False Positive Non-invasive Prenatal Testing Research Articles

False positive non-invasive prenatal testing (NIPT) for trisomy 21 in vanishing twin syndrome pregnancy: A comparison of the NIPT results performed in different gestations

False positive non-invasive prenatal testing (NIPT) for trisomy 21 in vanishing twin syndrome pregnancy: A comparison of the NIPT results performed in different gestations

Contribution of maternal mosaicism to false-positive chromosome X loss associated with noninvasive prenatal testing

Objective To report the frequency of maternal mosaicism contributing to false-positive chromosome X loss associated with noninvasive prenatal testing (NIPT) at a single center. Methods Pregnancies undergone NIPT using massively parallel sequencing at Guangzhou Women and Children’s Medical Center between February 2015 and May 2020 were included in this study. Fetal karyotyping, quantitative fluorescence PCR (QF-PCR) or microarray analysis was provided to patients with abnormal sex chromosomal aneuploidy (SCA) results for confirmatory testing, and QF-PCR was also employed to detect maternal sex chromosome status. Results cffDNA testing of 40682 pregnancies revealed 86 cases with NIPT results positive for chromosome X loss (0.21%). Among the 86 high-risk cases, 73 women had undergone confirmatory testing in our center, whereas 13 declined. Of the 73 women verified by invasive prenatal diagnosis, 27.4% (20/73) were true positive cases including six cases of monosomy X, two cases of microdeletion of Xp22.33, one case of deletion Xq27.2q28, one case of 47, XXX and ten cases with fetal sex chromosome mosaicism. Of the remaining 53 patients with fetal normal results, 30 cases had undergone QF-PCR analysis of maternal white blood cells. QF-PCR indicated that 36.7% (11/30) patients had an altered or mosaic maternal sex chromosome status. Statistical analysis indicated that cell-free fetal DNA (cffDNA) concentration estimated by chromosome X in maternal mosaic cases was significantly higher than that in the non-maternal mosaicism group (p < .05) and was related to maternal mosaicism rate (r = 0.88, p < .05). Conclusions Our findings indicated that maternal mosaicism of sex chromosome was not uncommon in false-positive NIPT chromosome X loss cases. We recommend that this information should be disclosed to pregnancies during clinical counseling and maternal sex chromosome status should be confirmed for the cases with NIPT chromosome X loss.

Genetic Counseling and Management: The First Study to Report NIPT Findings in a Romanian Population

Background and Objectives: Non-invasive prenatal testing (NIPT) has been confirmed as the most accurate screening test for trisomies 21, 18, 13, sex chromosomes aneuploidies and several microdeletions. This study aimed to assess the accuracy of cell free DNA testing based on low-level whole-genome sequencing to screen for these chromosomal abnormalities and to evaluate the clinical performance of NIPT. Materials and Methods: 380 consecutive cases from a single genetic center, from Western Romania were included in this retrospective study. Cell-free nucleic acid extraction from maternal blood, DNA sequencing and analysis of sequenced regions were performed by BGI Hong Kong and Invitae USA to determine the risk of specific fetal chromosomal abnormalities. In high-risk cases the results were checked by direct analysis of fetal cells obtained by invasive methods: 6 chorionic villus sampling and 10 amniocenteses followed by combinations of QF-PCR, karyotyping and aCGH. Results: NIPT results indicated low risk in 95.76% of cases and high risk in 4.23%. Seven aneuploidies and one microdeletion were confirmed, the other results were found to be a false-positive. A gestational age of up to 22 weeks had no influence on fetal fraction. There were no significant differences in fetal fraction across the high and low risk groups. Conclusions: This is the first study in Romania to report the NIPT results. The confirmation rate was higher for autosomal aneuploidies compared to sex chromosome aneuploidies and microdeletions. All cases at risk for trisomy 21 were confirmed. Only one large fetal microdeletion detected by NIPT has been confirmed. False positive NIPT results, not confirmed by invasive methods, led to the decision to continue the pregnancy. The main limitation of the study is the small number of patients included. NIPT can be used as a screening method for all pregnancies, but in high-risk cases, an invasive confirmation test was performed.

