High Risk for Rare Autosomal Trisomies Detected by Non-Invasive Prenatal Testing: Prenatal Diagnosis and Pregnancy Outcomes.

Genome-wide sequencing of maternal plasma cell-free DNA (cfDNA) enables non-invasive prenatal testing (NIPT) of all 24 chromosomes. An expanded NIPT approach can identify rare autosomal trisomies (RATs). However, this is not without interpretative challenges. More evidence is needed to determine pregnancy outcome in cases of RAT detected by NIPT. Between January 2016 and December 2017, a total of 42 924 maternal blood samples were received from Dongguan, Guangdong Province, China, for NIPT as a first-tier screening test as part of a government-funded pilot program. The suggested gestational age for NIPT was 12–22 weeks, and a nuchal translucency or second-trimester ultrasound examination before blood sampling was mandatory. We documented 72 (0.17%) NIPT results positive for RAT, 11 of which were excluded from this study because of loss to follow-up. Trisomy 7 was the most commonly detected RAT, followed equally by trisomies 3 and 16. Pregnancy characteristics, NIPT results and pregnancy outcome are summarized in Table 1. Placental karyotyping confirmed confined placental mosaicism in 54 cases, and the remaining seven had normal placental and maternal lymphocyte karyotype. Nineteen cases opted for amniocentesis, all of which showed normal karyotype; among these, only five (three at risk for trisomy 14 and two at risk for trisomy 15) opted for additional chromosomal microarray analysis, which did not show unbalanced rearrangements or uniparental disomy (UPD) in any case. At follow-up, isolated intrauterine growth restriction (IUGR) was identified in one case of trisomy 16 and one of compound trisomy 7/8. There was no case with a sonographic structural anomaly. All neonates were karyotyped, and none had evidence of true fetal mosaicism. In this study, we found no correlation between NIPT Z-score value and the percentage of abnormal cells observed in the placenta. However, these cell percentages might not represent the true condition of the affected placentas because of biopsy samplingbias. Several genome-wide cfDNA-based NIPT studies have reported RATs in association with a range of pregnancy complications, including miscarriage, true fetal mosaicism and UPD, as well as IUGR1–3. However, our results indicate favorable pregnancy outcome. Ninety-five percent of our cases had an uncomplicated pregnancy. Three reasons might contribute to this discrepancy. First, the a-priori risk in our population may explain the lower incidence of pregnancy complications. Our patients came from the general rather than a high-risk population, as in other studies. As a result, NIPT was positive for RAT in only 0.17% of cases, which is much lower than that (0.43%) in a previous study1. Second, the fact that all NIPT samples were taken only after a pretest ultrasound examination may also explain the relatively favorable outcome, as most of the affected fetuses would have been identified before performing NIPT. Third, the gestational age at NIPT in our study was relatively late, further reducing the incidence of finding an affected pregnancy. We observed a positive predictive value (PPV) of 0 for RATs. Expanding the range of aneuploidies that NIPT targets compounds this problem. Our results showed that RATs are most often mosaic and confined to the placenta. It seems that the risk of adverse pregnancy outcome associated with confined placental mosaicism for RATs is not higher than that in the general population, except for trisomy 164,5. Nevertheless, we found that the PPVs for RATs reflected closely those predicted by placental data. Eighty-eight percent of positive NIPT cases were confirmed by placental investigation. Although the clinical utility of expanding NIPT to include RATs has yet to be demonstrated and, as such, the clinical implementation of this testing is premature in the general population, screening for RATs using NIPT might benefit pregnancy management in some specific situations such as in cases of IUGR with a normal chromosomal microarray analysis result.

