Can Cell-Free DNA Testing Suffice for Isolated Fetal Nuchal Translucency Between the 95th – 99th Centile? A Retrospective Analysis in A Centre in North India

Can Cell-Free DNA Testing Suffice for Isolated Fetal Nuchal Translucency Between the 95th – 99th Centile? 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A Retrospective Analysis in A Centre in North India Neha Sethi, Anita Kaul, Rachna Gupta, Akshatha Prabhu This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4150794/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract All pregnancies with increased nuchal translucency (NT) > 95th percentile are traditionally offered invasive testing. This study aimed to determine whether cell-free DNA (cfDNA) could suffice in patients with an isolated increased NT between the 95th – 99th centile. A retrospectively analyzed prospective study was conducted in the Fetal Medicine Unit, Indraprastha Apollo Hospitals, New Delhi, between 2007 and 2021. Study criteria were singletons with increased NT between 95th – 99th centile without soft markers or structural abnormalities. cfDNA/follow-up was offered if patients declined invasive tests. Karyotyping was the primary genetic testing from 2007 to 2015, while microarray ± exome began thereafter. All outcomes were obtained over the phone or through patient records. There were 213 pregnancies with isolated findings of NT 95th – 99th centile. All were offered invasive testing, and 17 were lost to follow-up. In 196 cases with outcomes available, 7 (3.5%) had an abnormal genetic diagnosis. Four fetuses had trisomy 21, one trisomy 18, and two fetuses (1%) had other cytogenetic abnormalities. Five fetuses (2.5%) with chromosomal aneuploidy could be potentially detected with cfDNA. cfDNA may be a viable substitution to detect common aneuploidies in this clinical population. However, in 1% of cases, microdeletions may still be missed. Health sciences/Health care/Medical imaging/Ultrasonography Health sciences/Health care/Diagnosis Health sciences/Risk factors Health sciences/Health care/Paediatrics/Intrauterine growth Non-invasive Prenatal Testing Down Syndrome Trisomy 18 Syndrome Cell-Free Nucleic Acids Aneuploidy Figures Figure 1 Figure 2 1. Introduction Increased fetal nuchal translucency (NT) > 99th percentile (≥ 3.5 mm) thickness is a common phenotypical expression of chromosomal abnormalities between 11 + 0 to 13 + 6 weeks' gestation, particularly Down syndrome (DS; also referred to as T21), Edwards syndrome (T18), Patau syndrome (T13), and Turner syndrome 1 . Fetuses with increased NT thickness are also at increased risk of other chromosomal and cardiac abnormalities 1 – 3 . Combining NT measurements with maternal serum concentrations of pregnancy-associated plasma protein-A (PAPP-A) and free beta human chorionic gonadotrophin (free β-hCG) in the combined first-trimester screening test (FTS) between 11 + 0 to 13 + 6 weeks' gestation improves detection rates and is the UK National Health Service standard 3 – 5 . Many countries have offered cell-free DNA (cfDNA) or non-invasive prenatal testing (NIPT) since 2011. It is the most sensitive and specific screening test for common autosomal aneuploidies. It can be utilized as a first-tier screening test for all pregnant women or as a contingent screening program once FTS shows increased T21, T18, and T13 risk 5 . An invasive test is recommended if cfDNA indicates high aneuploidy risk 6 . Research on cfDNA in India shows equivalent results in all testing areas 7 , 8 . An universal consensus agrees invasive testing as first-line testing when NT > 99th centile (3.5mm). There is limited data on borderline increased nuchal translucency between the 95th – 99th centile, especially if the FTS shows a low aneuploidy risk. The incidence of chromosomal abnormalities in this cohort and whether cfDNA can substitute invasive testing remain questionable.The Indian Council of Medical Research promotes genetic screening for all pregnant women per National and Family Welfare Program recommendations 9 . Clinical practice in India is heterogeneous. Remote labs may lack resources, experience, and facilities for invasive diagnostic tests, but they still can run blood tests like cfDNA. With an ahcievable effective cost reduction strategies, cfDNA could benefits India's prenatal screening program due to its safety, accuracy, and ease of extension to peripheral areas. This study aimed to evaluate whether cfDNA may be used as the first-line test in fetuses with isolated increased NT between the 95th – 99th centile and which chromosomal aberrations would be overlooked. Another study objective was to determine the outcome of this cohort. 2. Materials and Methods 2.1. Study design This was a retrospective analysis of prospectively collected data retrieved from the electronic database (Astraia software; NEXUS / ASTRAIA GmbH, Munich, Ismaning, Germany) in the Fetal Medicine Unit in Apollo Centre for Fetal Medicine (ACFM), New Delhi, from March 2007 till April 2021. 2.2. Inclusion and exclusion criteria The data retrieved were of women with singleton pregnancies attending for first-trimester screening (11 + 0 – 13 + 6 weeks) where the crown-rump length (CRL) was between 45–84 mm. The NT was measured (in mm) using transabdominal or transvaginal sonography (GE Voluson® E8, Voluson® 730 Pro, and Voluson® P6; GE Healthcare, Chicago, IL, USA) according to the Fetal Medicine Foundation (FMF) United Kingdom criteria 10 . FMF-certified operators performed all the first-trimester scans. Those pregnancies with isolated increased NT between 95th – 99th percentile (99th centile taken as NT ≥ 3.5mm) on the FMF chart were identified and included in the study. To ensure a homogenous cohort, multiple pregnancies and pregnancies with the presence of other soft markers of aneuploidy, such as absent/hypoplastic nasal bone, tricuspid regurgitation, and abnormal ductus venosus, with or without structural abnormalities, were excluded. 2.3. Data collection All pregnancies were offered combined FTS with maternal serum PAPP-A, free β-hCG, with or without placental growth factor (PlGF). These pregnancies were later classified into high-risk (less than 1:150), intermediate-risk (between 1:151 to 1:1000), and low-risk (more than 1:1000), based on the FMF reference range of adjusted risk calculation. Irrespective of their combined risk, all women with isolated increased NT between 95th – 99th centile were offered invasive testing, and if they declined, they were offered cfDNA instead. Regardless of the decision made, the patients were given thorough pre-and post-test counselling with issues including but not limited to the testing process, risk benefits, testing limitations, and potential outcomes. Initially, the test sample was subjected to quantitative-fluorescence polymerase chain reaction (QF-PCR) or fluorescent in situ hybridization (FISH). If the QF-PCR/FISH result revealed aneuploidy, the sample was processed for karyotyping. When the QF-PCR/FISH was normal, chromosomal microarray analysis (CMA) was carried out using 750k array comparative genomic hybridization (Array-CGH). Results of both QF-PCR and CMA were evaluated to determine the rate of chromosomal or microdeletions that would theoretically be missed by cfDNA testing (cfDNA was not routinely performed in this cohort). It should be emphasized that, at the beginning of the study, karyotyping was the only test offered to all women from 2007 until 2015, when it was predominantly replaced with CMA. For fetuses with NT 95th – 99th centile, according to our clinical protocol, an early anomaly scan was performed at 16 weeks to assess the fetal anatomy and nuchal fold thickness (NFT). In cases with normal CMA, if the NFT was more than 6 mm or any other complex structural abnormality was found at the 16th week's scan or anomaly scan, an additional test in the form of exome sequencing (ES) was offered to rule out single gene mutations. In these cases, with a persistently thickened nuchal fold or structural abnormality at 16 weeks, if invasive testing was declined in the first trimester, it was offered again. The investigations offered included QF-PCR/FISH, CMA and ES. Detailed anatomical survey at 19th – 20th weeks and fetal echocardiography at 22nd – 24th weeks were offered as a part of the protocol to detect structural or cardiac abnormalities. Outcomes of these pregnancies were obtained from the hospital records or telephonic conversations with the patients. Demographic details like age, weight, body mass index, parity, and mode of conception were collected. The maternal medical and family histories were also reviewed for the outcomes. 