Impact of confined placental mosaicism on non-invasive prenatal testing and pregnancy outcomes

To assess the impact of confined placental mosaicism (CPM) on non-invasive prenatal testing (NIPT) and pregnancy outcomes. Copy number variation sequencing (CNV-seq) and single nucleotide polymorphism array (SNP-array) were carried out on placental specimen sampled from eight pregnancies with confirmed false-positive NIPT results. The impact of CPM on NIPT and pregnancy outcomes were analyzed based on the laboratory tests and clinical characteristics. Five of the eight cases with false-positive NIPT results were proven to be CPM involving trisomy 9, 13, 21, 22, and X, respectively. The mosaic ratios for different placental regions have varied from 4% to 80%. Two fetuses with confirmed CPM showed fetal growth restriction (FGR) and additional ultrasound abnormalities, 1 fetus showed only FGR. The remaining two fetuses showed normal growth. NIPT is highly sensitive to CPM, whilst CPM is an important cause for false-positive NIPT result. CPM may be associated with FGR. Investigation of the presence of CPM is important for both pre- and post-test genetic counseling and management of the pregnancy.

Temporal persistence of residual fetal cell-free DNA from a deceased cotwin after selective fetal reduction in dichorionic diamniotic twin pregnancies.

To determine the temporal persistence of the residual cell-free DNA (cfDNA) of the deceased cotwin in maternal circulation after selective fetal reduction and evaluate its long persistence in noninvasive prenatal testing (NIPT). Dichorionic diamniotic twins (N=5) undergoing selective fetal reduction because of a trisomy were recruited. After informed consent, maternal blood was collected immediately before reduction and periodically after reduction until birth. The plasma cfDNA of each sample was sequenced and analyzed for fetal aneuploidy and fetal fractions. In all pregnancies, the fetal fraction of the cfDNA of the deceased fetus increased to peak at 7-9weeks after fetal reduction, and subsequently decreased gradually to almost undetectable during the late third trimester. The NIPT T-scores persistently reflected the detection of fetal trisomy up to 16 (median 9.5) weeks after fetal reduction. Residual cfDNA from the deceased cotwin after selective reduction at 14-17 gestational weeks led to the persistent generation of false-positive NIPT results for up to 16weeks postdemise. Thus, providing NIPT for pregnancies with a cotwin demise in early second trimester is prone to misleading results and not recommended.

The role of ultrasound in women with a positive NIPT result for trisomy 18 and 13

ObjectiveThe aim of this study was to evaluate the usefulness of ultrasound in pregnancies with a positive non-invasive prenatal testing (NIPT) result for trisomy 18/13. Materials and methodsDuring a four-year period, the pregnant women who were referred for invasive genetic testing because of positive NIPT results for trisomy 18/13 were included in this study. An in-depth ultrasound was done for these patients before invasive procedures. The data of fetal ultrasound and cytogenetic results were collected. ResultsThere were 81 patients with a positive NIPT result for trisomy 18/13, including 39 (30 positive for trisomy 18; 9 positive for trisomy 13) within 12–14 weeks of gestation, and 42 (31 positive for trisomy 18; 11 positive for trisomy 13) within 15–22 weeks. The PPV of NIPT was 60.7% for trisomy 18, and 30% for trisomy 13, respectively. When adding ultrasound to NIPT, the new PPV for trisomy 18 was 100%, and the negative predictive value (NPV) was 92.3%, with a NPV of 85.7% in the first trimester and a NPV of 100% in the second trimester, respectively. The new PPV and NPV for trisomy 13 were 100% and 100%, respectively. ConclusionBy adding ultrasound to the NIPT, we achieved much higher PPVs and NPVs for trisomy 18/13. A normal scan can help to alleviate stress in parents caused by false positive NIPT results.

Noninvasive Prenatal Testing Using Next Generation Sequencing: Pilot Experience of the D.O. Ott Research Institute of Obstetrics, Gynecology and Reproductology

In recent years, noninvasive prenatal testing (NIPT) for fetal chromosomal abnormalities has come into wide use. NIPT allows detection of fetal chromosomal abnormalities without invasive sampling of fetal material: by analyzing cell-free fetal DNA in maternal blood. Here, we report on the pilot results of using NIPT at the D.O. Ott Research Institute of Obstetrics, Gynecology and Reproductology (St. Petersburg, Russia). The results obtained in 149 blood samples from pregnant women proved the clinical value of NIPT for detection of fetal trisomies 21, 18, and 13. Aneuploidy was detected in 20 out of 149 samples. Among these 20 aneuploid cases, trisomy 21 amounted to 60%, trisomy 18 – to 25%, and trisomy 13 to 5% of cases. In one sample, double aneuploidy involving chromosomes 13 and 21 was detected, and in one case, trisomy X was identified. In 100% of cases, aneuploidies were confirmed by prenatal karyotyping advocating for the absence of false positive NIPT results. The observed high specificity was consistent with the declared test specificity level of >99.9%. The sensitivity of the method was 100%. To summarize, NIPT is a new reliable technique that will probably soon replace the conventional first trimester combined screening. The only disadvantages of implementing NIPT in Russia are its relatively high cost and the lack of coverage by insurance companies. NGS-based NIPT is suitable for clinical practice and will be widely deployed due to a further cost decrease.