To investigate the prenatal diagnostic value and pregnancy outcomes of rare autosomal trisomies (RATs) as indicated by non-invasive prenatal testing (NIPT), and to contribute to good childbearing. This was a retrospective and cross-sectional analysis of 83,842 pregnant women who received NIPT in our hospital. Amniotic fluid karyotype analysis and SNP-array chip detection were used to evaluate the prenatal diagnostic value and pregnancy outcomes of pregnant women at high-risk of RATs. In 83,842 pregnant women, 331 were identified to be at high-risk of RATs; the positive screening rate was 0.39% (331/83,842). Among the 331 women at high risk of RATs, 245 received invasive prenatal diagnosis, and the referral reasons for NIPT were advanced maternal age (≥ 35 years), serological borderline risk, and single serological marker median (MoM) abnormality, with the constituent ratios of 27.35% (67/245), 19.18% (47/245) and 17.55% (43/245), respectively. In these 245 pregnant women at high-risk of RATs, the most common screening-risk results were trisomy 7 (33.88%), trisomy 8 (13.47%), trisomy 20 (11.43%), trisomy 3 (7.35%), and trisomy 10 (6.12%); the rarest conditions were trisomy 6 (0.41%), trisomy 12 (0.82%), trisomy 14 (0.82%), and trisomy 4 (1.22%). Trisomy 1, 17 and 19 weren’t screened. The positive predictive value (PPV) for RATs in women at high-risk was 13.06%, and the PPVs for trisomy 9, 16, 15, 10 and 2 were 42.86%, 40.00%, 25.00%, 20.00%, and 16.67%, respectively. NIPT indicated that for pregnant women at high-risk of RATs, the PPVs for trisomy 9, 16, 15 and 10 were relatively high, while the PPV for other trisomies were low. With the exception of trisomy 9, 15, 16 and 10, most RATs were associated with good pregnancy outcomes. In clinical genetic counseling for RATs, the principle of individualized medicine should be reflected, and different prenatal diagnostic measures should be adopted for different RATs to advise pregnant women on whether to continue or terminate the pregnancy.

Clinical impacts of genome-wide noninvasive prenatal testing for rare autosomal trisomy

To evaluate the impact of detailed late first-trimester ultrasound (LFTU) on the positive predictive value (PPV) of a high-risk non-invasive prenatal test (NIPT) result for various chromosomal abnormalities. This was a retrospective study of all cases undergoing invasive prenatal testing from three tertiary providers of obstetric ultrasound over 4 years, each using NIPT as a first-line screening test. Data were collected from pre-NIPT ultrasound, NIPT, LFTU, placental serology and later ultrasound examinations. Prenatal testing for chromosomal abnormalities was performed by microarray, initially using array comparative genomic hybridization and then single nucleotide polymorphism (SNP) array for the last 2 years. Uniparental disomy testing was performed by SNP array during all 4 years. The majority of NIPT tests were analyzed using the Illumina platform, initially confined to the assessment of the common autosomal trisomies, sex chromosome aneuploidies and rare autosomal trisomies (RAT), then extending to genome-wide analysis for the last 2 years. Amniocentesis or chorionic villus sampling (CVS) was performed on 2657 patients, 1352 (51%) of whom had undergone prior NIPT, with 612 (45%) of these returning a high-risk result and meeting the inclusion criteria for the study. LFTU findings significantly affected the PPV of the NIPT result for trisomies 13 (T13), 18 (T18) and 21 (T21), monosomy X (MX) and RAT but not for the other sex chromosomal abnormalities or segmental imbalances (> 7 Mb). Abnormal LFTU increased the PPV close to 100% for T13, T18, T21, MX and RAT. The magnitude of the change in PPV was highest for the most severe chromosomal abnormalities. When LFTU was normal, the incidence of confined placental mosaicism (CPM) was highest in those with a high-risk NIPT result for T13, followed by T18 and T21. After normal LFTU, the PPV for T21, T18, T13 and MX decreased to 68%, 57%, 5% and 25%, respectively. LFTU after a high-risk NIPT result can alter the PPV for many chromosomal abnormalities, assisting counseling regarding invasive prenatal testing and pregnancy management. The high PPVs of NIPT for T21 and T18 are not sufficiently modified by normal LFTU findings to alter management. These at-risk patients should be offered CVS for earlier diagnosis, particularly given the low rate of CPM associated with these aneuploidies. Patients with a high-risk NIPT result for T13 and normal LFTU findings often wait for amniocentesis or avoid invasive testing altogether given the low PPV and higher rate of CPM in this context. © 2023 The Authors. Ultrasound in Obstetrics & Gynecology published by John Wiley & Sons Ltd on behalf of International Society of Ultrasound in Obstetrics and Gynecology.

