The ratio of nasal bone length to prenasal thickness: a novel approach for prenatal ultrasound screening of Down syndrome in the second trimester

Objectives To evaluate the feasibility o f incorporating two-dim ensional ultrasound m easurem ents of nasal bone length (NBL) and prenasal thickness (PT) into the second-trim ester anom aly scan and to determ ine w hether the N BL: PT ratio could help in differentiating euploid and Down syndrome fetuses. Method Two-dim ensional m easurem ents o f NBL and PT were obtained from the midsagittal plane of the fetal head at 14-28 weeks of gestation in a Caucasian population at risk for aneuploidy. The screening perform ances o f NBL, PT, and the ratios N BL : PT and P T : NBL were analyzed in euploid (n = 1330) and Down syndrome (n = 33) fetuses. Results Nasal bone length and PT alone showed strong correlations with Down syndrome (sensitivity: 76% at 1.88% and 2.35% false positive rate, respectively). However, the N BL :P T ratio showed an even stronger correlation with Down syndrome (false positive rate: 0.9%, sensitivity: 97%). The m ean N BL : PT ratio showed a gradual increase from 1.48 to 1.79 (a 21.2% increase) betw een 14 and 28 weeks of gestation. Conclusion Two-dim ensional ultrasound m easurem ents of NBL and PT, particularly the N BL :P T ratio, are highly sensitive markers for Down syndrome fetuses. © 2014 John Wiley & Sons, Ltd. Funding sources: N one Conflicts of interest: N one declared INTRODUCTION Differences in nasal bone length (NBL), determined by ultrasound, have been suggested to differentiate second-trimester euploid and Down syndrome fetuses.1-6 From analyses of the facial profile, a thickening of the prenasal soft tissue [prenasal thickness (PT)] is also apparent in the vast majority of secondtrimester fetuses with Down syndrome. There is evidence that the combination of NBL and PT measurements as a ratio improves the detection of fetal Down syndrome by ultrasound.6-10 Despite the multitude of ultrasound soft markers for Down syndrome fetuses such as increased nuchal fold thickness, cystic hygroma, cardiac anomalies, echogenic intracardiac foci, nasal bone hypoplasia, ventriculomegaly, widened iliac crest angle, short femur/humerus PT, duodenal atresia, echogenic bowel, pyelectasis-hydronephrosis, sandal gap sign, choroid plexus cyst, and midphalanx hypoplasia of the fifth finger (Appendix 1) there are no very sensitive ultrasound markers in the second trimester that can be used either alone or in combination.11-13 Furthermore, these markers may not be present in all affected fetuses, and such as all soft markers, they can also be detected in euploid cases.11 Preliminary observations using 2D ultrasound measurements of NBL and PT at our tertiary referral center suggested the potential for the second-trimester identification of euploid and Down syndrome fetuses in a mixed-risk population.14 Therefore, we proposed that both markers and their ratios should be incorporated into the second-trimester fetal anomaly scan for a reliable, cheap, and efficient screening of Down syndrome. The current prospective study examined the clinical value of 2D ultrasound measurements of NBL, PT, and their ratios for differentiating euploid and Down syndrome fetuses in the second trimester (in an at-risk population). METHODS Women were referred for genetic counseling and secondtrimester anomaly scans to our regional prenatal genetics center because of advanced maternal age (>35 years); positive screening results; intermediate risk of combined, triple, or integrated tests; and the presence of one or more aneuploidy soft markers in previous ultrasound examinations. W omen were recruited for second-trimester assessment and measurem ent of the NBL and PT values between January 2008 and April 2013. Informed consent was obtained from the mothers before examination at the MEDISONO Fetal and Adult Health Research Center or at the Department of Obstetrics and Gynecology in Szeged, Hungary. The following criteria determined enrollment into the euploid group: singleton viable pregnancy, 14-28 weeks of gestation, a lack of maternal disease (such as hypertension, toxemia, renal disease, and diabetes mellitus), norm al fetal growth, norm al amniotic fluid volume, diagnosis of a norm al fetal anatomy, and newborns without chromosomal or structural abnormalities between the fifth and 95th percentile birth weight at delivery. The study included 1330 euploid and 33 Down syndrome fetuses. The protocol was approved by the ethics committee of the University of Szeged. A routine second-trimester anomaly scan in weeks 18-23 and a detailed examination of the fetal anatomy within 14-17 and 23-28 weeks of gestation were performed using a high-resolution 2D transabdominal ultrasound scanner (Voluson E8 Expert, GE Healthcare, Milwaukee, WI, USA). The facial profile was assessed at the beginning of the