Abnormal Noninvasive Prenatal Testing Research Articles

Genetic analysis of a fetus with with 45,X/46,X,idic(Y)(q11.2) mosaicism

To explore the genetic characteristics of a fetus with sex chromosome abnormality indicated by non-invasive prenatal testing (NIPT) at 25+ gestational weeks. A pregnant woman who was admitted to the Taizhou Hospital for abnormal NIPT result on January 6, 2023 was selected as the study subject. Relevant clinical data was collected. The fetus was subjected to chromosomal karyotyping analysis, copy number variation sequencing (CNV-seq), fluorescence in situ hybridization (FISH), and multiplex PCR assays. NIPT had suggested monosomy of X chromosome. The fetus was found to have a chromosomal karyotype of 45,X[59]/46,X,del(Y)(q11.2)[17] at 30+ weeks of gestational age. CNV-seq suggested the presence a 7.98 Mb deletion at Yq11.222q12 and a mosaicism 16.92 Mb deletion. FISH suggested that the fetus harbored two SRY genes and a mosaicism sex chromosomal abnormality, and multiplex PCR revealed that its AZF b+c region was completely deleted. C-banded karyotyping showed darkly stained dense mitotic granules at both ends of the Y chromosome. The fetus was ultimately determined as a 45,X/46,X,idic(Y)(q11.2) mosaicism. Following elected abortion, testing of the fetal tissue confirmed the presence of 45,X/46,XY mosaicism, and CNV-seq result of the placental tissue was compatible with that of NIPT. CNV-seq analysis of the couple revealed no obvious abnormality. With combined NIPT, karyotyping, CNV-seq, FISH and multiplex PCR assays, the fetus was diagnosed as a 45,X/46,X,idic(Y)(q11.2) mosaicism with deletion of the AZF b+c region. Above finding has enabled prenatal diagnosis for the fetus.

Chromosomal abnormalities detected by chromosomal microarray analysis and pregnancy outcomes of 4211 fetuses with high-risk prenatal indications

With the gradual liberalization of the three-child policy and the development of assisted reproductive technology in China, the number of women with high-risk pregnancies is gradually increasing. In this study, 4211 fetuses who underwent chromosomal microarray analysis (CMA) with high-risk prenatal indications were analysed. The results showed that the overall prenatal detection rate of CMA was 11.4% (480/4211), with detection rates of 5.82% (245/4211) for abnormal chromosome numbers and 5.58% (235/4211) for copy number variants. Additionally, the detection rates of clinically significant copy number variants were 3.78% (159/4211) and 1.8% (76/4211) for variants of uncertain significance. The detection rates of fetal chromosomal abnormalities were 6.42% (30/467) for pregnant women with advanced maternal age (AMA), 6.01% (50/832) for high-risk maternal serum screening (MSS) results, 39.09% (224/573) with abnormal non-invasive prenatal testing (NIPT) results, 9.21% (127/1379) with abnormal ultrasound results, and 5.1% (49/960) for other indications. Follow-up results were available for 4211 patients, including 3677 (3677/4211, 87.32%) whose infants were normal after birth, 462 (462/4211, 10.97%) who terminated their pregnancy, 51 (51/4211, 1.21%) whose infants were abnormal after birth, and 21 (21/4211, 0.50%) who refused follow-up. The results of this study demonstrate significant variation in the diagnostic rate of chromosomal microarray analysis across different indications, providing valuable guidance for clinicians to assess the applicability of CMA technology in prenatal diagnosis.

Limited additional value of karyotyping cultured amniotic fluid cell colonies in addition to microarray on uncultured cells for confirmation of abnormal non-invasive prenatal testing results.