2.4. Ethical consideration and statistical analysis The Institutional Review Board and Ethics Committee of Centre for Fetal Medicine, Apollo Hospital, approved the study protocol. All patients provided written informed consent for their data to be used for research. All the data collected were transferred to Microsoft Excel 2007 spreadsheet and analyzed using the Statistical Package for the Social Sciences (SPSS) software, version 14.0. We employed descriptive statistics for statistical analysis. 3. Results 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 95 th – 99 th 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 95 th – 99 th 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/m 2 (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 95 th – 99 th 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/m 2 ) 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 95 th – 99 th 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 95 th – 99 th centile. Among the 196 fetuses with isolated increased NT thickness between the 95 th – 99 th 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 manifestations 11-21 . 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 95 th – 99 th 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. 4. Discussion The NHS Fetal Anomaly Screening Programme (FASP) recommended adding cfDNA as a contingent test following a screen-positive result between 1:2 and 1:150, either from the combined or quadruple test in both singleton and twin pregnancies. However, both the American College of Obstetricians and Gynaecologists (ACOG) and the American College of Medical Genetics and Genomics (ACMG) strongly endorsed cfDNA as the first-line screening test for Patau, Edwards, and Down syndromes in the general obstetric population 22,23 . A shared decision making in early pregnancy is crucial to deliver evidence-based, value-congruent maternity care. The patient should understand the limitations of cfDNA and result ramifications. Invasive testing would help ascertain the residual risk but the associated miscarriage risk may outweigh its potential benefits 24,25 . Similar to ours, Miranda et al. evaluated the sustainability of cDNA testing as the sole screening test among 226 fetuses with increased NT > 99 th centile 26 . The cfDNA missed 12–19% of genetic abnormalities, making it unsuitable for this group. In contrast, our study showed very low incidence of genetic anomalies at 1.0% among fetuses with isolated increased NT between the 95 th – 99 th centile. The result showed that cfDNA is a practical test for this population, even with a low-risk FTS result. Although contingent use of cfDNA after a high-risk FTS result is cost-saving, there was no consensus regarding such application 27,28 . In our study, the only case where cfDNA showed a high T21 risk was accurately confirmed with an invasive test. The remaining cases showed a low aneuploidies risk, therefore all live newborns had normal perinatal outcomes. A total of 5 common aneuploidies cases were detected in this cohort (2.5%). The incidence of DS in isolated increased NT was 2.0% (4 out of 196), with 2 cases diagnosed postnatally. One T18 case was detected with invasive testing. Even though we assumed a theoretical 100% detection rate for cfDNA-detectable chromosomal abnormalities, it clinically ranges from 97% for T18 and T13 to 99% for T21. In fetuses with isolated increased NT between the 95 th – 99 th centile, we may still miss to detect common aneuploidies despite the low-risk from combined FTS that holds 92% accuracy rate. Hence, we can consider cfDNA testing in these fetuses that labelled low-risk on FTS. Meanwhile, for FTS-identified high-risk pregnancies, Srebniak et al. reported a significant reduction in cfDNA diagnostic yield. In such circumstances, invasive testing seemed preferable 25 . As we demonstrated that the cfDNA test can accurately predict common aneuploidy risk, we recommend offering cfNDA to women who decline invasive testing. Kagan et al. reported that a contingent screening strategy combining a serial detailed ultrasound scan, NT measurement, and cfDNA significantly reduces false positives rate compared to FTS alone 29 . Although cfDNA appears to be a better test alternative, many studies have shown that a small percentage of chromosome abnormalities may be missed 30,31 . The undetectable aneuploidies may associated with varying degrees of disability. In the two abnormal cytogenetic cases (oculocutaneous albinism and 2p15p16.1 microdeletion syndrome), both FTS risk were low, and the couples opted for invasive testing next. Hence, if theoretically only cfDNA was performed for isolated increased NT with low-risk FTS result, common aneuploidies are ruled out. As we would have suggested a follow-up with an early anomaly scan, potential structural abnormalities associated with the microdeletion syndrome could still be detected in time for a termination decision, if abnormal, within the realms of the abortion law. Based on our data, if only cfDNA is offered to these women, there is a potential 1.0% (1 in 98 fetuses) miss of non-autosomal chromosomal aberrations or a rare genetic syndrome. Our findings are also corroborated by Sagi-Dain et al., who shared a likewise missing rate of 1.5% (1 in 69 fetuses) to 1.9% (1 in 52 fetuses) in genome-wide NIPT and five-chromosomal NIPT, respectively 32 . Whereas, Petersen et al. reported a substantiallyhigher residual risk of microscopic chromosomal aberration at 3.0% – 4.8% (1 in 21 – 33 fetuses), which could be attributed to the inclusion of isolated and non-isolated cases 24 . Despite the low percentage, patients should be informed before opting for cfDNA testing because some microdeletions may be missed. Henceforth, offering only cfDNA in this cohort of patients remains a discussion and can be considered individually. Notably, an abnormal NIPT required a further confirmatory diagnostic test, which the wait for a definitive result increased the mother's anixety 24 . It is also worthwhile to consider the difficulty of resampling and longer turnaround time, especially for those staying in the peripheral area, if the result is "no-call" or discordant due to low fetal fraction 33 . The strength of this study includes a stringent study criterion, which considered only isolated increased NT between the 95 th – 99 th centile. With normal biomarker profiles and ultrasonography results, cfDNA predictability on aneuploidy risk can be tested without confounders. To our knowledge, our study is one of the pioneers that examines cfDNA utilizationin a cohort with isolated increased NT between the 95 th – 99 th centile. Further study with a large sample size across various countries and provinces is needed to reaffirm our outcome. Nevertheless, this study can be meta-analyzed. Another study's merits include adopting a tight protocol for FMF-certified ultrasonography and biochemistry analyses according to international standards. The results should be interpreted cautiously due to several study limitations. Despite the relatively large sample size, the study was commenced at a single centre in North India, which fairly limited the generalizability of the study findings to the rest of the country. A well-resourced specialized tertiary centre may have had selection and referral bias, therefore the study outcomes may not apply to other hospitals and regions 34 . Midway through the study, we have replaced karyotyping with CMA in year 2015 and routinely offered CMA as primary test since then. The increased resolution of CMA detects chromosomal abnormalities better than conventional karyotyping 35 . Thus, karyotyping may overlook certain chromosomal abnormalities in the first half of our study. 