To assess the value of non-invasive prenatal testing (NIPT) for the identification of sex chromosome aneuploidies (SCAs), copy number variants (CNVs) and rare autosomal trisomies (RATs). A total of 11 429 women with singleton pregnancy in Ningbo area were screened by NIPT. 106 women were subjected to invasive prenatal diagnosis due to high risk of chromosomal abnormalities other than 21, 18 and 13 aneuploidies. All cases were followed up for pregnancy outcome and postnatal status. Sixty-six women were signaled by NIPT for fetal SCAs, among whom 54 were willing to undergo prenatal diagnosis. Eighteen cases of fetal SCAs were verified as true positives and 4 were suspected positives, which yielded a positive predictive value (PPV) of 33.3%. Half of the women decided to continue their pregnancy. Forty women were signaled by NIPT for fetal CNVs, among which 32 underwent prenatal diagnosis. 19 cases of fetal CNVs were verified as true positives and 3 cases were suspected positives, which yielded a PPV of 46.8%. All women with pathological or possibly pathological CNVs decided to terminate their pregnancies. Thirty-one women were signaled for with fetal RATs. Two fetuses were confirmed to harbor mosaicism trisomies by prenatal diagnosis, and 1 case was suspected to be positive, which yielded a PPV of 9.7%. All of the three women have decided to terminate their pregnancy. In addition to aneuploidies of target chromosomes, NIPT also has important value for the detection of SCAs and CNVs. The results can help to further reduce birth defects. Nevertheless, in view of its low PPV, pregnant women with positive result still need appropriate genetic counseling and prenatal diagnosis to avoid unnecessary induced labor.

To analyze the result of prenatal diagnosis and outcome of pregnancy for fetuses with rare autosomal trisomies (RATs) suggested by non-invasive prenatal testing (NIPT). A total of 69 608 pregnant women who underwent NIPT at Genetics and Prenatal Diagnosis Center of the First Affiliated Hospital of Zhengzhou University from January 2016 to December 2020 were selected as study subjects. The result of prenatal diagnosis and outcome of pregnancy for those with a high risk for RATs were retrospectively analyzed. Among the 69 608 pregnant women, the positive rate of NIPT for high-risk RATs was 0.23% (161/69 608), with trisomy 7 (17.4%, 28/161) and trisomy 8 (12.4%, 20/161) being the most common, and trisomy 17 (0.6%, 1/161) being the rarest. For 98 women who had accepted invasive prenatal diagnosis, 12 fetal chromosomal abnormalities were confirmed, and in 5 cases the results were consistent with those of NIPT, which yielded a positive predictive value of 5.26%. Among the 161 women with a high risk for RATs, 153 (95%) were successfully followed up. 139 fetuses were ultimately born, with only one being clinically abnormal. Most women with a high risk for RATs by NIPT have good pregnancy outcomes. Invasive prenatal diagnosis or serial ultrasonography to monitor fetal growth, instead of direct termination of pregnancy, is recommended.

Objective: To analyze the report content, the methods and results of prenatal diagnosis of high risk of sex chromosome aneuploidy (SCA) in non-invasive prenatal testing (NIPT). Methods: A total of 227 single pregnancy pregnant women who received genetic counseling and invasive prenatal diagnosis at Drum Tower Hospital Affiliated to the Medical School of Nanjing University from January 2015 to April 2022 due to the high risk of SCA suggested by NIPT were collected. The methods and results of prenatal diagnosis were retrospectively analyzed, and the results of chromosome karyotype analysis and chromosome microarray analysis (CMA) were compared. The relationship between NIPT screening and invasive prenatal diagnosis was analyzed. Results: (1) Prenatal diagnosis methods for 277 SCA high risk pregnant women included 73 cases of karyotyping, 41 cases of CMA and 163 cases of karyotyping combined with CMA, of which one case conducted amniocentesis secondly for further fluorescence in situ hybridization (FISH) testing. Results of invasive prenatal diagnosis were normal in 166 cases (59.9%, 166/277), and the abnormal results including one case of 45,X (0.4%, 1/277), 18 cases of 47,XXX (6.5%, 18/277), 36 cases of 47,XXY (13.0%, 36/277), 20 cases of 47,XYY (7.2%, 20/277), 1 case of 48,XXXX (0.4%, 1/277), 20 cases of mosaic SCA (7.2%, 20/277), 5 cases of sex chromosome structural abnormality or large segment abnormality (1.8%, 5/277), and 10 cases of other abnormalities [3.6%, 10/277; including 9 cases of copy number variation (CNV) and 1 case of balanced translocation]. Positive predictive value (PPV) for SCA screening by NIPT was 34.7% (96/277). (2) Among the 163 cases tested by karyotyping combined with CMA, 11 cases (6.7%, 11/163) showed inconsistent results by both methods, including 5 cases of mosaic SCA, 1 case of additional balanced translocation detected by karyotyping and 5 cases of additional CNV detected by CMA. (3) NIPT screening reports included 149 cases of "sex chromosome aneuploidy"(53.8%, 149/277), 54 cases of "number of sex chromosome increased" (19.5%, 54/277), and 74 cases of "number of sex chromosome or X chromosome decreased" (26.7%, 74/277). The PPV of "number of sex chromosome increased" and "number of sex chromosome or X chromosome decreased" were 72.2% (39/54) and 18.9% (14/74), respectively, and the difference was statistically significant (χ2=34.56, P<0.01). Conclusions: NIPT could be served as an important prenatal screening technique of SCA, especially for trisomy and mosaicism, but the PPV is comparatively low. More information of NIPT such as the specific SCA or maternal SCA might help improving the confidence of genetic counseling and thus guide clinic management. Multi technology platforms including karyotyping, CMA and FISH could be considered in the diagnosis of high risk of SCA by NIPT.

