Secondary Logo

Journal Logo

Contents: Original Research

Risk of Clinically Significant Chromosomal Microarray Analysis Findings in Fetuses With Nuchal Translucency From 3.0 mm Through 3.4 mm

Sagi-Dain, Lena MD; Singer, Amihood MD; Ben Shachar, Shay MD; Josefsberg Ben Yehoshua, Sagi MD; Feingold-Zadok, Michal MD; Greenbaum, Lior MD, PhD; Maya, Idit MD

Author Information
doi: 10.1097/AOG.0000000000004195
  • Free
  • Journal Club

Numerous studies have demonstrated that increased nuchal translucency is associated with chromosomal disorders as well as submicroscopic copy number variants.1,2 However, the threshold of the nuchal translucency thickness above which invasive testing should be recommended is not uniformly defined, ranging from 2.5 mm to 3.5 mm (approximately the 99th percentile).3,4 In addition, there is evidence that abnormal nuchal translucency should be defined as above the 95th–99th percentile for gestational age and crown–rump length.5 A study that proclaims fetal nuchal translucency as a useful first-trimester marker for fetal chromosomal anomalies, published by Nicolaides et al6 in 1992, used a cutoff of 3.0 mm. An American College of Obstetricians and Gynecologists Practice Bulletin on screening for fetal aneuploidy published in 2007 recommended a threshold of 3.5 mm.7 However, in the updated guidelines published in 2020, it is noted that an enlarged nuchal translucency is defined as 3.0 mm or above the 99th percentile for the crown–rump length.8 Nevertheless, in a recent meta-analysis that examined the yield of chromosomal microarray analysis, of nine studies reporting the nuchal translucency cutoff, eight used the 3.5-mm threshold.2

The subgroup of fetuses with nuchal translucency from 3.0–3.4 mm might be associated with an increased risk for clinically significant chromosomal microarray analysis results. A recent prospective study of 211 pregnancies and meta-analysis of literature showed that, in the overall cohort of 522 fetuses with a nuchal translucency from 3.0–3.4 mm, the rate of abnormal chromosomal microarray analysis results was 13.5%.9 However, the main limitation noted by the investigators was inclusion of pregnant patients undergoing invasive testing owing to an abnormal first-trimester combined test result and not merely as a result of isolated increased nuchal translucency.

An additional important issue is the use of noninvasive prenatal testing (NIPT), which has rapidly expanded in recent years. Several studies have demonstrated that nuchal translucency measurement has limited added clinical value in the setting of NIPT,10,11 because the vast majority of fetuses with chromosomal abnormalities theoretically could be identified by NIPT, die in utero, or present with major ultrasonographic anomalies at the second-trimester anomaly scan. However, these studies did not perform uniform fetal chromosomal microarray analysis and mostly relied on a nuchal translucency threshold of 3.5 mm.

The primary objective of our study was to examine, in a large cohort, the risk for clinically significant chromosomal microarray analysis results in fetuses with isolated nuchal translucency from 3.0–3.4 mm. Our secondary aim was to define the yield of NIPT in such pregnancies.

METHODS

This retrospective cohort study included results of all chromosomal microarray analysis tests performed owing to a nuchal translucency from 3–3.4 mm without ultrasonographic anomalies, retrieved from the Israeli Ministry of Health computerized database (data entered by head of Community Genetics, Public Health Services). In Israel, nuchal translucency measurement is routinely performed at a gestational age of 11 0/7–13 6/7 weeks (fetal crown-rump length of 38–84 mm). There are no binding guidelines for measurement of ductus venosus or tricuspid valve Doppler, or for evaluation of the presence or size of the nasal bone. A nuchal translucency measurement of 3.0 mm or more constitutes an indication for genetic counseling and invasive testing by chromosomal microarray analysis. All invasive procedures performed owing to abnormal ultrasonographic findings are nationally funded and documented by the Ministry of Health. The data included type of invasive testing (chorionic villous sampling [CVS] vs amniocentesis, the ultrasonographic anomaly), maternal age, and chromosomal microarray analysis results (genomic coordinates of the detected copy number variants and the classification by the performing laboratory).