5. Conclusions This study shows the incidence rate of chromosomal abnormalities in the North Indians with isolated increased NT between the 95 th and 99 th centiles. Our study reported that these 2.5% chromosomal aneuploidies could also be potentially detected with cfDNA. Therefore, cfDNA may be a reasonable alternative to invasive diagnostics for common aneuploidies in this clinical group. Nevertheless, albeit small, cfDNA may still miss 1% of cases of microdeletions or rare genetic syndromes. Declarations Author Contributions: Conceptualization, N.S. and A.K.; methodology, A.K.; validation, N.S., A.K., R.G., and A.P.; formal analysis, N.S. and A.K.; investigation, N.S.; resources, N.S.; data curation, N.S.; writing—original draft preparation, N.S.; writing—review and editing, N.S., R.G., and A.P.; supervision, A.K.abb; project administration, N.S., and A.K.. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Institutional Review Board Statement: The study was conducted in accordance with the Declaration of Helsinki, and approved by the Institutional Ethics Committee of Indraprastha Apollo Hospitals (protocol code IAH-BMR-057/11-21 and date of approval 2 nd March 2022). Data Availability Statement: The data from this study are available on request from the corresponding author. The data are not publicly available due to patient confidentiality and are used under license for the current study. 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Ultrasound in Obstetrics & Gynecology 51, 437–444, doi: 10.1002/uog.18905 (2018). Norton, M. E., Jelliffe-Pawlowski, L. L. & Currier, R. J. Chromosome Abnormalities Detected by Current Prenatal Screening and Noninvasive Prenatal Testing. Obstetrics & Gynecology 124, 979–986, doi: 10.1097/aog.0000000000000452 (2014). Yang, L. & Tan, W. C. Prenatal screening in the era of non-invasive prenatal testing: a Nationwide cross-sectional survey of obstetrician knowledge, attitudes and clinical practice. BMC Pregnancy and Childbirth 20, 579, doi: 10.1186/s12884-020-03279-y (2020). Sagi-Dain, L. et al. Risk of Clinically Significant Chromosomal Microarray Analysis Findings in Fetuses With Nuchal Translucency From 3.0 mm Through 3.4 mm. Obstetrics & Gynecology 137, 126–131, doi: 10.1097/aog.0000000000004195 (2021). Massa, J. D. et al. Current Status of Noninvasive Prenatal Testing and Counselling Considerations: An Indian Perspective. Journal of Fetal Medicine 7, 9–16, doi: 10.1007/s40556-019-00228-4 (2020). Delgado-Rodríguez, M. & Llorca, J. Bias. Journal of Epidemiology and Community Health 58, 635–641, doi: 10.1136/jech.2003.008466 (2004). Hay, S. B. et al. ACOG and SMFM guidelines for prenatal diagnosis: Is karyotyping really sufficient? Prenatal Diagnosis 38, 184–189, doi: 10.1002/pd.5212 (2018). Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About In Review Editorial Policies Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4150794","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":286836598,"identity":"643bd382-e51a-4b82-9c9d-fd47a3139802","order_by":0,"name":"Neha Sethi","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAzklEQVRIiWNgGAWjYJACZgYGGzl+MJONeC1pxpINJGo5nGhwgFgt8g28Bx8X1DAnGN/ITmD4UHaYQXdGAn4tBgf4ko1nHGPLM7uRu4FxxrnDDGY3CGlh4DGT5mHjKQZpYeZtI0KLfAOP+W+efxKJm2cAtfwlRgvDAR4zoOEGiRskgFoYidFicIDHWJq3L8FY4szbDQd7zqXzmJ15QNBhhp95vv2X42/P3fjgR5m1nNlxQg6TRzLzABDzMAgQ0oIJ+A+QrGUUjIJRMAqGNwAAbLxCKK0c8h8AAAAASUVORK5CYII=","orcid":"","institution":"University Malaya Medical Centre","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Neha","middleName":"","lastName":"Sethi","suffix":""},{"id":286836599,"identity":"4abbfe2c-5cf8-41ff-bc67-1ae25a08050c","order_by":1,"name":"Anita Kaul","email":"","orcid":"","institution":"Indraprastha Apollo Hospitals","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Anita","middleName":"","lastName":"Kaul","suffix":""},{"id":286836600,"identity":"3580eba9-53c5-46ac-aba5-562c68d15d49","order_by":2,"name":"Rachna Gupta","email":"","orcid":"","institution":"Indraprastha Apollo Hospitals","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Rachna","middleName":"","lastName":"Gupta","suffix":""},{"id":286836601,"identity":"11399889-decc-4ed2-8e54-a29bd79a17b6","order_by":3,"name":"Akshatha Prabhu","email":"","orcid":"","institution":"Indraprastha Apollo Hospitals","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Akshatha","middleName":"","lastName":"Prabhu","suffix":""}],"badges":[],"createdAt":"2024-03-22 15:31:32","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4150794/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4150794/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":54192038,"identity":"7251dd8d-b0a3-48d5-b7b0-8a2f37d5e32f","added_by":"auto","created_at":"2024-04-05 21:01:55","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":45398,"visible":true,"origin":"","legend":"\u003cp\u003eFlow sheet explaining the data of women included in the study.\u003c/p\u003e\n\u003cp\u003eAbbreviations: ACFM, Apollo Centre for Fetal Medicine; FTS, first-trimester screening; NT, nuchal translucency\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-4150794/v1/1194a7df2fc682c0a429756c.png"},{"id":54192024,"identity":"ebae7fb4-590f-411e-a64d-307304ed2504","added_by":"auto","created_at":"2024-04-05 21:01:51","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":52898,"visible":true,"origin":"","legend":"\u003cp\u003eOutcome of pregnancies with isolated NT 95\u003csup\u003eth\u003c/sup\u003e – 99\u003csup\u003eth\u003c/sup\u003e centile.\u003c/p\u003e\n\u003cp\u003eAbbreviations: NT, nuchal translucency; cfDNA, cell-free DNA\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-4150794/v1/16ad21dbdf4331414985caa6.png"},{"id":60372686,"identity":"b37eb627-4477-4462-80b4-a332f37fa36a","added_by":"auto","created_at":"2024-07-16 05:27:04","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":518320,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4150794/v1/f7c8500e-3bc4-466c-8b6a-8b6da5499ce7.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Can Cell-Free DNA Testing Suffice for Isolated Fetal Nuchal Translucency Between the 95th – 99th Centile? A Retrospective Analysis in A Centre in North India","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eIncreased fetal nuchal translucency (NT)\u0026thinsp;\u0026gt;\u0026thinsp;99th percentile (\u0026ge;\u0026thinsp;3.5 mm) thickness is a common phenotypical expression of chromosomal abnormalities between 11\u003csup\u003e+\u0026thinsp;0\u003c/sup\u003e to 13\u003csup\u003e+\u0026thinsp;6\u003c/sup\u003e weeks' gestation, particularly Down syndrome (DS; also referred to as T21), Edwards syndrome (T18), Patau syndrome (T13), and Turner syndrome\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Fetuses with increased NT thickness are also at increased risk of other chromosomal and cardiac abnormalities\u003csup\u003e\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Combining NT measurements with maternal serum concentrations of pregnancy-associated plasma protein-A (PAPP-A) and free beta human chorionic gonadotrophin (free β-hCG) in the combined first-trimester screening test (FTS) between 11\u003csup\u003e+\u0026thinsp;0\u003c/sup\u003e to 13\u003csup\u003e+\u0026thinsp;6\u003c/sup\u003e weeks' gestation improves detection rates and is the UK National Health Service standard\u003csup\u003e\u003cspan additionalcitationids=\"CR4\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eMany countries have offered cell-free DNA (cfDNA) or non-invasive