To evaluate the value of non-invasive prenatal testing(NIPT)for detecting rare autosomal trisomies in fetuses. We retrospectively analyzed the data of cases with rare autosomal trisomies detected by NIPT in our hospital from January, 2019 to April, 2023.Invasive prenatal diagnostic tests including chromosome karyotype analysis, chromosome microarray analysis, copy number variation sequencing, and fluorescence in situ hybridization were performed in all the cases after clinical counseling, and their test results and pregnancy outcomes were analyzed. Among 25 282 women receiving NIPT, 56(0.22%)were found to have high risks for rare autosomal trisomies in circulating plasma DNA.Trisomy 7 was the most frequently detected trisomy, accounting for 45% of the total cases(25/56), while trisomies 1, 4, 17, and 19 were not detected.Among the 46 cases with genetic results of the fetuses, 10 were identified to have true fetal mosaicism.The overall positive predictive value of NIPT was 22%(10/46)for rare autosomal trisomies, and 10% for trisomy 7(2/20).Of the 52 cases followed up for pregnancy outcomes, 33(63%)cases without fetal mosaicism resulted in normal live births, while 10 had unfavorable outcomes including fetal growth restriction, preterm birth, and maternal complications, and among them fetal growth restriction was the most typical and the earliest condition observed in these cases.Among the 22 followed up cases of non-true mosaicism for trisomy 7, 82% resulted in normal live births. NIPT increases the detection rate of true fetal mosaicism but with a low positive predictive value.Most of the cases with non-true mosaicism, particularly trisomy 7, can have favorable outcomes.NIPT can also be useful in identifying causes of fetal growth restriction in the second and third trimesters when invasive prenatal testing does not reveal abnormalities.

Copy number variation (CNVs) can result in various genomic diseases and variable clinical phenotypes. This study aimed to assess the feasibility and reliability of noninvasive prenatal testing (NIPT) for prenatal screening of maternal copy number variation (CNVs) involving the Xq28 recurrent region. In this retrospective, single-center study, we analyzed the NIPT data of 80,371 pregnant women to detect the maternal CNVs from 2017 to 2022. Maternal CNVs involving the int22h1/int22h2-mediated chromosome Xq28 recurrent region detected by NIPT were focused on. Chromosomal microarray analysis (CMA) was then performed to validate NIPT results if the remaining maternal lymphocytes were available. A total of 48 (0.060%, 95% CI 0.045% ~ 0.079%) maternal CNVs involving int22h1/int22h2-mediated chromosome Xq28 recurrent region were identified by NIPT, including 29 (0.036%, 95% CI 0.025% ~ 0.052%) pathogenic deletions sizing from 128.1kb to 93,244.3kb, 17 (0.021%, 95% CI 0.013% ~ 0.034%) pathogenic duplications sizing from 442.5kb to 2059.1kb and two (0.002%, 95% CI 0.000% ~ 0.009%) variants of uncertain significance (VOUS) sizing from 215.2kb to 326.1kb. The detection rates of typical Xq28 recurrent microdeletions and microduplications were 0.005% (4/80,371, 95% CI 0.002% ~ 0.013%) and 0.006% (5/80,371, 95% CI 0.003% ~ 0.015%), respectively. Chromosomal microarray analysis (CMA) was performed to validate NIPT results in 22 cases for which remaining maternal lymphocytes were available. The positive predictive value (PPV) of NIPT for maternal CNV's detection in the 22 cases was 100.0% (95% CI 85.1% ~ 100.0%). This study illustrated the feasibility and potential of NIPT to detect maternal CNVs involving the Xq28 recurrent region.