The study was approved by the Institutional Review Board Committee (Helsinki committee) for Human Subjects (date of issue—September 6, 2016, registration number—MOH2016). All chromosomal microarray analyses performed owing to isolated nuchal translucency from 3–3.4 mm between January 2013 and September 2018 were included.

Chromosomal microarray analysis testing in Israel is performed in 12 laboratories. The most widely used platform is CytoScan 750K array; a minority of centers use Infinium OmniExpress-24 v1.2 BeadChip and GenetiSure Unrestricted CGH+SNP (4x180K) P/N G5976A Agilent.

The interpretation of the copy number variants is performed according to the American College of Medical Genetics' standards and guidelines for the interpretation and reporting of postnatal constitutional copy number variants.12,13 Each copy number variant is classified as benign, pathogenic, or variant of unknown significance, and the latter group is further subclassified as likely benign, NOS (not otherwise specified), or likely pathogenic. Using the American College of Medical Genetics' metric, copy number variants that scored 0.99 points or higher are defined as “pathogenic,” and from 0.90 through 0.98 points are defined as “likely pathogenic.”13 Several parameters significantly affect the pathogenicity score; for instance, partial or whole inclusion of more than 35 protein-coding genes in the copy-number loss imparts the variant a score of 0.9. Most cytogenetically visible alterations (more than 3–5 Mb) are usually defined as pathogenic.14 Each copy number variant classification was reviewed by one author (I.M.) and reclassified, as needed, based on the accumulated experience and updated evidence in literature and public databases, including ClinGen (https://www.clinicalgenome.org/), DECIPHER (http://decipher.sanger.ac.uk/), Database of Genomic Variants (http://dgv.tcag.ca/dgv/app/home), ClinVar database (https://www.ncbi.nlm.nih.gov/clinvar/), and Genome Aggregation Database (http://gnomad.broadinstitute.org/).

After database and literature review, chromosomal microarray analysis results were classified into one of three groups:

  • 1. Clinically significant—defined as pathogenic and likely pathogenic chromosomal microarray analysis results.
  • 2. Variants of unknown significance. This category included findings of unclear significance as well as copy number variants with clinical penetrance lower than 10% (such as duplications at 15q13.3, 16p11.2 and 16p11.13 loci).15,16
  • 3. Normal results—including no copy number variants, benign, and likely benign CNVs, as well as NOS findings below the report threshold of 1 Mb for deletions and 2 Mb for duplications, in accordance with Israeli Society of Medical Genetics guidelines.

Clinically significant results were further subclassified into:

  • 1. Submicroscopic findings (detectable only by chromosomal microarray analysis and not by karyotype)—copy number variants sized less than 10 Mb.
  • 2. Noninvasive prenatal testing-detectable—trisomies 13, 18, 21, and sex chromosome aneuploidies.
  • 3. Karyotype-detectable findings (or genome-wide NIPT-detectable)—aberrations equal to or larger than 10 Mb.

The relative risk (RR) for clinically significant chromosomal microarray analysis results in fetuses with increased nuchal translucency was calculated and compared with a historical local control population of 2,752 women with low-risk pregnancies (ie, younger than 35 years, with normal ultrasound findings and nuchal translucency of less than 3.0 mm) undergoing chromosomal microarray analysis at maternal request.11 In this group, chromosomal microarray analysis testing yielded 21 clinically significant results (0.76%), four of these NIPT-detectable (47, XXY; 45, X mosaicism; trisomy 21 and trisomy 13 mosaicism). Of note, the final interpretation of chromosomal microarray analysis findings in the current study and the control cohort13 was done by the same author (I.M.).

Continuous variables are presented as mean±SDs and compared by t test. Categorical data were presented as numbers and proportions (absolute risks) with 95% CIs. The effect estimates were calculated as RR with 95% CI. P<.05 (using chi-square with Yates' correction) was considered statistically significant.

RESULTS

Between January 2013 and September 2018, 619 pregnant women with fetuses with isolated nuchal translucency of from 3.0–3.4 mm were referred for invasive chromosomal microarray analysis testing. Mean (±SD) maternal age was 31.6±4.9 years, not significantly different from the control population (31.4±2.1 years). The average timing of invasive testing was 17.1±3.8 weeks of gestation. In 179 of the cases (32.2%), the testing was performed by CVS with an average timing of 12.8±0.8 weeks of gestation; amniocentesis was done in 440 cases (67.8%), at 18.9±3.0 weeks of gestation.

