3.1. Subsection Patient recruitment and clinical profile
Data of 11230 women with singleton pregnancies attending for first-trimester scans were screened during the study period. Overall, 251 women were identified to have increased NT between the 95th – 99th centile. With that, 38 women were excluded as they had other abnormal soft markers and/or structural abnormality(-ies), and later 17 were lost to follow-up. Hence, a total of 196 pregnancies with isolated increased NT between the 95th – 99th centile were analyzed for outcomes. Among these, 20 pregnancies were in the high-risk category on FTS, 23 in the intermediate-risk category and 153 in the low-risk category for T21. The flow-sheet diagram in Figure 1 depicts data selection from 11230 women screened.
Figure 1: Flow sheet explaining the data of women included in the study.
The demographic and baseline characteristics of 196 patients are presented in Table 1. The mean maternal age was 30 years, with the majority (n=169, 86.22%) of women being ≤ 35 years of age. The mean BMI was 24.6 kg/m2 (95% CI: 24.04, 25.15). The average gestational age of first-trimester screening was 12+4 weeks. The mean NT thickness was noted to be 2.7mm, with a mean CRL of 64.25mm (95% CI: 63.08, 65.42).
Table 1: Demographic and baseline characteristics of 196 patients with isolated increased NT between the 95th – 99th centile in the FTS.
Characteristics
|
Mean ± SD (95% confidence interval)
|
Maternal age (years)
|
|
All (n= 196)
|
30.94 ± 4.182 (30.35 – 31.53)
|
≤ 35 years (n= 169; 86.22%)
|
29.83 ± 3.266 (29.33 – 30.32)
|
> 35 years (n= 27; 13.78%)
|
37.93 ± 1.880 (37.18 – 38.67)
|
BMI (kg/m2)
|
24.60 ± 3.892 (24.04 – 25.15)
|
Gestational age at FTS (weeks)
|
12.62 ± 0.6686 (12.52 – 12.71)
|
Crown-rump length (mm)
|
64.25 ± 8.298 (63.08 – 65.42)
|
Nuchal translucency (mm)
|
2.76 ± 0.343 (2.71 – 2.81)
|
3.2. Pregnancy outcome
As per protocol, all women were offered invasive testing to rule out chromosomal abnormalities following the diagnosis of isolated increased NT between the 95th – 99th centile in the first-trimester screening, irrespective of the FTS risk. However, only 75 (38.20%) women accepted further testing in the study, with 63 opting for invasive and 12 for the cfDNA as their preferred screening test. In contrast, the rest of the patients (n=121, 61.7%) continued pregnancy without testing (Figure 2).
Figure 2: Outcome of pregnancies with isolated NT 95th – 99th centile.
Among the 196 fetuses with isolated increased NT thickness between the 95th – 99th centile analyzed, 7 fetuses (3.5%) were found to have chromosomal abnormalities (Table 2). Out of these, five fetuses had chromosomal aneuploidy, which could potentially be detectable by cfDNA analysis (2.5%; 5 out of 196). However, 2 cases with normal FISH results were found to have other cytogenetic abnormalities (1%; 2 out of 196). The first case had a low risk for T21 on FTS and had a normal microarray. However, further testing was carried out due to a family history of albinism, which later identified a homozygous mutation on exon2 consistent with the oculocutaneous albinism type 1A gene. An incidental note of increased NT was made on this fetus during the first-trimester screening. The second case revealed a loss of 179kbp on chromosome 2, identified as 2p15p16.1 microdeletion syndrome. Both these pregnancies with abnormal cytogenetic results had a termination of pregnancy.
The first case was a pathogenic variant of albinism, which was a rare cytogenetic finding. Albinism, a recessive genetic condition. Hence, this was an infrequent finding where it could be missed if only microarray was done. The parents chose to terminate the pregnancy and were referred for genetic consultation and parental carrier testing. It should be noted that in the absence of family history, this case would have been missed without ES.
The other case, however, was a loss of 179kbp on chromosome 2 identified as 2p15p16.1 microdeletion syndrome on the microarray, which is often characterized by developmental, intellectual disability, microcephaly, and dysmorphic facial features. There have been 12 cases of 2p15-p16.1 microdeletions reported in the literature since the first study by Rajcan-Separovic et al. (2007), and they shared similar clinical manifestations11-21. In all the reported cases, the patients were diagnosed with a genotype-first approach using chromosomal microarray testing; all deletions were of de novo origin, indicating that the copy number losses in this region were pathogenic. The ultrasound features described with this condition included facial abnormalities like flattened nasal bridge, retrognathia and structural abnormalities in the brain such as microcephaly, cortical dysplasia/pachygyria, renal anomalies (multicystic kidney, hydronephrosis), and digital camptodactyly. These findings may be apparent in certain fetuses, and some would have presented with fetal growth restriction closer to delivery. Considering that the ultrasound for this fetus was unremarkable, although its genotype is associated with the abnormalities as mentioned earlier, we suggested parental testing correlation to the couple. Eventually, they opted to discontinue the pregnancy.