Non-invasive prenatal testing (NIPT) allows the detection of placental chromosome aberrations. To verify whether the fetus also has the chromosome aberration, diagnostic follow-up testing is required. The aim of this retrospective study was to assess the added value of analyzing amniotic fluid (AF) cell cultures in addition to uncultured AF cells for the detection of fetal mosaicism. NIPT was performed as part of the Dutch TRIDENT study. Cytogenetic studies in uncultured AF were performed using single nucleotide polymorphism (SNP)-array. Cultured AF cell colonies (in situ method) were investigated with fluorescent in situ hybridization and/or karyotyping. Clinical outcome data were collected in cases with discordant results. Between April 2014 and December 2021, 368 amniocenteses were performed after a chromosomal aberration was detected with NIPT. Excluding 134 cases of common aneuploidies (confirmed by quantitative fluorescence polymerase chain reaction), 29 cases with investigation of uncultured cells only and 1 case without informed consent, 204 cases were eligible for this study. In 196 (96%) cases, the results in uncultured and cultured cells were concordant normal, abnormal or mosaic. Five cases (2%) showed mosaicism in cultured AF cells, whereas uncultured AF cells were normal. Two (1%) of these, one mosaic trisomy 13 and one mosaic trisomy 16, were considered true fetal mosaics. The added value of investigating AF cell cultures in addition to uncultured cells is limited to two of 204 (1%) cases in which true fetal mosaicsm would otherwise be missed. The clinical relevance of one (trisomy 13) remained unknown and the other case also showed ultrasound anomalies, which determined pregnancy management. This seems to justify limiting prenatal cytogenetic confirmatory testing to SNP arrays on uncultured AF cells, considerably shortening the reporting time.

Detection of Maternal Malignancy after Abnormal Noninvasive Prenatal Testing (NIPT)

Introduction: Noninvasive prenatal testing (NIPT) is routinely used to screen for fetal aneuploidies in chromosomes 13, 18 and 21. NIPT uses NGS based methods to detect fetal cfDNA in maternal circulation. In 2016, the American College of Obstetricians and Gynecologists recommended offering NIPT testing for all pregnant patients regardless of maternal age. Reports of maternal malignancy detected by NIPT have been increasing since its wide adoption into clinical practice, but subsequent management has not been standardized. We therefore report the outcomes of patients with abnormal NIPT evaluated and managed at a single institution. Methods: A case series ofpatients referred to Dana-Farber Cancer Institute between 2014 and 2022 for evaluation following an abnormal NIPT or for treatment of a cancer identified through NIPT. Results: Fourteen cases were included in this series. The median age at NIPT was 34 (range 29-42). Most patients (79%) had multiple chromosomal aneuploidy while 3 patients had ≤2 chromosomal abnormalities reported on their NIPT. Twelve patients (86%) underwent subsequent invasive prenatal testing: results were normal in all but one whose trisomy 12 finding led to early termination. Eleven patients (79%) underwent a whole-body MRI (WBMRI), 6 (55%) of which identified a suspicious finding that required biopsy. Of the 6 patients biopsied for a finding on WBMRI, 5 (83%) were found to have a malignancy. All patients who had a negative WBMRI did not have an identified malignancy. Of the total 14 cases, 10 (71%) underwent biopsies for abnormal findings and 8 (80%) of biopsies led to diagnosis of malignancy (or 57% of overall cohort). This was found to be Hodgkin lymphoma in 4 (29%), primary mediastinal large B cell lymphoma in 2 (14%) and triple negative breast cancer in 2 (14%) of the patients. Other causes of abnormal NIPT were uterine fibroids in 3 (21%), hepatic adenoma in 1 (7%), ovarian dermoid cyst in 1 (7%) and no abnormal findings in 1 (7%). Half of the patients received cancer treatment during pregnancy and the other half after pregnancy. Treatment included chemotherapy 6 (75%), chemotherapy/radiation 1 (12.5%) and surgery/chemotherapy 1 (12.5%). Post treatment, 6/8 (75%) patients achieved a complete metabolic response (CMR), one patient is still undergoing treatment and one patient (breast cancer) is deceased. Overall, there were 12 (86%) live births, one pregnancy termination and one patient with missing data. The median birthweight was 7.2 lbs (6.1-8.5 lbs). Two patients had NIPT with previous pregnancies. One patient had a normal NIPT and the other had an abnormal NIPT that was similar in two pregnancies, but she had a hepatic adenoma. Two patients with PMBCL had subsequent pregnancies and repeat NIPT after treatment were both normal . Conclusions: Specific abnormal NIPT results may be caused by maternal malignancy, especially where there is discordance with the fetal karyotype. A WBMRI without contrast is a safe and feasible initial approach for diagnosis and staging in these patients. Most tumors are lymphomas, for which treatment during and soon after pregnancy is possible with favorable maternal and fetal outcomes.