Overall, clinically significant chromosomal microarray analysis results were found in 29 (4.67%) of the fetuses (Appendix 1, available online at https://links.lww.com/AOG/C136). This rate was significantly higher compared with the 0.76% in the control cohort (RR 3.3, 95% CI 2.6–4.2). Except for 3.0 mm, for each millimeter increment in the range, the risk of clinically significant chromosomal microarray analysis results was significantly increased (Table 1).

T1
Table 1.:
Clinically Significant Copy Number Variants in 619 Fetuses With Nuchal Translucency Measurements From 3 mm Through 3.4 mm

The risk for abnormal chromosomal microarray analysis findings was twice as high in pregnant women in whom CVS was performed (13/179, 7.3%) compared with those in whom amniocentesis was performed (16/440, 3.6%), but this difference was not statistically significant (P=.06). In both groups, the risk was significantly increased compared with the control cohort (RR 6.7, 95% CI 4.2–10.5 for CVS; RR 3.2, 95% CI 2.2–4.7 for amniocentesis).

Of the 29 fetuses with abnormal chromosomal microarray analysis findings, trisomy 21 was diagnosed in 12 cases (one in a mosaic form) and trisomy 18 in three cases (one mosaic). Thus, common autosomal trisomies involved 51.7% of the fetuses. In addition, two fetuses with sex chromosome aneuploidies were detected (47, XXX and 47, XXY). Thus, NIPT aimed at the five common aneuploidies could have theoretically detected 17 cases (58.6%).

Of the remaining 12 fetuses with findings undetectable by NIPT (41.4%), three could have been diagnosed by fetal karyotyping or genome-wide NIPT: trisomy 2 mosaicism, 7p22.3q36.3 mosaic duplication sized 159 Mb, and a 13q21.33q31.1 deletion sized 18.4 Mb. Nine cases of submicroscopic copy number variants only could have been detected by chromosomal microarray analysis. These included 1.7 Mb 1q21.1q21.2 duplication, a case with two adjacent duplications at 4p, an Xq21.21-q21.33 duplication sized 8.9 Mb, a recurrent 8p23.1 deletion sized 3.8 Mb, three recurrent copy number variants at 16p11.2 locus (one deletion and two duplications), and two recurrent copy number variants at 22q11.2 locus (one deletion and one duplication). Thus, the NIPT aimed for the five common chromosome aneuploidies would have missed clinically significant copy number variants in 12 of the 619 (1.9%) fetuses in the entire cohort, whereas genome-wide NIPT could have missed clinically significant copy number variants in 9 of 619 (1.5%) fetuses.

In 440 pregnant women who underwent amniocentesis, 10 of the 16 clinically significant chromosomal microarray analysis findings could have been detected by NIPT aimed at the three common autosomal trisomies and sex chromosome aberrations; 6 of 440 (1.4%) were NIPT-undetectable (1/73 of the entire cohort). Genome-wide NIPT, as well as traditional karyotyping, would have missed 5 of 16 (31.3%) abnormal findings in the amniocentesis group, constituting 1.1% of the entire cohort (Table 2).

T2
Table 2.:
Characteristics of Clinically Significant Microarray Results According to Type of Invasive Prenatal Testing

In 179 pregnant women who underwent CVS, 7 of 13 (53.8%) clinically significant chromosomal microarray analysis findings were detectable by NIPT aimed at five common aneuploidies. Thus, 6 of 179 cases (1/30, 3.4%) would be missed by this test. Genome-wide NIPT could have missed 4 of the 13 abnormal findings (30.7%), or 2.2% (1/45) in the overall CVS subgroup (Table 2).

Finally, five (0.8%) NOS and three (0.5%) low-penetrant copy number variants were demonstrated (Appendix 2, available online at https://links.lww.com/AOG/C136).

