%), T18 (69.44%) and moderate for T13 (46.67%) SCAs (42.66%). Conclusion: Our findings support the application of NIPT for reliable and accurate testing of the general population of reproductive women for clinically significant fetal aneuploidies. This article is protected by copyright. All rights reserved.
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First-trimester screening between 11 + 0 and 13 + 6 weeks with qualified prenatal counseling, detailed ultrasound, biochemical markers and maternal factors has become the basis for decisions about further examinations. It detects numerous structural and genetic anomalies. The inclusion of uterine artery Doppler and PlGF screens for preeclampsia and fetal growth restriction. Low-dose aspirin significantly reduces the prevalence of severe preterm eclampsia. Cut-off values define groups of high, intermediate and low probability. Prenatal counseling uses detection and false-positive rates to work out the individual need profile and the corresponding decision: no further diagnosis/screening - cell-free DNA screening - diagnostic procedure and genetic analysis. In pre-test counseling it must be recognized that the prevalence of trisomy 21, 18 or 13 is low in younger women, as in submicroscopic anomalies in every maternal age. Even with high specificities, the positive predictive values of screening tests for rare anomalies are low. In the general population trisomies and sex chromosome aneuploidies account for approximately 70 % of anomalies recognizable by conventional genetic analysis. Screen positive results of cfDNA tests have to be proven by diagnostic procedure and genetic diagnosis. In cases of inconclusive results a higher rate of genetic anomalies is detected. Procedure-related fetal loss rates after chorionic biopsy and amniocentesis performed by experts are lower than 1 to 2 in 1000. Counseling should include the possible detection of submicroscopic anomalies by comparative genomic hybridization (array-CGH). At present, existing studies about screening for microdeletions and duplications do not provide reliable data to calculate sensitivities, false-positive rates and positive predictive values. © Georg Thieme Verlag KG Stuttgart · New York.
Article
Objectives: To assess the accuracy of non-invasive prenatal testing (NIPT) for sex chromosome aneuploidy (SCA) in routine clinical practice and to review counselling and sonographic issues arising in SCA cases. Methods: Three specialist Australian obstetric ultrasound and prenatal diagnosis practices offering NIPT after 10 weeks' gestation participated in this study. NIPT was reported for chromosomes 21, 18, 13, X, and Y. Results: NIPT screening was performed in 5,267 singleton pregnancies. The odds of being affected given a positive screening result (OAPR) was lowest for SCAs, most notably for monosomy X (20%). Fewer women underwent invasive prenatal testing when counselled regarding a high risk for SCA (65.5%) compared with those who had a high risk for another aneuploidy (85%). The positive screening rate of NIPT including SCA was 2.3%, but 1.2% if only the autosomal trisomies were included in the panel. Conclusion: The addition of SCA testing to NIPT doubles the positive screening rate. The OAPR for SCAs (most notably for monosomy X) is reduced compared with the autosomal trisomies. Clinicians need a more extensive discussion with women prior to the inclusion of the X and Y chromosomes in the NIPT panel, given the complexity in counselling regarding further management and the additional anxiety that these abnormal results may cause. A benefit of sex chromosome analysis is an improvement in antenatal diagnosis of some disorders of sexual development.
Article
Objective: To review clinical validation or implementation studies of maternal blood cell-free (cf) DNA analysis and define the performance of screening for fetal trisomies 21, 18 and 13 and sex chromosome aneuploidies. Data sources: Searches of PubMed, Embase and the Cochrane library were performed to identify all peer-reviewed articles on cfDNA testing in screening for aneuploidies between January 2011, when the first such study was published and 31 December 2016. Results: In total, 35 relevant studies were identified and these reported cfDNA results in relation to fetal karyotype from invasive testing or clinical outcome. In the combined total of 1,963 cases of trisomy 21 and 225,032 non-trisomy 21 singleton pregnancies the pooled weighted detection rate (DR) and false positive rate (FPR) were 99.7% (95% CI 99.1-99.9%) and 0.04% (95% CI 0.02-0.08%), respectively. In a total of 560 cases of trisomy 18 and 212,019 unaffected singleton pregnancies the pooled weighted DR and FPR were 98.2% (95% CI 95.5-99.2%) and 0.05% (95% CI 0.03-0.07%). In a total of 119 cases of trisomy 13 and 212,883 unaffected singleton pregnancies the pooled weighted DR and FPR were 99.0% (95% CI 65.8-100%) and 0.04% (95% CI 0.02-0.07%). In a total of 36 cases of monosomy X and 7,677 unaffected singleton pregnancies the pooled weighted DR and FPR were 95.8% (95% CI 70.3-99.5%) and 0.14% (95% CI 0.05-0.38%). In a combined total of 17 cases of sex chromosome abnormalities other than monosomy X and 5,383 unaffected singleton pregnancies the pooled weighted DR and FPR were 100% (95% CI 83.6-100%) and 0.003% (95% CI 0-0.07%). For twin pregnancies, in a total of 24 cases of trisomy 21 and 1,111 unaffected cases the DR was 100% (95% 95.2-100%) and FPR was 0% (95% CI 0-0.003%). Conclusion: Screening by analysis of cfDNA in maternal blood in singleton pregnancies could detect >99% of fetuses with trisomy 21, 98% of trisomy 18 and 99% of trisomy 13 at a combined FPR of 0.13%. The number of reported cases of sex chromosome abnormalities is too small for accurate assessment of performance of screening. In twin pregnancies performance of screening for trisomy 21 is encouraging but the number of cases reported is small.