Prenatal diagnosis of non-mosaic sex chromosome abnormalities: a 10-year experience from a tertiary referral center.

The aim of this study was to explore the frequency and profile of non-mosaic sex chromosome abnormalities detected in prenatal diagnosis over the past 10 years. We retrospectively reviewed pregnancies diagnosed with non-mosaic sex chromosome abnormalities between January 2012 and December 2021, using karyotyping and/or single nucleotide polymorphism (SNP) array. Maternal age, indications for testing, and outcomes were recorded. Traditional karyotyping identified 269 (0.90 %) cases of non-mosaic sex chromosome abnormalities among 29,832 fetuses, including 249 cases of numerical abnormalities, 15 unbalanced structural abnormalities, and 5 balanced structural abnormalities. The overall detection rate of common sex chromosome aneuploidies (SCAs) was 0.81 %, with 47,XXY, 47,XXX, 47,XYY, and 45,X accounting for 0.32 , 0.19, 0.17, and 0.13 % respectively. All showed a fluctuating upward trend over the study period, except for 45,X. During the first five years (2012-2016), the major indication for testing was advanced maternal age (AMA), followed by abnormal ultrasound, abnormal noninvasive prenatal testing (NIPT), and abnormal maternal serum screening (MSS). In the second five years (2017-2021), the most frequent indication was abnormal NIPT, followed by AMA, abnormal ultrasound, and abnormal MSS. Among the 7,780 cases that underwent SNP array in parallel, an additional 29 clinically significant aberrations were detected. The most frequent aberration was a microdeletion in the Xp22.31 region, which was associated with X-linked ichthyosis. Fetal sex chromosome abnormalities are important findings in prenatal diagnosis. The application of NIPT and SNP array technology has greatly improved the detection of SCAs and submicroscopic aberrations associated with sex chromosomes.

Mosaic trisomy 21 at amniocentesis in a twin pregnancy associated with a favorable fetal outcome, maternal uniparental disomy 21 and postnatal decrease of the trisomy 21 cell line

ObjectiveWe present mosaic trisomy 21 at amniocentesis in a twin pregnancy associated with a favorable fetal outcome, maternal uniparental disomy (UPD) 21 and postnatal decrease of the trisomy 21 cell line. Case reportA 36-year-old woman underwent elective amniocentesis at 16 weeks of gestation because of advanced maternal age, and an abnormal non-invasive prenatal testing (NIPT) result suggesting trisomy 21. Amniocentesis revealed the karyotype of 46, XX in co-twin A and the karyotype of 47,XY,+21[12]/46,XY[21] in co-twin B in the cultured amniocytes by in situ culture method. Simultaneous array comparative genomic hybridization (aCGH) analysis on uncultured amniocytes revealed the result of arr (21) × 3 [0.40] in co-twin B, consistent with 40% mosaicism for trisomy 21. Prenatal ultrasound was unremarkable, and the parental karyotypes were normal. Following genetic counseling, the parents decided to continue the pregnancy. At 36 weeks of gestation, a 2140-g female co-twin A and a 1800-g male co-twin B were delivered without any phenotypical abnormality. The karyotypes of the umbilical cord and placenta of co-twin B were 47,XY,+21[16]/46,XY[24] and 47,XY,+21 (40/40 cells), respectively. Quantitative fluorescence polymerase chain reaction (QF-PCR) analysis on the DNA extracted from parental bloods and umbilical cord, cord blood and placenta and peripheral blood at age five months of co-twin B confirmed a maternal origin of trisomy 21 and maternal uniparental isodisomy 21. aCGH analysis on the cord blood revealed the result of arr 21q11.2q22.3 × 2.25 consistent with 20%–25% (log2 ratio = 0.15–0.2) mosaicism for trisomy 21. When follow-up at age five months, the co-twin B was phenotypically normal. His peripheral blood had a karyotype of 47,XY,+21[3]/46,XY[37]. Interphase fluorescence in situ hybridization (FISH) on 100 buccal mucosal cells detected no trisomy 21 signals. The peripheral blood had uniparental isodisomy 21. ConclusionMosaic trisomy 21 at amniocentesis can be a transient and benign condition and should alert the possibility of UPD 21. The abnormal trisomy 21 cell line in mosaic trisomy 21 at amniocentesis may decrease and disappear after birth.