DISCUSSION

According to our results, fetuses with isolated nuchal translucency measurements from 3.0–3.4 mm are associated with 4.7% rate of clinically significant copy number variants, a significantly increased risk compared with the control group with normal ultrasound findings and similar maternal age. These findings roughly agree with those in previous studies—Maya and collaborators found pathogenic copy number variants in 11 (6.5%) of 170 fetuses with isolated nuchal translucency from 3.0–3.4 mm,17 and Zhao et al18 identified 18 (16.4%) abnormal findings in 110 fetuses with nuchal translucency of similar thickness (the latter also including additional ultrasonographic anomalies). Although many previous studies have used a cutoff of 3.5 mm to define increased nuchal translucency,2 we present additional evidence that nuchal translucency from 3.0–3.4 mm increases the risk for clinically significant chromosomal microarray analysis findings by 3.3, supporting the Israeli national policy to perform invasive testing at nuchal translucency of 3.0 mm and above. It must be noted that in the subgroup of 198 fetuses with a nuchal translucency measurement of 3.0 mm, the risk for abnormal chromosomal microarray analysis results was not significantly increased, which suggests that the cutoff should be placed at 3.1 mm.

It was interesting to note that NIPT would have missed more than 40% of fetuses with clinically significant copy number variants (1.94%, or 1 in 52 fetuses with nuchal translucency from 3.0–3.4 mm). Even genome-wide NIPT could have missed 1 in 69 fetuses (1.5%) with abnormal findings. These rates are lower compared with the meta-analysis by Petersen et al,9 which reports a 2.8% missing out rate for five-chromosomal NIPT and 0.2% for genome-wide NIPT. These differences could be explained by inclusion of fetuses with a lower risk for macroscopic chromosomal aberrations in our study, that is, only those referred for invasive testing owing to isolated nuchal translucency from 3.0–3.4 mm, in which the majority (67.8%) also had normal findings on the routine 14–16-week anatomical ultrasound screening, whereas the meta-analysis included isolated as well as nonisolated cases.

Previous studies have suggested that NIPT combined with advanced ultrasonographic survey could fairly replace nuchal translucency measurement.10,11,19 However, these studies had several drawbacks, undermining the stated conclusions. The percentage of aneuploidy ranged from 21.5% to 74%, suggesting varying proportions of fetuses with additional ultrasonographic findings. The cutoff to define abnormal nuchal translucency also differed (using 3.0 or 3.5 mm), and no comparison with fetuses with normal ultrasound findings was done. Most importantly, chromosomal microarray analysis was not undertaken for all of the fetuses in the above-mentioned studies: some used fetal karyotyping only,10 whereas the others used chromosomal microarray analysis in some but not all cases.11,19 Thus, it is possible that in cases with normal karyotype, defined as “normal liveborns,” submicroscopic chromosomal microarray analysis anomalies could have been missed.

Thus, previous studies do not offer clear evidence that NIPT with subsequent ultrasonographic survey can adequately replace nuchal translucency measurement with chromosomal microarray analysis in cases with abnormal findings. Indeed, CVS and amniocentesis are associated with increased risk for miscarriage; however, according to recent studies, this risk is estimated to be negligible,20,21 especially in a population at an increased risk for trisomy 21.22

Our study has its own limitations. The main drawback is lack of data regarding further pregnancy follow-up, such as second-trimester anatomical survey or a possible subsequent fetal death. We had no data regarding the presence or size of the nasal bone at the time of nuchal translucency measurement, or Doppler of ductus venosus or tricuspid valve. In addition, we had no information about postnatal evaluation and neurocognitive development. Furthermore, we did not examine the value of next-generation sequencing methods, such as whole exome sequencing or a genetic panel for potentially relevant conditions (such as Noonan syndrome). As exome testing can detect up to 3% of additional genetic anomalies,23 the rate of fetuses with molecular diagnosis might be even higher than the demonstrated 4.7%. In addition, the clinical relevance of several copy number variants finding to increased nuchal translucency is not trivial, as some detected chromosomal microarray analysis aberrations (such as 16p11.2 copy number variants or 22q11.2 duplication) are mainly associated with neurocognitive disorders with moderate-to-high penetrance and normal ultrasonographic survey.24,25

Nevertheless, we describe a large cohort of fetuses with isolated nuchal translucency from 3–3.4 mm undergoing uniform chromosomal microarray analysis. Our results support 3.1 mm as the cutoff for abnormal nuchal translucency. In addition, because more than 40% of the fetuses with clinically significant chromosomal microarray analysis results would be missed by NIPT, it seems that cell-free DNA testing may not adequately replace invasive testing for chromosomal microarray analysis in such cases.