scanning sessions to avoid effects of fetal movements that could alter the fetal position. Three image acquisitions were obtained during one scan session, and the best one was used for analysis. I f it was not successful, then the patient came back for another scanning session 30-40 min later. The sonographer was blind to the fetal karyotype, and each ultrasound examination was performed before the chromosomal study. Nasal bone length1,10 and PT9 m easurem ents can be obtained on the sam e image if the face of the transducer was positioned parallel to the nasal bone. The insonation angle should be close to 45°. The following im age settings were used: low gain, m edium dynam ic contrast, and m axim um m agnification so that the fetal h ea d occup ied the entire screen. Im ages were ad justed to ensure correct m idsagittal plane and sharp m argins o f the skin and the nasal bone. The diencephalon, nasal bone, lips, maxilla, and m andible were used as reference points for the correct m easurem ents of NBL and PT in the m idsagittal p lane.3,9 Briefly, PT was m easured as the shortest d istance from the low er m argin of the frontal bone to the outer surface of the overlying skin. The margins o f the nasal bone are the proxim al and the distal ends o f the white ossification line. The NBL and PT were m easured using the sam e view (Figure 1A and B). M aternal data and sonographic findings were recorded in a database (Astraia Software GmbH, M unich, Germany). The ultrasound imaging data were stored in the local Digital Imaging and Communications in Medicine (DICOM) format via Astraia. Values of NBL and PT were exported to Microsoft Excel (Microsoft Corp., Redmond, WA, USA). Statistical analyses were performed using SigmaPlot (Systat Software Inc., San Jose, CA, USA). Scatter plots of NBL and PT with linear polynomial regression lines and percentile curves (third and 97th) were created. Similarly, scatter plots o f N BL:PT and P T:N BL ratios with linear polynomial regression lines and percentile curves (fifth and 95th) were produced. Comparisons between euploid and Down syndrome m easurem ents for NBL, PT [in millimeters (mm) and in multiple o f medians (MoMs)], and their ratios (N BL:PT and PT:N BL) were performed using the M ann­ W hitney U independent samples test. NBL, PT, and P T : NB and N B :P T ratio correlations were analyzed. No analysis of correlation was performed between any other markers. RESULTS The total num ber of the screened patients was 1470. The mean maternal age in euploid and Down syndrome cases was 30.6 years (16.6-47.1 years) and 31 .5years (21.1-42.3years). The mean gestational age was 19.6 weeks (14.0-28.9 weeks) for euploid and 20.3 weeks (15.0-25.6 weeks) for Down syndrom e cases. Those excluded were the following: fetal structural abnormalities (24), multiple pregnancy (35), maternal conditions listed in the m ethod (41), and chromosomal abnormalities, such as Turner syndrome (n = 1), trisomy 18 (n = 4), and trisomy 13 (n = 2) (Appendix 2). Ultrasound markers found in the Down syndrome group were increased nuchal fold thickness (n =10), cystic hygroma (n = 2), cardiac defects (n = 9), echogenic intracardiac focus (n = 4), mild ventriculomegaly (n = 4), short femur (n = 3), duodenal atresia (n = 1), hyperechogenicbowel (n = 3), pyelectasis-hydronephrosis (n = 3), choroid plexus cyst (n = 4), sandal gap sign (n = 3), and midphalanx hypoplasia of the fifth finger (n = 4) (Appendix 3). All invasive tests were amniocenteses, either because maternal age (>35 years) (18 cases), a positive combined test (> 1 :2 5 0 ) (12 cases), and second-trimester ultrasound soft markers (three cases). The three consecutive NBL and PT m easurem ents lasted 3 to 6 min and were com pleted during the first, the second, and the third attempts in 77%, 19%, and 4% o f the cases, respectively. There was a statistically significant difference (p < 0.0001) in the N BL : PT ratio betw een the euploid and Down syndrome groups. Both the NBL and PT alone were found to be strong markers (sensitivity of 76% for both markers) for Down syndrome (Figure 2A and B). A linear increase was observed in the m ean NBL, the m ean PT, and the m ean N BL: PT ratio according to increasing gestational age between the 14th and 28th weeks. The m ean N BL: PT ratio showed a gradual increase from 1.48 to 1.79 between the 14th and 28th weeks of gestation (a 21.2% increase) (Table 1). A total of 14 out of the 1330 euploid pregnancies and 32 out of the 33 Down syndrome cases were under the fifth percentile, with 97% sensitivity, 0.9% false positive rate, and 99% specificity (Table 2.) Figure 1 Examples of nasal bone length and prenasal thickness measurements obtained in euploid (A) and Down syndrome (B) fetuses Evaluating the perform ance of the ratios, there were 32 true positive and one false negative Down syndrome cases identified. However, using the