False positive non-invasive prenatal testing results due to vanishing twins

To explore the mechanism and duration of false positive results of non-invasive prenatal testing (NIPT) caused by vanishing twins. To detect the variation of cell-free fetal DNA fraction before and after the fetal death and explore its influence on the results of NIPT at different gestational weeks. Prenatal diagnosis was also carried out on amniotic fluid sample derived from the survivor twin. After birth, the two placentas and papyraceous fetus were obtained to ascertain the definitive genetic diagnosis and pathological changes through fluorescence in situ hybridization, fluorescence quantitative PCR and histopathological examination. Eight cases of vanishing twins leading to discordant NIPT results were reviewed for determining the duration of this influence. The vanishing twin has led to immediate flooding of cfDNA into the maternal plasma due to necrotic cytotrophoblasts, which in turn caused increased release of fetal DNA in a short time. However, this did not change the NIPT result for a period of time. The tissue and chorionic villi of perished fetus presented extensive degenerative necrosis. The false positive NIPT result caused by vanishing twins may be attributed to continuous release of DNA fragments into the maternal plasma by the fetuses. The influence of the vanished fetuses, which may lead to discordant NIPT results, can last for at least 7-8 weeks but no more than 12-14 weeks during the first and second trimester.

The Combining Effects of Cell-Free Circulating Tumor DNA of Breast Tumor to the Noninvasive Prenatal Testing Results: A Simulating Investigation.

Massively parallel sequencing of circulating fetal DNA in the plasma of pregnant women is a common method for noninvasive prenatal testing (NIPT) of fetal trisomy 13, 18, and 21. However, circulating DNA is not restricted to pregnant women, with increased levels of plasma DNA also frequently detected in the plasma of cancer patients. Among pregnant women whose NIPT results were inconsistent with the fetal karyotype, a small number of patients have subsequently been diagnosed with a previously undetected malignancy. However, the extent to which circulating tumor DNA (ctDNA) affects the results of NIPT is still unclear. We examined serum from 50 nonpregnant women with breast tumors by NIPT. These samples were then added to serum containing trisomy 13, 18, and 21 fetal DNA to figure out the extent to which maternal tumors can interrupt NIPT results in pregnant women with breast tumors. Concentrations of cell-free DNA (cfDNA) were higher in both pregnant women and breast tumor patients, relative to nonpregnant healthy controls. Among the 50 samples evaluated, 3 produced false positive NIPT results for trisomy 13, 18, or 21, indicating that genomic copy number variations (CNVs) had occurred. Simulation testing also showed that ctDNA can increase the standard deviation of the associated z-scores, which lower absolute z-scores by decreasing the proportion of circulating fetal DNA relative to total DNA. Of the 50 samples tested, 9 fell within the equivocal range and 8 produced false negative results for trisomy 13, 18, or 21. Data presented here show for the first time that ctDNA is able to affect NIPT results in two ways. First, ctDNA can lead to false positive results due to the detection of genomic CNVs in tumor DNA. Alternatively, ctDNA can increase the likelihood of a false negative by decreasing the proportion of circulating fetal DNA in serum.

Sex chromosome aneuploidy screened by non-invasive prenatal testing from 35 827 singletons: prenatal diagnosis and pregnancy determinations