REFERENCES

1. Snijders RJ, Noble P, Sebire N, Souka A, Nicolaides KH. UK multicentre project on assessment of risk of trisomy 21 by maternal age and fetal nuchal-translucency thickness at 10-14 weeks of gestation. Fetal Medicine Foundation First Trimester Screening Group. Lancet 1998;352:343–6. doi: 10.1016/s0140-6736(97)11280-6
2. Grande M, Jansen FA, Blumenfeld YJ, Fisher A, Odibo AO, Haak MC, et al. Genomic microarray in fetuses with increased nuchal translucency and normal karyotype: a systematic review and meta-analysis. Ultrasound Obstet Gynecol 2015;46:650–8. doi: 10.1002/uog.14880
3. Oloyede OA, Abbey M, Oloyede AA, Nwachukwu O. Fetal nuchal translucency scan in Nigeria. Pan Afr Med J 2014;18:62. doi: 10.11604/pamj.2014.18.62.3291
4. Egloff M, Herve B, Quibel T, Jaillard S, Le Bouar G, Uguen K, et al. Diagnostic yield of chromosomal microarray analysis in fetuses with isolated increased nuchal translucency: a French multicenter study. Ultrasound Obstet Gynecol 2018;52:715–21. doi: 10.1002/uog.18928
5. Kaul A, Radhakrishnan P. Performance of common Down syndrome screening methods used in India with construction of an Indian normogram for nuchal translucency/crown-rump length measurements in 14,337 subjects. J Obstet Gynaecol India 2019;69(suppl 2):142–6. doi: 10.1007/s13224-018-1196-3
6. Nicolaides KH, Azar G, Byrne D, Mansur C, Marks K. Fetal nuchal translucency: ultrasound screening for chromosomal defects in first trimester of pregnancy. BMJ 1992;304:867–9. doi: 10.1136/bmj.304.6831.867
7. Screening for fetal chromosomal abnormalities. ACOG Practice Bulletin No. 77 [withdrawn]. American College of Obstetricians and Gynecologists. Obstet Gynecol 2007;109:217–27. doi: 10.1097/00006250-200701000-00054
8. Screening for fetal chromosomal abnormalities. ACOG Practice Bulletin No. 226. American College of Obstetricians and Gynecologists. Obstet Gynecol 2020;136:e48–69. doi: 10.1097/AOG.0000000000004084
9. Petersen OB, Smith E, Van Opsta LD, Polak M, Knapen M, Diderich KEM, et al. Nuchal translucency of 3.0-3.4 mm an indication for NIPT or microarray? Cohorts analysis and literature review. Acta Obstet Gynecol Scand 2020;99:765–74. doi: 10.1111/aogs.13877
10. Holzer I, Husslein PW, Bettelheim D, Scheidl J, Kiss H, Farr A. Value of increased nuchal translucency in the era of noninvasive prenatal testing with cell-free DNA. Int J Gynaecol Obstet 2019;145:319–23. doi: 10.1002/ijgo.12808
11. Lichtenbelt KD, Diemel BD, Koster MP, Manten GT, Siljee J, Schuring-Blom GH, et al. Detection of fetal chromosomal anomalies: does nuchal translucency measurement have added value in the era of non-invasive prenatal testing? Prenat Diagn 2015;35:663–8. doi: 10.1002/pd.4589
12. South ST, Lee C, Lamb AN, Higgins AW, Kearney HM, Working Group for the American College of Medical Genetics and Genomics Laboratory Quality Assurance. ACMG standards and guidelines for constitutional cytogenomic microarray analysis, including postnatal and prenatal applications: revision 2013. Genet Med 2013;15:901–9. doi: 10.1038/gim.2013.129
13. Riggs ER, Andersen EF, Cherry AM, Kantarci S, Kearney H, Patel A, et al. Technical standards for the interpretation and reporting of constitutional copy-number variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics (ACMG) and the Clinical Genome Resource (ClinGen). Genet Med 2020;22:245–57. doi: 10.1038/s41436-019-0686-8