The role of non-invasive prenatal testing and ultrasound in prenatal screening of fetal chromosomal abnormalities in singleton: a retrospective study.

Non-invasive prenatal testing (NIPT) has good screening performance for common chromosomes, but it may have false positive (FP) and false negative (FN) results for various reasons. For abnormal NIPT results, the combination of fetal ultrasound phenotypes will provide more fetal information for prenatal diagnosis. The aim of this study was to combine NIPT and ultrasound phenotypes to analyze their complementary roles in prenatal screening of fetal chromosome abnormalities. From January 2018 to December 2021, 12,803 pregnant women with singleton who successfully underwent NIPT/expanded NIPT (NIPT-Plus) at Xiangya Hospital of Central South University, of which 111 cases were positive results and one case was FN result. We retrospectively collected the clinical features, ultrasonographic findings, prenatal diagnosis, and pregnancy outcomes of these 112 pregnant women and analyzed the ultrasonic manifestations of different chromosomal abnormalities in detail. The positive predictive values (PPVs) of NIPT/NIPT-Plus for trisomy (T)21, T18, sex chromosome abnormality (SCA), microdeletion/microduplication syndrome (MMS), T13, and rare autosomal trisomy (RAT) were 100.0%, 85.7%, 57.1%, 44.4%, 40.0%, and 7.7%, respectively. The total termination rates of pregnancy for T21, T18, T13, SCA, pathogenic MMS, and RAT were 93.5%, 100.0%, 100.0%, 66.7%, 100.0%, and 100.0%, respectively. From the karyotypes of SCA live-born fetuses, 47,XYY and 47,XXX were more likely to be selected for continued pregnancy. The ultrasound phenotypes of T21 were diverse, including normal, soft marker, and structural malformation. Both T18 and T13 had structural malformations as the main phenotypes. Most ultrasound phenotypes of FP T21, T18, and T13 were normal but occasionally manifested as fetal growth restriction (FGR). The ultrasound phenotypes of SCA, MMS, and RAT were relatively mild and manifested as normal, soft marker, FGR, or polyhydramnios, and the ultrasound phenotypes were similar between FP and true positive (TP) cases. Ultrasound phenotypes are helpful in identifying FP NIPT/NIPT-Plus results, especially for T18 and T13. Given its mild ultrasound phenotypes, NIPT-Plus has important clinical significance in reducing the missed diagnosis of SCA, MMS, and RAT, but its screening performance needs to be further improved.

Non-Invasive Prenatal Testing in Germany.

In the short 10 years following the introduction of non-invasive prenatal testing (NIPT), it has been adapted in many countries around the world as a standard screening test. In this review, this development was analyzed with a special focus on Germany. As a result, it can be stated that all known advantages of NIPT apart from "compensating for having no access to centers offering invasive diagnostics" are valid for Germany. In addition, following a review of the international literature, all documented issues with NIPT are also observed in Germany. However, the German Gene Diagnostics Act (GenDG) addresses a number of these issues, for example, the regulations by GenDG hamper induced abortions, based exclusively on an abnormal NIPT result. At the same time, GenDG has created new problems, as a possible collusion between the "right not to know with regard to parts of the examination result" may occur, or that the sex of the fetus must not be reported to the pregnant woman before the 12th week of gestation. Main conclusions drawn are that appropriate training and the continuing education of the physicians providing NIPT-related counseling are needed, as well as the provision of balanced and comprehensive information for the pregnant woman or the couple that is imperative.