prenatal testing (NIPT) since 2011. It is the most sensitive and specific screening test for common autosomal aneuploidies. It can be utilized as a first-tier screening test for all pregnant women or as a contingent screening program once FTS shows increased T21, T18, and T13 risk\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. An invasive test is recommended if cfDNA indicates high aneuploidy risk\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Research on cfDNA in India shows equivalent results in all testing areas\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eAn universal consensus agrees invasive testing as first-line testing when NT\u0026thinsp;\u0026gt;\u0026thinsp;99th centile (3.5mm). There is limited data on borderline increased nuchal translucency between the 95th \u0026ndash; 99th centile, especially if the FTS shows a low aneuploidy risk. The incidence of chromosomal abnormalities in this cohort and whether cfDNA can substitute invasive testing remain questionable.The Indian Council of Medical Research promotes genetic screening for all pregnant women per National and Family Welfare Program recommendations\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Clinical practice in India is heterogeneous. Remote labs may lack resources, experience, and facilities for invasive diagnostic tests, but they still can run blood tests like cfDNA. With an ahcievable effective cost reduction strategies, cfDNA could benefits India's prenatal screening program due to its safety, accuracy, and ease of extension to peripheral areas. This study aimed to evaluate whether cfDNA may be used as the first-line test in fetuses with isolated increased NT between the 95th \u0026ndash; 99th centile and which chromosomal aberrations would be overlooked. Another study objective was to determine the outcome of this cohort.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Study design\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThis was a retrospective analysis of prospectively collected data retrieved from the electronic database (Astraia software; NEXUS / ASTRAIA GmbH, Munich, Ismaning, Germany) in the Fetal Medicine Unit in Apollo Centre for Fetal Medicine (ACFM), New Delhi, from March 2007 till April 2021.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Inclusion and exclusion criteria\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe data retrieved were of women with singleton pregnancies attending for first-trimester screening (11\u003csup\u003e+\u0026thinsp;0\u003c/sup\u003e \u0026ndash; 13\u003csup\u003e+\u0026thinsp;6\u003c/sup\u003e weeks) where the crown-rump length (CRL) was between 45\u0026ndash;84 mm. The NT was measured (in mm) using transabdominal or transvaginal sonography (GE Voluson\u0026reg; E8, Voluson\u0026reg; 730 Pro, and Voluson\u0026reg; P6; GE Healthcare, Chicago, IL, USA) according to the Fetal Medicine Foundation (FMF) United Kingdom criteria\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. FMF-certified operators performed all the first-trimester scans.\u003c/p\u003e \u003cp\u003eThose pregnancies with isolated increased NT between 95th \u0026ndash; 99th percentile (99th centile taken as NT\u0026thinsp;\u0026ge;\u0026thinsp;3.5mm) on the FMF chart were identified and included in the study. To ensure a homogenous cohort, multiple pregnancies and pregnancies with the presence of other soft markers of aneuploidy, such as absent/hypoplastic nasal bone, tricuspid regurgitation, and abnormal ductus venosus, with or without structural abnormalities, were excluded.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Data collection\u003c/h2\u003e \u003cp\u003eAll pregnancies were offered combined FTS with maternal serum PAPP-A, free β-hCG, with or without placental growth factor (PlGF). These pregnancies were later classified into high-risk (less than 1:150), intermediate-risk (between 1:151 to 1:1000), and low-risk (more than 1:1000), based on the FMF reference range of adjusted risk calculation. Irrespective of their combined risk, all women with isolated increased NT between 95th \u0026ndash; 99th centile were offered invasive testing, and if they declined, they were offered cfDNA instead. Regardless of the decision made, the patients were given thorough pre-and post-test counselling with issues including but not limited to the testing process, risk benefits, testing limitations, and potential outcomes.\u003c/p\u003e \u003cp\u003eInitially, the test sample was subjected to quantitative-fluorescence polymerase chain reaction (QF-PCR) or fluorescent in situ hybridization (FISH). If the QF-PCR/FISH result revealed aneuploidy, the sample was processed for karyotyping. When the QF-PCR/FISH was normal, chromosomal microarray analysis (CMA) was carried out using 750k array comparative genomic hybridization (Array-CGH). Results of both QF-PCR and CMA were evaluated to determine the rate of chromosomal or microdeletions that would theoretically be missed by cfDNA testing (cfDNA was not routinely performed in this cohort). It should be emphasized that, at the beginning of the study, karyotyping was the only test offered to all women from 2007 until 2015, when it was predominantly replaced with CMA.\u003c/p\u003e \u003cp\u003eFor fetuses with NT 95th \u0026ndash; 99th centile, according to our clinical protocol, an early anomaly scan was performed at 16 weeks to assess the fetal anatomy and nuchal fold thickness (NFT). In cases with normal CMA, if the NFT was more than 6 mm or any other complex structural abnormality was found at the 16th week's scan or anomaly scan, an additional test in the form of exome sequencing (ES) was offered to rule out single gene mutations. In these cases, with a persistently thickened nuchal fold or structural abnormality at 16 weeks, if invasive testing was declined in the first trimester, it was offered again. The investigations offered included QF-PCR/FISH, CMA and ES. Detailed anatomical survey at 19th \u0026ndash; 20th weeks and fetal echocardiography at 22nd \u0026ndash; 24th weeks were offered as a part of the protocol to detect structural or cardiac abnormalities.\u003c/p\u003e \u003cp\u003eOutcomes of these pregnancies were obtained from the hospital records or telephonic conversations with the patients. Demographic details like age, weight, body mass index, parity, and mode of conception were collected. The maternal medical and family histories were also reviewed for the outcomes.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Ethical consideration and statistical analysis\u003c/h2\u003e \u003cp\u003eThe Institutional Review Board and Ethics Committee of Centre for Fetal Medicine, Apollo Hospital, approved the study protocol. All patients provided written informed consent for their data to be used for research.\u003c/p\u003e \u003cp\u003eAll the data collected were transferred to Microsoft Excel 2007 spreadsheet and analyzed using the Statistical Package for the Social Sciences (SPSS) software, version 14.0. We employed descriptive statistics for statistical analysis.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cp\u003e3.1. Subsection\u0026nbsp;Patient recruitment and clinical profile\u003c/p\u003e\n\u003cp\u003eData 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 95\u003csup\u003eth\u003c/sup\u003e \u0026ndash; 99\u003csup\u003eth\u003c/sup\u003e 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 95\u003csup\u003eth\u003c/sup\u003e \u0026ndash; 99\u003csup\u003eth\u003c/sup\u003e 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 \u003cstrong\u003eFigure 1\u0026nbsp;\u003c/strong\u003edepicts data selection from 11230 women screened.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFigure 1:\u0026nbsp;\u003c/strong\u003eFlow sheet explaining the data of women included in the study.