There were three pregnancies with a high risk of T21 on FTS where FISH revealed T21 in one and T18 in another. The third case of T21 was diagnosed postnatally, where the couple declined any further testing despite the high risk on FTS and unremarkable ultrasound assessment. Interestingly, in the remaining four fetuses that had a low risk on FTS, two fetuses were detected with T21, and one had a high risk on cfDNA, which was then confirmed with FISH. The other was diagnosed with T21 postnatally and had an otherwise uneventful pregnancy.
In the 63 pregnancies that underwent invasive testing, apart from 5 cases that showed chromosomal aberrations, 58 had normal FISH and microarray. In this cohort of patients, if the nuchal fold was found to be prominent at the 16-week scan or anomaly scan, exome sequencing was offered to rule out single gene mutations such as Noonan syndrome. In 12 cases where cfDNA was used as the primary testing method, 11 cases reported a low risk of common aneuploidies and ten normal live babies were born. In one case, it indicated high risk, which was subsequently confirmed by an invasive test, and the mother opted for termination of pregnancy. Although cfDNA test showed low risk in the remaining one case, the pregnancy, unfortunately, ended with a miscarriage, and no cytogenetic analysis was performed for this fetus. Thereby, cfDNA accurately predicted the risk assessment for the detection of common trisomies in 11 cases mentioned above, and one case was not subjected to confirmatory analysis due to miscarriage.
Of the 185 fetuses with no genetic anomalies identified, an early anomaly scan and a detailed anatomical scan identified structural defects in 6 (3.2%) fetuses, 3 (1.6%) with complex cardiac abnormalities, 1 with agenesis of corpus callosum, 1 with skeletal dysplasia, and 1 with fetal ascites where parents chose to terminate the pregnancies. The remaining 173/196 (88.3%) fetuses with isolated increased NT between 95th – 99th centile in the first trimester had a normal perinatal outcome.
Table 2: Abnormal results in the cohort
No.
|
GA (weeks)
|
CRL (mm)
|
NT (mm)
|
Serum biochemistry (MoM)
|
Risk
|
Age risk
|
Indication
|
Invasive test
|
FISH
|
Karyotype
|
Microarray
|
Outcome
|
PAPP-A
|
β-hCG
|
PlGF
|
1
|
12+3
|
61
|
2.5
|
0.86
|
1.20
|
-
|
3431
|
1:396
|
Increased NT
|
CVS
|
Normal
|
-
|
Oculocutaneous albinism type 1A gene
DNA analysis: Homozygous for mutation on exon2 (G278X/G278X)
|
Termination
|
2
|
13+4
|
67.5
|
2.7
|
0.43
|
0.69
|
-
|
9622
|
1:985
|
Increased NT
|
Not done
|
-
|
-
|
-
|
T21 livebirth
|
3
|
11+6
|
53
|
2.7
|
0.79
|
1.79
|
0.74
|
1094
|
1:609
|
High risk on cfDNA for T21
|
CVS
|
T21
|
T21
|
-
|
Termination
|
4
|
13+6
|
70.5
|
2.8
|
0.30
|
0.60
|
-
|
155
|
1:988
|
Increased NT
|
CVS
|
T18
|
-
|
-
|
Termination
|
5
|
11+1
|
64
|
2.8
|
0.74
|
0.61
|
-
|
19303
|
1:965
|
Increased NT
|
Amniocentesis
|
Normal
|
-
|
Loss of 179Kb at 2p15; overlaps with 2p15p16.1 microdeletion syndrome
|
Termination
|
6
|
12+5
|
66.8
|
3.0
|
0.50
|
2.69
|
0.67
|
175
|
1:118
|
Increased NT
|
CVS
|
T21
|
-
|
-
|
Termination
|
7
|
13+0
|
74
|
3.1
|
0.16
|
1.32
|
0.45
|
3
|
1:783
|
Increased NT
|
Not done
|
-
|
-
|
-
|
T21 livebirth
|
Abbreviations: GA, gestational age; CRL, crown rump length; NT, nuchal translucency; MoM, multiple of median; PAPP-A, pregnancy associated plasma protein-A; β-hCG, beta human chorionic gonadotrophin; PlGF, placental growth factor; FISH, fluorescent in situ hybridisation; CVS, chorionic villus sampling, cfDNA, Cell-free DNA; T21, trisomy 21; T18, trisomy 18.