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Factors influencing inconclusive sex chromosomal aneuploidies results on non-invasive prenatal testing
*Corresponding author: Priya Kadam, MedGenome Labs Ltd, Bengaluru, Karnataka, India. priya.k@medgenome.com
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Received: ,
Accepted: ,
How to cite this article: Kadam P, Devanboo A. Factors influencing inconclusive sex chromosomal aneuploidies results on non-invasive prenatal testing. Wadia J Women Child Health. 2025;4:106-9. doi: 10.25259/WJWCH_40_2025
Abstract
Non-invasive prenatal testing (NIPT) has been gaining wider acceptance as the preferred screening test for detecting common fetal chromosomal aneuploidies. In addition, NIPT can also screen for sex chromosomal aneuploidies (SCAs), namely Turner syndrome (45,X), Klinefelter syndrome (47,XXY), Triple X syndrome (47,XXX), and Jacobs syndrome (47,XYY). Recently, the American College of Medical Genetics and Genomics 2022 guidelines recommend that NIPT should be offered to screen for SCAs in pregnant women with singleton pregnancies. In our experience, in 99.8% of pregnant women, the NIPT gave a conclusive result i.e. either low-risk or high-risk for SCAs. However, in the remaining 0.2%, an inconclusive result was released as the test parameters did not fall within the expected low-risk or high-risk score. In such cases, detailed ultrasound and further diagnostic testing with appropriate genetic counselling are recommended. In 30 of 183 samples with an inconclusive result, follow-up information from a diagnostic test was obtained. In most cases, i.e., in 40% (12/30), the diagnostic results did not reveal any abnormal findings. About 30% (9/30) of the cases presented with a history of vanishing twin. In about 10% (3/30) of the cases, the fetus had sex chromosomal abnormality (Monosomy X, 20% Mosaic XXX and XXY). In addition, in about 10% (3/30) of cases, the maternal biological background was confounding the NIPT result for the fetus, i.e., the pregnant woman was identified to have an SCA (2 XXX and Mosaic MX). Adverse outcomes such as intrauterine fetal demise and spontaneous miscarriage were observed in 6.7% (2/30) of cases (the reason for the adverse outcome could not be ascertained) and pregnancy was continued without any further investigations in 3.3% (1/30) of the cases. In conclusion, this study highlights the importance of careful analysis of NIPT results when reporting SCAs. While NIPT offers superior sensitivity (99.6%) and specificity (99.8%) for screening SCAs, it is critical that pregnant women receive appropriate pre-test and post-test counseling to understand the clinical implications of inconclusive results, and the importance of further investigations to determine the fetus’s chromosomal status and in some cases of the mother.
Keywords
Biological factors
Inconclusive results
Non-invasive prenatal testing
Sex chromosome aneuploidies
INTRODUCTION
Sex chromosomal aneuploidies (SCAs) are a common group of conditions characterized by an abnormal number of sex chromosomes in individuals, namely Turner syndrome or Monosomy X (MX) (45,X), Klinefelter syndrome (47,XXY), Triple X syndrome (47,XXX), and Jacobs syndrome (47,XYY). SCAs can be associated with variable phenotypes, including normal or mildly affected phenotypes, possible infertility, atypical stature, heart defects, moderate reduction in IQ that may not result in intellectual disability, and other physical characteristics.[1] Turner syndrome occurs in about 1 in 2,000 females, Klinefelter syndrome in 1 in 660 males, Triple X syndrome in 1 in 1,000 females, and Jacob’s syndrome in 1 in 1,000 males.[1] Although common, most cases remain undiagnosed and are often identified in adulthood following a clinical evaluation for infertility or neurodevelopmental disorders.[1] In the prenatal period, except for Turner syndrome, these conditions are not always associated with clinical features or sonographic abnormalities.[1] Non-invasive prenatal testing (NIPT) has been established as the most sensitive screening test for common chromosomal abnormalities. NIPT, unlike the traditional serum markers, can detect sex chromosome abnormalities. The overall screening detection rate for any SCA was 99.6%, with specificity of 99.8%, with positive predictive values (PPVs) ranging from 29.5% (MX) to 74.5% (XYY), depending on the type of SCA. [2] In our laboratory 99.8% of the NIPT samples, the test provided a conclusive result for SCAs, i.e., 99.7% of low-risk cases and 0.1% of high-risk cases for SCAs. The PPVs for the SCAs observed were 41.7% (MX), 100% (XXY), 90% (XXX), and 75% (XYY). However, in the remaining 0.2%, the result was inconclusive as the parameters did not fall within the expected low-risk or high-risk score. In cases with an inconclusive result further diagnostic testing with appropriate genetic counselling along with comprehensive ultrasound was recommended and follow-up information was obtained. The objective of this study was to understand the reasons for the inconclusive results for SCAs on NIPT.