\u003c/p\u003e\n\u003cp\u003eThe demographic and baseline characteristics of 196 patients are presented in \u003cstrong\u003eTable 1\u003c/strong\u003e. The mean maternal age was 30 years, with the majority (n=169, 86.22%) of women being \u0026le; 35 years of age. The mean BMI was 24.6 kg/m\u003csup\u003e2\u0026nbsp;\u003c/sup\u003e(95% CI: 24.04, 25.15). The average gestational age of first-trimester screening was 12\u003csup\u003e+4\u003c/sup\u003e weeks. The mean NT thickness was noted to be 2.7mm, with a mean CRL of 64.25mm (95% CI: 63.08, 65.42).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1:\u0026nbsp;\u003c/strong\u003eDemographic and baseline characteristics of 196 patients with isolated increased NT between the 95\u003csup\u003eth\u003c/sup\u003e \u0026ndash; 99\u003csup\u003eth\u003c/sup\u003e centile in the FTS.\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"50%\" valign=\"top\"\u003e\n \u003cp\u003eCharacteristics\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"50%\" valign=\"top\"\u003e\n \u003cp\u003eMean \u0026plusmn; SD (95% confidence interval)\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"50%\" valign=\"top\"\u003e\n \u003cp\u003eMaternal age (years)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"50%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"50%\" valign=\"top\"\u003e\n \u003cp\u003eAll (n= 196)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"50%\" valign=\"top\"\u003e\n \u003cp\u003e30.94 \u0026plusmn; 4.182 (30.35 \u0026ndash; 31.53)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"50%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026le; 35 years (n= 169; 86.22%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"50%\" valign=\"top\"\u003e\n \u003cp\u003e29.83 \u0026plusmn; 3.266 (29.33 \u0026ndash; 30.32)\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"50%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026gt; 35 years (n= 27; 13.78%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"50%\" valign=\"top\"\u003e\n \u003cp\u003e37.93 \u0026plusmn; 1.880 (37.18 \u0026ndash; 38.67)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"50%\" valign=\"top\"\u003e\n \u003cp\u003eBMI (kg/m\u003csup\u003e2\u003c/sup\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"50%\" valign=\"top\"\u003e\n \u003cp\u003e24.60 \u0026plusmn; 3.892 (24.04 \u0026ndash; 25.15)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"50%\" valign=\"top\"\u003e\n \u003cp\u003eGestational age at FTS (weeks)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"50%\" valign=\"top\"\u003e\n \u003cp\u003e12.62 \u0026plusmn; 0.6686 (12.52 \u0026ndash; 12.71)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"50%\" valign=\"top\"\u003e\n \u003cp\u003eCrown-rump length (mm)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"50%\" valign=\"top\"\u003e\n \u003cp\u003e64.25 \u0026plusmn; 8.298 (63.08 \u0026ndash; 65.42)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"50%\" valign=\"top\"\u003e\n \u003cp\u003eNuchal translucency (mm)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"50%\" valign=\"top\"\u003e\n \u003cp\u003e2.76 \u0026plusmn; 0.343 (2.71 \u0026ndash; 2.81)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e3.2. Pregnancy outcome\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAs per protocol, all women were offered invasive testing to rule out chromosomal abnormalities following the diagnosis of isolated increased NT between the 95\u003csup\u003eth\u003c/sup\u003e \u0026ndash; 99\u003csup\u003eth\u003c/sup\u003e 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 (\u003cstrong\u003eFigure 2\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFigure 2:\u003c/strong\u003e Outcome of pregnancies with isolated NT 95\u003csup\u003eth\u003c/sup\u003e \u0026ndash; 99\u003csup\u003eth\u003c/sup\u003e centile.\u003c/p\u003e\n\u003cp\u003eAmong the 196 fetuses with isolated increased NT thickness between the 95\u003csup\u003eth\u003c/sup\u003e \u0026ndash; 99\u003csup\u003eth\u003c/sup\u003e centile analyzed, 7 fetuses (3.5%) were found to have chromosomal abnormalities (\u003cstrong\u003eTable 2\u003c/strong\u003e). 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.\u003c/p\u003e\n\u003cp\u003eThe 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.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe 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 manifestations\u003csup\u003e11-21\u003c/sup\u003e. In all the reported cases, the patients were diagnosed with a genotype-first approach using chromosomal microarray testing; all deletions were of \u003cem\u003ede novo\u0026nbsp;\u003c/em\u003eorigin, 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.\u003c/p\u003e\n\u003cp\u003eThere 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.\u003c/p\u003e\n\u003cp\u003eIn 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.\u003c/p\u003e\n\u003cp\u003eOf 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 95\u003csup\u003eth\u003c/sup\u003e \u0026ndash; 99\u003csup\u003eth\u003c/sup\u003e centile in the first trimester had a normal perinatal outcome.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2:\u003c/strong\u003e Abnormal results in the cohort\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"100%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"3.260869565217391%\" rowspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003eNo.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.434782608695652%\" rowspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003eGA (weeks)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"4.3478260869565215%\" rowspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003eCRL (mm)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"4.3478260869565215%\" rowspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003eNT (mm)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.47826086956522%\" colspan=\"3\" valign=\"top\"\u003e\n \u003cp\u003eSerum biochemistry (MoM)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"4.3478260869565215%\" rowspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003eRisk\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"4.3478260869565215%\" rowspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003eAge risk\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.782608695652174%\" rowspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003eIndication\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.782608695652174%\" rowspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003eInvasive test\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.434782608695652%\" rowspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003eFISH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.521739130434782%\" rowspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003eKaryotype\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.217391304347826%\" rowspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003eMicroarray\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.695652173913043%\" rowspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003eOutcome\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"33.333333333333336%\" valign=\"top\"\u003e\n \u003cp\u003ePAPP-A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.333333333333336%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026beta;-hCG\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.333333333333336%\" valign=\"top\"\u003e\n \u003cp\u003ePlGF\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"3.3333333333333335%\" valign=\"top\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.555555555555555%\" valign=\"top\"\u003e\n \u003cp\u003e12\u003csup\u003e+3\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"4.444444444444445%\" valign=\"top\"\u003e\n \u003cp\u003e61\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"4.444444444444445%\" valign=\"top\"\u003e\n \u003cp\u003e2.