Efficiency of Noninvasive Prenatal Testing for Sex Chromosome Aneuploidies

Objective: This study was designed to investigate the efficiency of noninvasive prenatal testing (NIPT) for screening fetal sex chromosome aneuploidies (SCAs) through sequencing of cell-free DNA in maternal plasma. Methods: This is a retrospective study on the positive NIPT results for SCAs collected from our hospital between January 2012 and December 2018. Samples with positive NIPT results for SCAs were then confirmed by prenatal or postnatal karyotyping analysis. Results: After cytogenetic analysis, abnormal karyotypes were confirmed in 104 cases and the overall positive predictive value (PPV) of NIPT for SCAs was 43.40% (102/235). The most frequently detected karyotypes included 47,XXY (n = 42), 47,XXX (n = 20), 47,XYY (n = 16), and 45,X (n = 2). Meanwhile, 10 cases were confirmed with mosaic karyotype 45,X/46,XX and 14 cases with numerical or structural chromosome abnormalities, including a double trisomy 48,XXX,+18. Cytogenetic results from the other 131 cases showed normal XX or XY, which were discordant with NIPT results. Upon analysis of parental karyotypes, 29 (12.34%) showed false positivity in NIPT results that were caused by maternal sex chromosome abnormalities. Conclusion: NIPT is an effective screening tool for SCA with a PPV of 43.40%. Maternal karyotype abnormalities occurred in 12.34% of the cases with abnormal NIPT. Diagnostic testing of the fetus and the mother are recommended.

Evaluation of the Z-score accuracy of noninvasive prenatal testing for fetal trisomies 13, 18 and 21 at a single center.

To assess the correlation between Z-scores of positive noninvasive prenatal testing (NIPT) results and the positive predictive value (PPV) of NIPT. Pregnancies with positive NIPT results at Guangzhou Women and Children's Medical Centre between July 2017 and May 2020 were included in this study. Fetal karyotyping or microarray analysis was provided to patients with abnormal NIPT results for confirmatory testing. Logistic regression analyses was applied to study the relationship between the Z scores and the PPV performance. The optimal cutoff values for indicating fetal common trisomies were obtained based on receiver operating characteristic (ROC) curve analysis, and then the PPV were calculated in pregnancies with positive NIPT results at Z-score greater than or equal to cutoff value and in patients with a Z-score between 3 and cutoff value respectively. A total of 214 pregnancies with positive NIPT results for fetal common trisomies were validated by invasive prenatal diagnosis and follow up in this study. Of these, NIPT indicated trisomy 13 in 25 cases, trisomy 18 in 54 cases and trisomy 21 in 135 patients. Logistic regression analyses showed a significant association (p<0.05) between the Z-scores and true positive results for T21 and T18. For T13, the significant association was not observed (p>0.05). The ROC curve analysis showed that the optimal cutoff Z-score for indicating fetal trisomies 13, 18, and 21 were 6.889, 7.574 and 6.612 respectively, and the corresponding area under curve were 0.706, 0.916, and 0.954. In this cohort with abnormal NIPT results, the cutoff values revealed a sensitivity of 96.8% and a specificity of 90% for indicating trisomies 21, and a sensitivity of 88.9% and a specificity of 92.6% for trisomies 18. However, probably due to the sample size, the sensitivity and specificity for indicating trisomy 13 were lower (85.7% and 61.1%) than that for trisomies 21 and 18. The PPVs in pregnancies with positive NIPT results at Z-score greater than or equal to cutoff value were 99.18% (121/122) for trisomy 21, 92.31% (24/26) for trisomy 18 and 46.15% (6/13) for trisomy 13. In patients with a Z-score between 3 and cutoff Z-score, the PPV of NIPT for trisomies 21, 18, and 13 were 30.77% (4/13), 10.71% (3/28), and 8.33% (1/12) respectively. Moreover, by classifying Z scores as 3≤Z<5, 5≤Z<10, and Z≥10, the majority of Z scores were above 10 with a PPV of 99% for T21 and just 5.2% were between 3 and 5 with a PPV of 14.3%. In contrast for T18, over a third of tests had Z scores between 3 and 5. The PPV in this group is just over 5%. The present results show that the PPV performance of NIPT for fetal trisomies 13, 18, and 21 are closely associated with Z-score. The higher the Z-score, the greater the likelihood that the aneuploidy result is correct. Our experience in evaluating the Z-score accuracy of NIPT in this study could be of use in similar work.

Clinical Utility of SNP Array Analysis in Prenatal Diagnosis: A Cohort Study of 5000 Pregnancies.