Objective To assess the positive predictive value (PPV) of fetal sex chromosome aneuploidy (SCA) identified by non-invasive prenatal testing (NIPT) and investigate families' acceptance of SCA fetus. Methods All suspected SCA cases screened by NIPT from singletons were reviewed in Prenatal Diagnosis Center of Shanghai First Maternity and Infant Hospital from April 1, 2015 to October 31, 2017. Maternal age, NIPT indications, prenatal diagnosis protocols, testing results and their pregnancy determinations were analyzed. Results NIPT was provided to 35 827 singletons and 86 suspected SCA cases were identified out of 35 823 successful ones, giving a positive detection rate of 0.24%. The average maternal age was (31.5±5.0) years. After genetic counseling, 20 patients declined prenatal diagnosis, the rest 66 cases proceeded with aminiocentesis and fetal chromosomal testing, of which 32 were cytogenetically diagnosed as SCA with the PPV of 48.5% . The SCA fetus consisted of 25 sex chromosome trisomies (seven cases of 47,XXX, three cases of 47,XYY and 15 cases of 47,XXY), one monosomy X (45,X), three mosacisms (47,XXY/48,XXYY, 47,XXX/45,X, 45,X/46,XX, one for each) and three microdeletions/microduplications. Besides, two false positive NIPT cases were proved to be low level of maternal mosacism (45,X/46,XX, 5% and 10% for each). After genetic counseling, 17 out of 20 who declined prenatal diagnosis and 9 out of 32 who diagnosed fetal SCAs continued their pregnancies, with a combined proportion of continued pregnancy of 50%. Thirty-four pregnancies were also continued after exclusion of SCA. Interestingly, the proportion of continued pregnancy among those sex chromosomal trisomy fetuses was only 32%(8/25). Conclusions As a safe and rapid prenatal testing for common autosomal aneuploidies, NIPT could also identify some types of SCA, but with relatively low PPV. More long-term researches are required to determine its sensitivity and specificity. For some types of SCA with mild phenotypes, some family would continue the pregnancy. Therefore, limitations of NIPT should be appropriately explained during both pre- and post-testing counseling. Key words: Non-invasive prenatal testing; Sex chromosome aberrations; Prenatal diagnosis; Aneuploidy; Chimera; Predictive value of tests

Confined placental mosaicism of trisomy 16 detected by non-invasive prenatal testing and multiple abnormalities

This study aimed to investigate a case of confined placental trisomy 16 mosaicism (CPM16) with abnormal amniotic fluid, placental lake, and other abnormalities. Maternal serum screening was performed to assess the risk of foetal aneuploidy, and massively parallel sequencing was used to detect cell-free fetal DNA. The abnormalities of the fetus, amniotic fluid, and placenta were detected by ultrasonic inspection. Maternal serum screening indicated a high risk for trisomy 21, and the non-invasive prenatal testing (NIPT) result was positive for trisomy 16. The pregnancy was terminated and karyotype analysis of fetal heart blood revealed a 46, XX karyotype. Copy number variation (CNV) sequencing of placental tissues indicated that CPM16 is the main cause of false-positive NIPT results and intrauterine growth retardation (IUGR) diagnoses. Combining molecular genetics technologies, such as CNV sequencing, can be complementary, and provide an effective strategy to determine the cause of such abnormalities.

“False Positive” Noninvasive Prenatal Testing Result? Consider Maternal Malignancy

Not long after the introduction of noninvasive prenatal testing (NIPT) for fetal aneuploidy, case reports emerged of women with false-positive NIPT