A new comprehensive paradigm for prenatal diagnosis: seeing the forest through the trees.

Value of noninvasive prenatal testing in the detection of rare fetal autosomal abnormalities

To establish the positive predictive values (PPV) of cfDNA testing based on data from a nationwide survey of independent clinical cytogenetics laboratories. Prenatal diagnostic test results obtained by Italian laboratories between 2013 and March 2020 were compiled for women with positive non-invasive prenatal tests (NIPT), without an NIPT result, and cases where there was sex discordancy between the NIPT and ultrasound. PPV and other summary data were reviewed. Diagnostic test results were collected for 1327 women with a positive NIPT. The highest PPVs were for Trisomy (T) 21 (624/671, 93%) and XYY (26/27, 96.3%), while rare autosomal trisomies (9/47, 19.1%) and recurrent microdeletions (8/55, 14.5%) had the lowest PPVs. PPVs for T21, T18, and T13 were significantly higher when diagnostic confirmation was carried out on chorionic villi (97.5%) compared to amniotic fluid (89.5%) (p<0.001). In 19/139 (13.9%), of no result cases, a cytogenetic abnormality was detected. Follow-up genetic testing provided explanations for 3/6 cases with a fetal sex discordancy between NIPT and ultrasound. NIPT PPVs differ across the conditions screened and the tissues studied in diagnostic testing. This variability, issues associated with fetal sex discordancy, and no results, illustrate the importance of pre- and post-test counselling.

Rare autosomal trisomies (RATs) other than common aneuploidies can be detected using noninvasive prenatal testing (NIPT). However, conventional karyotyping is insufficient for evaluating diploid fetuses with uniparental disomy (UPD) due to trisomy rescue. Using the diagnostic process for Prader-Willi syndrome (PWS), we aim to describe the need for additional prenatal diagnostic testing for confirming UPD in fetuses diagnosed with RATs via NIPT and its clinical implications. NIPT was performed using the massively parallel sequencing (MPS) method, and all pregnant women with RATs underwent amniocentesis. After confirming the normal karyotype, short tandem repeat (STR) analysis, methylation-specific PCR (MS-PCR), and methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) were performed to detect UPD. Overall, six cases were diagnosed with RATs. There was a suspicion of trisomies of chromosomes 7, 8, and 15 in two cases each. However, these cases were confirmed to have a normal karyotype using amniocentesis. In one of six cases, PWS caused by maternal UPD 15 was diagnosed using MS-PCR and MS-MLPA. We propose that in cases where RAT is detected by NIPT, UPD should be considered following trisomy rescue. Even if amniocentesis confirms a normal karyotype, UPD testing (such as MS-PCR and MS-MLPA) should be recommended for accurate assessment, as an accurate diagnosis can lead to appropriate genetic counseling and improved overall pregnancy management.

Counseling for non-invasive prenatal testing (NIPT): what pregnant women may want to know

Data from high-income countries have shown that expanded non-invasive prenatal testing (NIPT) is highly reliable for detecting common autosomal trisomies (CATs), whereas its diagnostic accuracy for rare autosomal trisomies (RATs) remains relatively low. Information on the clinical performance of expanded NIPT in middle- and low-income countries is still limited. The objective of this study was to evaluate the clinical validity and utility of an expanded NIPT for CATs, sex chromosome aneuploidies (SCAs), and RATs following its nationwide clinical implementation. The results of NIPT were retrospectively reviewed. Those with high-risk test results were followed up via phone and/or by reviewing medical records regarding the downstream prenatal diagnosis and the pregnancy outcomes. The NIPT results of 19,714 samples were analyzed. We found screening positive rate of 0.95%; sensitivity at 97.92%, 94.12%, 85.71%, and 90% for T21, T18, T13, and SCAs; and PPV at 87.04%, 69.57%, 50%, and 54.55% for T21, T18, T13, and SCAs respectively. As for RATs, clinical validity included sensitivity at 100% and PPV of 13.3%. Over 95% of the participants with high-risk NIPT results underwent prenatal diagnosis. The majority (86.52%) of those with a confirmed diagnosis chose termination of the pregnancy. This large-scale study of mixed-risk pregnancies revealed that expanded NIPT had a good sensitivity for CATs (specifically for T21 and T18) and RATs; and fair sensitivity for T13 and SCAs. The PPV is acceptable for CATs and SCAs, but poor for RATs. These data indicated that expanded NIPT results strongly influenced pregnancy outcomes in a real-world experience.