14. Kearney HM, Thorland EC, Brown KK, Quintero-Rivera F, South ST, Working Group of the American College of Medical Genetics and Genomics Laboratory Quality Assurance. American College of Medical Genetics standards and guidelines for interpretation and reporting of postnatal constitutional copy number variants. Genet Med 2011;13:680–5. doi: 10.1097/GIM.0b013e3182217a3a
15. Maya I, Sharony R, Yacobson S, Kahana S, Yeshaya J, Tenne T, et al. When genotype is not predictive of phenotype: implications for genetic counseling based on 21,594 chromosomal microarray analysis examinations. Genet Med 2018;20:128–31. doi: 10.1038/gim.2017.89
16. Rosenfeld JA, Coe BP, Eichler EE, Cuckle H, Shaffer LG. Estimates of penetrance for recurrent pathogenic copy-number variations. Genet Med 2013;15:478–81. doi: 10.1038/gim.2012.164
17. Maya I, Yacobson S, Kahana S, Yeshaya J, Tenne T, Agmon-Fishman I, et al. Cut-off value of nuchal translucency as indication for chromosomal microarray analysis. Ultrasound Obstet Gynecol 2017;50:332–5. doi: 10.1002/uog.17421
18. Zhao XR, Gao L, Wu Y, Wang YL. Application of chromosomal microarray in fetuses with increased nuchal translucency. J Matern Fetal Neonatal Med 2020;33:1749–54. doi: 10.1080/14767058.2019.1569622
19. Huang LY, Pan M, Han J, Zhen L, Yang X, Li DZ. What would be missed in the first trimester if nuchal translucency measurement is replaced by cell free DNA foetal aneuploidy screening? J Obstet Gynaecol 2018;38:498–501. doi: 10.1080/01443615.2017.1391755
20. Wulff CB, Gerds TA, Rode L, Ekelund CK, Petersen OB, Tabor A, et al. Risk of fetal loss associated with invasive testing following combined first-trimester screening for Down syndrome: a national cohort of 147,987 singleton pregnancies. Ultrasound Obstet Gynecol 2016;47:38–44. doi: 10.1002/uog.15820
21. Salomon LJ, Sotiriadis A, Wulff CB, Odibo A, Akolekar R. Risk of miscarriage following amniocentesis or chorionic villus sampling: systematic review of literature and updated meta-analysis. Ultrasound Obstet Gynecol 2019;54:442–51. doi: 10.1002/uog.20353
22. Malan V, Bussieres L, Winer N, Jais JP, Baptiste A, Le Lorc'h M, et al. Effect of cell-free DNA screening vs direct invasive diagnosis on miscarriage rates in women with pregnancies at high risk of trisomy 21: a randomized clinical trial. JAMA 2018;320:557–65. doi: 10.1001/jama.2018.9396
23. Lord J, McMullan DJ, Eberhardt RY, Rinck G, Hamilton SJ, Quinlan-Jones E, et al. Prenatal exome sequencing analysis in fetal structural anomalies detected by ultrasonography (PAGE): a cohort study. Lancet 2019;393:747–57. doi: 10.1016/S0140-6736(18)31940-8
24. Miller DT, Chung W, Nasir R, Shen Y, Steinman J, Wu BL, et al. 16p11.2 recurrent microdeletion. In: Adam MP, Ardinger HH, Pagon RA, editors. GeneReviews. Accessed December 10, 2015. https://www.ncbi.nlm.nih.gov/books/NBK11167/
25. Firth HV. 22q11.2 duplication. In: Adam MPAH, Pagon RA, editors. GeneReviews. Accessed November 21, 2013. Available at: https://www.ncbi.nlm.nih.gov/books/NBK3823/

Supplemental Digital Content

© 2020 by the American College of Obstetricians and Gynecologists. Published by Wolters Kluwer Health, Inc. All rights reserved.