METHODS
Retrospective analysis was performed on samples processed in the laboratory from September 2019 to December 2024 (98,051 samples). All the samples were processed under Illumina VeriSeq v2 NIPT, which employs a counting-based genome-wide paired-end sequencing method. The follow-up information for inconclusive cases obtained by further diagnostic testing such as quantitative fluorescent polymerase chain reaction/fluorescence in situ hybridization/Karyotype/chromosomal microarray was analyzed.
RESULTS
NIPT could not provide a conclusive result for SCA in 183/98,051 (0.2%) samples. Follow-up information was obtained for 30/183 samples, and the outcomes are illustrated in Figure 1. Figure 2 depicts the various inconclusive results by this NIPT technology, Figure 2a represents the normal sex chromosome while Figure 2b and 2d illustrate examples of cases that did not fall in either low risk or high risk SCA category. Follow-up confirmatory testing is shown in figure 2c and 2e respectively. Samples with a history of vanishing twin (VT) underwent further genetic testing, and the results obtained did not reveal any abnormalities in the sex chromosomes of the live fetus. Two pregnancies resulted in intrauterine fetal demise and spontaneous miscarriage. However, information on whether testing was performed on the product of conception could not be obtained. One patient chose to continue the pregnancy without further testing, but information on the birth outcome is unavailable. Two patients preferred to have repeat NIPT because they did not want to undergo an invasive procedure such as amniocentesis. However, the results of the repeat NIPT also returned inconclusive. Prenatal genetic testing was then performed on both pregnancies, and the results did not reveal any abnormalities in the fetus.
- Outcomes of inconclusive sex chromosomal abnormalities results on Non-invasive prenatal testing and the type of fetal and maternal SCAs detected on further genetic testing. SCAs: Sex chromosomal aneuploidies, NIPT: Non-invasive prenatal testing.
- The different scenarios with inconclusive sex chromosomal aneuploidies result on non-invasive prenatal testing and corresponding diagnostic results. (a) The graph indicates the expected range of values to provide a conclusive low-risk or high-risk result. (b) The value falls in the MX range, but other parameters were not consistent with MX. The red arrow indicates deviation. (c) The chromosomal microarray of the fetus detected 20% Mosaic XXX. (d) The graph is skewed, the values didn’t fall in any expected range, and the test could not interpret the results. (e) The Quantitative fluorescent polymerase chain reaction results revealed maternal XXX and a normal fetus.
DISCUSSION
NIPT is widely accepted as the preferred screening test worldwide. The test has been commercially available since 2011, allowing patients to screen for SCAs in addition to Trisomy 21, 18, and 13. Screening for SCAs presents with challenges and raises ethical concerns, especially when interpreting the high-risk results on NIPT. The variable phenotypes associated with SCAs and the uncertain outcomes of mosaic SCAs create challenges for clinicians and prospective parents. Furthermore, the PPVs for screening SCAs are lower compared to common trisomies in particular for Monosomy X. Biological factors such as confined placental mosaicism and maternal mosaicism can cause false-positive SCA results on NIPT.[2] NIPT screening for SCA can potentially lead to detection previously undiagnosed cases of SCA in pregnant women [Figure 2d]. In this study, we highlight the importance of careful analysis of sex chromosomes when reporting results for SCAs on NIPT. In 40% of cases with normal genetic testing results, a biological factor could not be determined. This uncertainty might be due to technical aspects of the test, preventing a conclusive result. Factors such as Vanishing Twins (VTs) and maternal mosaicisms are known to cause false positive results on NIPT.[2,3] In our experience, we have observed that history of VTs (including anembryonic twin) and maternal SCA might impact the analysis of SCA result on NIPT.
CONCLUSION
This study suggests that screening for SCAs largely produces conclusive results, but in a small percentage of cases, inconclusive results may be obtained. Therefore, patients should receive appropriate pre-test genetic counseling before deciding to screen for SCAs along with common trisomies. Further investigations are recommended to determine the fetal sex chromosomal status following an inconclusive SCA result on NIPT, as there is a possibility of detecting fetal abnormalities.
Ethical approval:
Institutional Review Board approval is not required.
Declaration of patient consent:
The authors certify that they have obtained all appropriate patient consent forms. In the form, the patients have given their consent for clinical information to be reported in the journal. The patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed.
Conflicts of interest:
Priya Kadam and Angela Devanboo are from MedGenome Labs Ltd, Bengaluru, Karnataka, India.
Use of artificial intelligence (AI)-assisted technology for manuscript preparation:
The authors confirm that there was no use of artificial intelligence (AI)-assisted technology for assisting in the writing or editing of the manuscript and no images were manipulated using AI.
Financial support and sponsorship: Nil
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