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.555555555555555%\" valign=\"top\"\u003e\n \u003cp\u003e0.86\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.555555555555555%\" valign=\"top\"\u003e\n \u003cp\u003e1.20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.555555555555555%\" valign=\"top\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"4.444444444444445%\" valign=\"top\"\u003e\n \u003cp\u003e3431\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"4.444444444444445%\" valign=\"top\"\u003e\n \u003cp\u003e1:396\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10%\" valign=\"top\"\u003e\n \u003cp\u003eIncreased NT\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10%\" valign=\"top\"\u003e\n \u003cp\u003eCVS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.555555555555555%\" valign=\"top\"\u003e\n \u003cp\u003eNormal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.666666666666667%\" valign=\"top\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.555555555555555%\" valign=\"top\"\u003e\n \u003cp\u003eOculocutaneous albinism type 1A gene\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eDNA analysis: Homozygous for mutation on exon2 (G278X/G278X)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.88888888888889%\" valign=\"top\"\u003e\n \u003cp\u003eTermination\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"3.3333333333333335%\" valign=\"top\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.555555555555555%\" valign=\"top\"\u003e\n \u003cp\u003e13\u003csup\u003e+4\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"4.444444444444445%\" valign=\"top\"\u003e\n \u003cp\u003e67.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"4.444444444444445%\" valign=\"top\"\u003e\n \u003cp\u003e2.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.555555555555555%\" valign=\"top\"\u003e\n \u003cp\u003e0.43\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.555555555555555%\" valign=\"top\"\u003e\n \u003cp\u003e0.69\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.555555555555555%\" valign=\"top\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"4.444444444444445%\" valign=\"top\"\u003e\n \u003cp\u003e9622\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"4.444444444444445%\" valign=\"top\"\u003e\n \u003cp\u003e1:985\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10%\" valign=\"top\"\u003e\n \u003cp\u003eIncreased NT\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10%\" valign=\"top\"\u003e\n \u003cp\u003eNot done\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.555555555555555%\" valign=\"top\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.666666666666667%\" valign=\"top\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.555555555555555%\" valign=\"top\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.88888888888889%\" valign=\"top\"\u003e\n \u003cp\u003eT21 livebirth\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"3.3333333333333335%\" valign=\"top\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.555555555555555%\" valign=\"top\"\u003e\n \u003cp\u003e11\u003csup\u003e+6\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"4.444444444444445%\" valign=\"top\"\u003e\n \u003cp\u003e53\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"4.444444444444445%\" valign=\"top\"\u003e\n \u003cp\u003e2.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.555555555555555%\" valign=\"top\"\u003e\n \u003cp\u003e0.79\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.555555555555555%\" valign=\"top\"\u003e\n \u003cp\u003e1.79\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.555555555555555%\" valign=\"top\"\u003e\n \u003cp\u003e0.74\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"4.444444444444445%\" valign=\"top\"\u003e\n \u003cp\u003e1094\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"4.444444444444445%\" valign=\"top\"\u003e\n \u003cp\u003e1:609\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10%\" valign=\"top\"\u003e\n \u003cp\u003eHigh risk on cfDNA for T21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10%\" valign=\"top\"\u003e\n \u003cp\u003eCVS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.555555555555555%\" valign=\"top\"\u003e\n \u003cp\u003eT21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.666666666666667%\" valign=\"top\"\u003e\n \u003cp\u003eT21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.555555555555555%\" valign=\"top\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.88888888888889%\" valign=\"top\"\u003e\n \u003cp\u003eTermination\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"3.3333333333333335%\" valign=\"top\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.555555555555555%\" valign=\"top\"\u003e\n \u003cp\u003e13\u003csup\u003e+6\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"4.444444444444445%\" valign=\"top\"\u003e\n \u003cp\u003e70.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"4.444444444444445%\" valign=\"top\"\u003e\n \u003cp\u003e2.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.555555555555555%\" valign=\"top\"\u003e\n \u003cp\u003e0.30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.555555555555555%\" valign=\"top\"\u003e\n \u003cp\u003e0.60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.555555555555555%\" valign=\"top\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"4.444444444444445%\" valign=\"top\"\u003e\n \u003cp\u003e155\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"4.444444444444445%\" valign=\"top\"\u003e\n \u003cp\u003e1:988\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10%\" valign=\"top\"\u003e\n \u003cp\u003eIncreased NT\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10%\" valign=\"top\"\u003e\n \u003cp\u003eCVS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.555555555555555%\" valign=\"top\"\u003e\n \u003cp\u003eT18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.666666666666667%\" valign=\"top\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.555555555555555%\" valign=\"top\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.88888888888889%\" valign=\"top\"\u003e\n \u003cp\u003eTermination\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"3.3333333333333335%\" valign=\"top\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.555555555555555%\" valign=\"top\"\u003e\n \u003cp\u003e11\u003csup\u003e+1\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"4.444444444444445%\" valign=\"top\"\u003e\n \u003cp\u003e64\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"4.444444444444445%\" valign=\"top\"\u003e\n \u003cp\u003e2.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.555555555555555%\" valign=\"top\"\u003e\n \u003cp\u003e0.74\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.555555555555555%\" valign=\"top\"\u003e\n \u003cp\u003e0.61\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.555555555555555%\" valign=\"top\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"4.444444444444445%\" valign=\"top\"\u003e\n \u003cp\u003e19303\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"4.444444444444445%\" valign=\"top\"\u003e\n \u003cp\u003e1:965\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10%\" valign=\"top\"\u003e\n \u003cp\u003eIncreased NT\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10%\" valign=\"top\"\u003e\n \u003cp\u003eAmniocentesis\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.555555555555555%\" valign=\"top\"\u003e\n \u003cp\u003eNormal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.666666666666667%\" valign=\"top\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.555555555555555%\" valign=\"top\"\u003e\n \u003cp\u003eLoss of 179Kb at 2p15; overlaps with 2p15p16.1 microdeletion syndrome\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.88888888888889%\" valign=\"top\"\u003e\n \u003cp\u003eTermination\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"3.3333333333333335%\" valign=\"top\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.555555555555555%\" valign=\"top\"\u003e\n \u003cp\u003e12\u003csup\u003e+5\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"4.444444444444445%\" valign=\"top\"\u003e\n \u003cp\u003e66.