N BL: PT ratio, the false positive rate was 50% o f those using the P T : NBL ratio (Figure 3A and B). The positive and negative cases with the calculated sensitivity, specificity, and false negative rate, using NBL, PT, the NBL : PT ratio, and the PT : NBL ratio for screening Down syndrome are summarized in Table 2. No correlation has been found between PT and NBL with Spearman Rank Order Correlation test (SROC = 0.830 at p < 0.05) supporting that both markers are independent variables. The PT (PT mean: 2.0-5.8 mm) has lower values than the NBL (NBL mean: 3 .0-10.0 mm), and PT (axPT average = 1.066) and NBL (axNBL average = 1.084) elevation are also different during the second trimester. Their ratios have different reference ranges because o f the inverted counterparts, and the reference range of the N BL: PT ratio is wider than that o f the P T : NBL ratio. DISCUSSION This 2D ultrasound study demonstrates that NBL and PT measurem ents can easily be incorporated into routine secondtrimester anatomy scans. We confirmed in a potentially high risk Caucasian population that both NBL and PT alone are strong markers of Down syndrome, with both having a sensitivity of 76%. The com bination o f these two markers as a ratio increased the detection rate to 97% with a 0.9% false positive rate. Furtherm ore, the N BL : PT ratio performs slightly better than its inverse counterpart. This is new that the N BL: PT ratio is a better m arker than the PT : NBL ratio for detecting Down syndrome fetuses, primarily because it produced less false positive cases, and it can be used in cases where the nasal bone is absent. Moreover, the N BL : PT ratio can easily be calculated during the scan. If the N BL :P T ratio is less than the fifth percentile, a search for other aneuploidy soft markers and invasive fetal karyotyping should be considered. In euploid fetuses, NBL, PT, and the N BL : PT ratio showed a linear increase with advancing gestational age. However, our data do not support previous observations7,15 that the ratio is constant throughout the second trim ester because the increase is m ore accelerated in NBL than in PT, and their ratio seem s to be dependent on the gestational age (Table 1).15 The correlation betw een nasal bone hypoplasia, absent nasal bone, and the correct m easurem ent of NBL in Down syndrome fetuses betw een 15 and 22 weeks of gestation was published in 2002.1-3 The im portance of increased PT in second-trim ester screening for Down syndrome was first reported by M aymon etal. in 2005, and this technique has a sensitivity of 70%.7 They com bined PT and NBL m easurem ents, yielding a 27% higher screening detection rate than NBL alone (43%). Three subsequent studies confirm ed the association.8,15,16 De Jong-P leije tal., in a retrospective study, first reported that the PT : NBL ratio is a strong marker for Down syndrome. In their analysis of 3D volumes of 106 euploid and 30 Down syndrome cases (20 cases on 3D volumes and ten cases on 2D volumes), the detection rate was 100% with a 5% false positive rate.16 G enetic sonography can substantially increase detection rates for com bined and quadruple tests, with a m ore m odest increase in sequential p rotocols.17 Com bining PT and biochem ical m arkers yields an 85% detection rate with a 5% false positive rate. W hen nuchal fold thickness is added to PT, NBL, and serum m arkers, the sensitivity increases to 93% .18 W hen PT, NBL, and their ratios, all in MoM s, are com bined with the lengths o f the second and third digits, a 76% detection rate is achieved w ith a 6.7% false positive rate using a 1-in-200 risk cutoff.19 The com bination o f quadruple tests with the m easurem ents of nuchal fold thickness and long bones can yield 90% sensitivity at a 3.1% false positive rate.20 Two-dimensional measurements of NBL21 and PT are feasible in the first trimester22; therefore, the markers examined in that study could also be beneficial for earlier Down syndrome detection. Using a m arker similar to PT (e.g., frontonasal fold thickness), one 2D study showed that the ratio o f frontonasal fold thickness to NBL in a Latin American low-risk population (1922 pregnancies with four cases of Down syndrome) can easily be obtained during the second-trim ester anatom y scan.15 This study presents novel evidence that the N BL: PT ratio is a better marker than the P T : NBL ratio for detecting Down syndrome fetuses. Our data indicate that the N BL: PT ratio is Figure 2 (A) Gestational-age-dependent nasal bone length values in 1330 euploid (black filled circles) and 33 Down syndrome (black open circles) fetuses. Approximately 76% of cases with Down syndrome fell under the third percentile. (B) Gestational-age-dependent prenasal thickness values in 1330 euploid (black filled circles) and 33 Down syndrome (black open circles) fetuses. 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