Noninvasive Prenatal Testing and Detection of Maternal Cancer

Noninvasive prenatal testing (NIPT) to detect fetal aneuploidy (an abnormal number of chromosomes) usingmassive parallel sequencing of cell-free DNA (cfDNA) from maternal blood is gaining rapid acceptance in obstetrics, given its high sensitivity and specificity for the detection of trisomies 21, 18, and 13. Placental DNA, a surrogate of fetal DNA, enters the maternal bloodstream via apoptosis of trophoblasts in the intervillous space of the placenta,where itmixes with cfDNA of maternal origin. The fetal fraction in this mixture usually varies between 3% and 15% in the late first and second trimesters.NIPT represents a formofadvancedscreening, and positive results consistent with aneuploidy require confirmation by a diagnostic test usingmaterial obtained via amniocentesis or chorionic villus sampling (CVS).1,2 The sensitivity of NIPT for the detection of trisomies 21, 18, and 13 ranges between 91% and 99.2%; the false-positive rates range between 0.09% and 0.13%.3 False-positive results havebeen attributed to confinedplacentalmosaicism,maternal chromosomalmosaicism,maternalDNAcopynumbervariants, andmaternal organ transplant fromamale donor.4 Two case reports (in 2013 and 2015) suggested that the discordant results (between those of NIPT and amniocentesis) could be due to undiagnosed maternal cancer.5,6 The first case described a 37-year-old pregnant woman with an uncomplicated pregnancy who had NIPT performed at 13 weeks of gestation. The findings were suggestive of fetal aneuploidy for chromosomes 13 and 18 in 2 different maternal blood samples. Yet the fetal karyotype was 46,XY without evidence of submicroscopic genomic imbalances with wholegenome oligonucleotide microarray. After delivering a healthy infant, the patient reported pelvic pain found to be due to a pathologic fracture of the pubis; a biopsy showed metastatic neuroendocrine carcinoma of unknown origin. A separate lesion on the anterior vaginal wall proved to be a high-grade neuroendocrine small cell carcinoma. Tumor cells showed aneuploidy for chromosomes 13 and 18, consistent with the findings from the analysis of maternal blood assessed with NIPT.5 The second patient was a 27-year-old asymptomatic woman at 12 weeks of gestation whose NIPT results had abnormal genomic findings for chromosomes 8, 9, and 14 that were discordantwith a normal fetal karyotype from amniotic fluid cells. Moreover, maternal lymphocytes did not have evidence of aneuploidy ormosaicism. Given the genomic imbalances, amalignancywas suspected, and thepatientunderwent a whole-body diffusion magnetic resonance imaging (MRI) scan, which showed an anterior mediastinal mass and enlarged lymph nodes in the cervical and retroclavicular regions. The diagnosis of nodular sclerosis Hodgkin lymphoma wasmade,basedonabiopsyof themediastinalmass.Hodgkin/ Reed-Sternbergcells showedfindingsconsistentwith thoseobservedwith the analysis of cfDNA inmaternal blood.Basedon these observations, the authors investigated whether cfDNA couldbe informative innewlydiagnosedcasesofnodular sclerosis Hodgkin lymphoma in nonpregnant women (n = 4) and in nonpregnant patients who had a first relapse after therapy (n = 5). The results in 8 of the 9 cases showed genomic imbalances, and such findingswere validated by fluorescent in situ hybridization on Hodgkin/Reed-Sternberg cells in tissue specimens.6 Commonfeaturesof the2caseswere that thepatientswere youngasymptomaticpregnantwomen,hadabnormalNIPT results discrepant with the fetal karyotype (“false-positive NIPT”),were subsequently diagnosed tohave cancer, and the malignant cells showedevidenceofmultiple aneuploidies that mirrored the findings reported by maternal cfDNA analyzed for NIPT. A recent study reported that amongmore than4000pregnant women undergoing NIPT, 3 patients were found to have aberrant genome representation profiles that were reminiscent of cancer-related copy number variation. These women underwent whole-body diffusion-weighted MRI and were found to have cancer—the first had a high-grade serous ovarian carcinoma (stage IV with multiple metastases); the second, a follicular lymphoma; and the third, Hodgkin lymphoma. Analyses of the genome representation profile of the tumors showed that the copy-number variations detected in maternal plasma were present in the tumors.7 The report by Bianchi et al8 in this issue of JAMA is the first large-scale study that strengthens the premise that a false-positive NIPT result could be due to the presence of maternal malignancy based on the results of a case series of pregnant women who had NIPT and were subsequently diagnosed with cancer. This work leverages the information of a laboratory that had processed 125 426 samples from NIPT over 2.5 years with a test to detect the presence of aneuploidy for chromosomes 21, 18, and 13 (testing for sex chromosome aneuploidy was optional). Of these samples, 3% (3757) had NIPT results consistent with aneuploidy. The practice of the laboratory was to discuss all abnormal NIPT results with the referring clinician and recommend a diagnostic test, and clinicians were asked to inform the laboratory if the results Related article page 162 Opinion

Advantages and Disadvantages of Different Implementation Strategies of Non-Invasive Prenatal Testing in Down Syndrome Screening Programmes

Background: Implementation of non-invasive prenatal testing (NIPT) in Down syndrome screening programmes requires health policy decisions about its combination with other tests and its timing in pregnancy. Aim: Our aim was to aid health policy decision makers by conducting a quantitative analysis of different NIPT implementation strategies. Methods: Decision trees were created to illustrate all plausible alternatives in a theoretical cohort of 100,000 pregnant women in five screening programmes: classical screening by the first-trimester combined test (FCT), pre-selection of high-risk women prior to NIPT by the FCT, NIPT as the first screening test at 10 weeks and at 13 weeks, and the simultaneous conductance of NIPT and the FCT. Results: Pre-selection by FCT prior to NIPT reduces the number of amniocenteses to a minimum because of a reduction of false-positive NIPT results. If NIPT is the first screening test, it detects almost all fetal Down syndrome cases. NIPT at 10 weeks reassures women early in pregnancy, while NIPT at 13 weeks prevents unnecessary tests due to spontaneous miscarriages and allows for immediate confirmation by amniocentesis. Conclusion: Every implementation strategy has its advantages and disadvantages. The most favourable implementation strategy may be NIPT as the first screening test at 13 weeks, offering the most accurate screening test for Down syndrome, when the risk for spontaneous miscarriage has declined remarkably and timely confirmation by amniocentesis can be performed.