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"4.444444444444445%\" valign=\"top\"\u003e\n \u003cp\u003e3.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.555555555555555%\" valign=\"top\"\u003e\n \u003cp\u003e0.50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.555555555555555%\" valign=\"top\"\u003e\n \u003cp\u003e2.69\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.555555555555555%\" valign=\"top\"\u003e\n \u003cp\u003e0.67\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"4.444444444444445%\" valign=\"top\"\u003e\n \u003cp\u003e175\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"4.444444444444445%\" valign=\"top\"\u003e\n \u003cp\u003e1:118\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10%\" valign=\"top\"\u003e\n \u003cp\u003eIncreased NT\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10%\" valign=\"top\"\u003e\n \u003cp\u003eCVS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.555555555555555%\" valign=\"top\"\u003e\n \u003cp\u003eT21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.666666666666667%\" valign=\"top\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.555555555555555%\" valign=\"top\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.88888888888889%\" valign=\"top\"\u003e\n \u003cp\u003eTermination\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"3.3333333333333335%\" valign=\"top\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.555555555555555%\" valign=\"top\"\u003e\n \u003cp\u003e13\u003csup\u003e+0\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"4.444444444444445%\" valign=\"top\"\u003e\n \u003cp\u003e74\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"4.444444444444445%\" valign=\"top\"\u003e\n \u003cp\u003e3.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.555555555555555%\" valign=\"top\"\u003e\n \u003cp\u003e0.16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.555555555555555%\" valign=\"top\"\u003e\n \u003cp\u003e1.32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.555555555555555%\" valign=\"top\"\u003e\n \u003cp\u003e0.45\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"4.444444444444445%\" valign=\"top\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"4.444444444444445%\" valign=\"top\"\u003e\n \u003cp\u003e1:783\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10%\" valign=\"top\"\u003e\n \u003cp\u003eIncreased NT\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10%\" valign=\"top\"\u003e\n \u003cp\u003eNot done\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.555555555555555%\" valign=\"top\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.666666666666667%\" valign=\"top\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.555555555555555%\" valign=\"top\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.88888888888889%\" valign=\"top\"\u003e\n \u003cp\u003eT21 livebirth\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eAbbreviations: GA, gestational age; CRL, crown rump length; NT, nuchal translucency; MoM, multiple of median; PAPP-A, pregnancy associated plasma protein-A; \u0026beta;-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.\u003c/p\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eThe NHS Fetal Anomaly Screening Programme (FASP)\u0026nbsp;\u0026nbsp;recommended adding cfDNA as a contingent test\u0026nbsp;following a screen-positive result between 1:2 and 1:150, either from the combined or quadruple test in both singleton and twin pregnancies.\u0026nbsp;However,\u0026nbsp;both the American College of Obstetricians and Gynaecologists (ACOG) and the American College of Medical Genetics and Genomics (ACMG) strongly endorsed cfDNA as the first-line screening test for Patau, Edwards, and Down syndromes in the general obstetric population\u003csup\u003e22,23\u003c/sup\u003e. A shared decision making in early pregnancy is crucial to deliver evidence-based, value-congruent maternity care. The patient should understand the limitations of cfDNA and result ramifications. \u0026nbsp;Invasive testing would help ascertain the residual risk but the associated miscarriage risk may\u0026nbsp;outweigh its potential benefits\u003csup\u003e24,25\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSimilar to ours, Miranda et al. evaluated the sustainability of cDNA testing as the sole screening test among 226 fetuses with increased NT \u0026gt; 99\u003csup\u003eth\u003c/sup\u003e centile\u003csup\u003e26\u003c/sup\u003e. The cfDNA missed 12\u0026ndash;19% of genetic abnormalities, making it unsuitable for this group. In contrast, our study showed very low incidence of genetic anomalies at 1.0% among fetuses with isolated increased NT between the 95\u003csup\u003eth\u003c/sup\u003e \u0026ndash; 99\u003csup\u003eth\u003c/sup\u003e centile. The result showed that cfDNA is a practical test for this population, even with a low-risk FTS result.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAlthough contingent use of cfDNA after a high-risk FTS result is cost-saving, there was no consensus regarding such application\u003csup\u003e27,28\u003c/sup\u003e. In our study, the only case where cfDNA showed a high T21 risk was accurately confirmed with an invasive test. The remaining cases showed a low aneuploidies risk, therefore all live newborns had normal perinatal outcomes. A total of 5 common aneuploidies cases were detected in this cohort (2.5%). The incidence of DS in isolated increased NT was 2.0% (4 out of 196), with 2 cases diagnosed postnatally. One T18 case was detected with invasive testing. Even though we assumed a theoretical 100% detection rate for cfDNA-detectable chromosomal abnormalities, it clinically ranges from 97% for T18 and T13 to 99% for T21.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn fetuses with isolated increased NT between the 95\u003csup\u003eth\u003c/sup\u003e \u0026ndash; 99\u003csup\u003eth\u003c/sup\u003e centile, we may still miss to detect common aneuploidies despite the low-risk from combined FTS that holds 92% accuracy rate. Hence, we can consider cfDNA testing in these fetuses that labelled low-risk on FTS.\u0026nbsp;Meanwhile, for FTS-identified high-risk pregnancies, Srebniak et al. reported a significant reduction in cfDNA diagnostic yield. In such circumstances, invasive testing seemed preferable\u003csup\u003e25\u003c/sup\u003e.\u0026nbsp;As we demonstrated that the cfDNA test can accurately predict common aneuploidy risk, we recommend offering cfNDA to women who decline invasive testing. \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eKagan et al. reported that a contingent screening strategy combining a serial detailed ultrasound scan, NT measurement, and cfDNA significantly reduces false positives\u0026nbsp;rate compared to FTS alone\u003csup\u003e29\u003c/sup\u003e. Although cfDNA appears to be a better test alternative, many studies have shown that a small percentage of chromosome abnormalities may be missed\u003csup\u003e30,31\u003c/sup\u003e. The undetectable aneuploidies may associated with varying degrees of disability. In the two abnormal cytogenetic cases (oculocutaneous albinism\u0026nbsp;and 2p15p16.1 microdeletion syndrome), both FTS risk were low, and the couples opted for invasive testing next. Hence, if theoretically only cfDNA was performed for isolated increased NT with low-risk FTS result, common aneuploidies are ruled out. As we would have suggested a follow-up with an early anomaly scan, potential structural abnormalities associated with the microdeletion syndrome could still be detected in time for a termination decision, if abnormal, within the realms of the abortion law.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eBased on our data, if only cfDNA is offered to these women, there is a potential 1.0% (1 in 98 fetuses) miss of non-autosomal chromosomal aberrations or a rare genetic syndrome. Our findings are also corroborated by Sagi-Dain et al., who shared a likewise missing rate of 1.5% (1 in 69 fetuses) to 1.9% (1 in 52 fetuses) in genome-wide NIPT and five-chromosomal NIPT, respectively\u003csup\u003e32\u003c/sup\u003e. Whereas, Petersen et al. reported a substantiallyhigher residual risk of microscopic chromosomal aberration at 3.0% \u0026ndash; 4.8% (1 in 21 \u0026ndash; 33 fetuses), which could be attributed to the inclusion of isolated and non-isolated cases\u003csup\u003e24\u003c/sup\u003e.\u0026nbsp;Despite the low percentage, patients should be informed before opting for cfDNA testing because some microdeletions may be missed. Henceforth, offering only cfDNA in this cohort of patients remains a discussion and can be considered individually. Notably, an abnormal NIPT required\u0026nbsp;a further confirmatory diagnostic test, which the wait for a definitive result increased the mother\u0026apos;s anixety\u003csup\u003e24\u003c/sup\u003e. It is also worthwhile to consider the difficulty of resampling and longer turnaround time, especially for those staying in the peripheral area, if the result is \u0026quot;no-call\u0026quot; or discordant due to low fetal fraction\u003csup\u003e33\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eThe strength of this study includes a stringent study criterion, which considered only isolated increased NT between the 95\u003csup\u003eth\u003c/sup\u003e \u0026ndash; 99\u003csup\u003eth\u003c/sup\u003e centile. With normal biomarker profiles and ultrasonography results, cfDNA predictability on aneuploidy risk can be tested without confounders. To our knowledge, our study is one of the pioneers \u0026nbsp;that examines cfDNA utilizationin a cohort with isolated increased NT between the 95\u003csup\u003eth\u003c/sup\u003e \u0026ndash; 99\u003csup\u003eth\u003c/sup\u003e centile.\u0026nbsp;Further study with a large sample size across various countries and provinces is needed to reaffirm our outcome. Nevertheless, this study can be meta-analyzed.\u0026nbsp;Another study\u0026apos;s merits include adopting a tight protocol for FMF-certified ultrasonography and biochemistry analyses according to international standards.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe results should be interpreted cautiously due to several study limitations. Despite the relatively large sample size, the study was commenced at a single centre in North India, which fairly limited the generalizability of the study findings to the rest of the country. A well-resourced specialized tertiary centre may have had selection and referral bias, therefore the study outcomes may not apply to other hospitals and regions\u003csup\u003e34\u003c/sup\u003e. Midway through the study, we have replaced karyotyping with CMA in year 2015 and routinely offered CMA as primary test since then. The increased resolution of CMA detects chromosomal abnormalities better than conventional karyotyping\u003csup\u003e35\u003c/sup\u003e. Thus, karyotyping may overlook certain chromosomal abnormalities in the first half of our study.\u003c/p\u003e"},{"header":"5. Conclusions","content":"\u003cp\u003eThis study shows the incidence rate of chromosomal abnormalities in the North Indians with isolated increased NT between the 95\u003csup\u003eth\u003c/sup\u003e and 99\u003csup\u003eth\u003c/sup\u003e centiles. Our study reported that these 2.5% chromosomal aneuploidies could also be potentially detected with cfDNA. Therefore, cfDNA may be a reasonable alternative to invasive diagnostics for common aneuploidies in this clinical group. Nevertheless, albeit small, cfDNA may still miss 1% of cases of microdeletions or rare genetic syndromes.\u003c/p\u003e\n"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor Contributions:\u003c/strong\u003e Conceptualization, N.S. and A.K.; methodology, A.K.; validation, N.S., A.K., R.G., and A.P.; formal analysis, N.S. and A.K.; investigation, N.S.; resources, N.S.; data curation, N.S.; writing\u0026mdash;original draft preparation, N.S.; writing\u0026mdash;review and editing, N.S., R.G., and A.P.; supervision, A.K.abb; project administration, N.S., and A.K.. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e This research received no external funding.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInstitutional Review Board Statement:\u0026nbsp;\u003c/strong\u003eThe study was conducted in accordance with the Declaration of Helsinki, and approved by the Institutional Ethics Committee of Indraprastha Apollo Hospitals (protocol code IAH-BMR-057/11-21 and date of approval 2\u003csup\u003end\u003c/sup\u003e March 2022).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement:\u003c/strong\u003e The data from this study are available on request from the corresponding author. The data are not publicly available due to patient confidentiality and are used under license for the current study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of Interest:\u003c/strong\u003e The authors declare no conflict of interest.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eHassold, T. J. \u0026amp; Jacobs, P. A. Trisomy in Man. Annual Review of Genetics 18, 69\u0026ndash;97, doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1146/annurev.ge.18.120184.000441\u003c/span\u003e\u003cspan address=\"10.1146/annurev.ge.18.120184.000441\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (1984).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHassold, T. \u003cem\u003eet al.\u003c/em\u003e A cytogenetic study of 1000 spontaneous abortions. 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This study aimed to determine whether cell-free DNA (cfDNA) could suffice in patients with an isolated increased NT between the 95th \u0026ndash; 99th centile. A retrospectively analyzed prospective study was conducted in the Fetal Medicine Unit, Indraprastha Apollo Hospitals, New Delhi, between 2007 and 2021. Study criteria were singletons with increased NT between 95th \u0026ndash; 99th centile without soft markers or structural abnormalities. cfDNA/follow-up was offered if patients declined invasive tests. Karyotyping was the primary genetic testing from 2007 to 2015, while microarray\u0026thinsp;\u0026plusmn;\u0026thinsp;exome began thereafter. All outcomes were obtained over the phone or through patient records. There were 213 pregnancies with isolated findings of NT 95th \u0026ndash; 99th centile. All were offered invasive testing, and 17 were lost to follow-up. In 196 cases with outcomes available, 7 (3.5%) had an abnormal genetic diagnosis. Four fetuses had trisomy 21, one trisomy 18, and two fetuses (1%) had other cytogenetic abnormalities. Five fetuses (2.5%) with chromosomal aneuploidy could be potentially detected with cfDNA. cfDNA may be a viable substitution to detect common aneuploidies in this clinical population. However, in 1% of cases, microdeletions may still be missed.\u003c/p\u003e","manuscriptTitle":"Can Cell-Free DNA Testing Suffice for Isolated Fetal Nuchal Translucency Between the 95th – 99th Centile? A Retrospective Analysis in A Centre in North India","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-04-05 21:00:59","doi":"10.21203/rs.3.rs-4150794/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"9129786a-111e-4ece-bcbd-ff4cbd451364","owner":[],"postedDate":"April 5th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":30193148,"name":"Health sciences/Health care/Medical imaging/Ultrasonography"},{"id":30193149,"name":"Health sciences/Health care/Diagnosis"},{"id":30193150,"name":"Health sciences/Risk factors"},{"id":30193151,"name":"Health sciences/Health care/Paediatrics/Intrauterine growth"}],"tags":[],"updatedAt":"2024-07-16T05:10:58+00:00","versionOfRecord":[],"versionCreatedAt":"2024-04-05 21:00:59","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4150794","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4150794","identity":"rs-4150794","version":["v1"]},"buildId":"pf3fE39SIOqb-0xH_OWvX","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}