BackgroundSingle nucleotide polymorphism array (SNP-array) has been introduced for prenatal diagnosis. We aimed to evaluate the clinical value of SNP-array in the diagnosis of fetal chromosomal anomalies.MethodsA retrospective study was conducted on 5000 cases tested by SNP-array, and the results of 4022 cases analyzed by both karyotyping and SNP-array were compared.ResultsSNP-array analysis of 5000 samples revealed that the overall abnormality detection rate by SNP-array was 12.3%, and the overall detection rate of clinically significant copy number variations (CNVs) by SNP-array was 2.6%. SNP-array identified clinically significant submicroscopic CNVs in 4.5% fetuses with anomaly on ultrasonography, in 1.6% of fetuses with advanced maternal age (AMA), in 2.5% of fetuses with abnormal result on maternal serum screening, in 2.9% of fetuses with abnormal non-invasive prenatal testing (NIPT) results and in 3.0% of fetuses with other indications. Of the 4022 samples analyzed by both karyotyping and SNP-array, SNP-array could identify all the aneuploidy and triploidy detected by karyotyping but did not identify balanced structural chromosomal abnormalities and low-level mosaicism detected by karyotyping.ConclusionSNP-array could additionally identify clinically significant submicroscopic CNVs, and we recommend the combination of SNP-array analysis and karyotyping in prenatal diagnosis.

Clinical management of pregnancies with positive screening results for rare autosomal aneuploidies at a single center.

ObjectiveTo review our experiences on clinical management of pregnancies with positive noninvasive prenatal testing (NIPT) results for rare autosomal aneuploidies (RAAs) at a single center.MethodsWe performed a retrospective study and reviewed data from 18,016 pregnancies undergoing NIPT at a single center in China from March 2017 to February 2020. Depending on the patient’s choice, women with positive screening results for RAAs underwent chromosomal microarray analysis for invasive prenatal diagnosis.ResultsThirty-three positive cases for RAAs were identified, with a positive screening rate of 0.18%. The most common RAA was trisomy 7 (33.3%), while trisomies for other chromosomes were less frequent. Monosomies involving chromosomes 16, 14, and 22 were observed. Twenty-eight cases of RAAs underwent invasive diagnosis. Abnormal pregnancy outcomes were observed in four cases, including true fetal mosaicism (n=1), partial uniparental disomy (n=1), miscarriage (n=1), and structural anomalies on ultrasound (n=1).ConclusionsRAAs at NIPT might be associated with fetal uniparental disomy, mosaic aneuploidy, and poor pregnancy outcomes, but most positive cases have normal pregnancy outcomes. For RAAs, genetic counseling on the potential risks of abnormal NIPT results, as well as on benefits and limitations of invasive prenatal diagnosis, might help guide clinical management.

Cytogenetic discrepancy between uncultured amniocytes and cultured amniocytes in mosaic trisomy 15 at amniocentesis

ObjectiveWe present mosaic trisomy 15 at amniocentesis. Materials and methodsA 41-year-old woman underwent amniocentesis at 16 weeks of gestation because of an abnormal non-invasive prenatal testing (NIPT) result suspicious of trisomy 15. Amniocentesis revealed a karyotype of 46,XY. Array comparative genomic hybridization (aCGH) on uncultured amniocytes revealed 26% mosaicism for trisomy 15. She was referred for repeat amniocentesis. aCGH, interphase fluorescence in situ hybridization (FISH), quantitative fluorescent polymerase chain reaction (QF-PCR) assays and/or conventional cytogenetic analysis were applied on various cells and tissues including uncultured amniocytes, cultured amniocytes, cord blood, placenta, parental bloods and/or buccal mucosal cells. ResultsRepeat amniocentesis at 21 weeks of gestation revealed a karyotype of 46, XY in cultured amniocytes, and 30% mosaicism for trisomy 15 by aCGH and 32% mosaicism for trisomy 15 by FISH in uncultured amniocytes. Repeat amniocentesis at 29 weeks of gestation revealed a karyotype of 46, XY in cultured amniocytes, and 15% mosaicism for trisomy 15 by aCGH and 7.2% mosaicism for trisomy 15 by FISH in uncultured amniocytes. QF-PCR on cultured amniocytes excluded uniparental disomy (UPD) 15. A phenotypically normal baby was delivered subsequently with a karyotype of 46, XY in cord blood and 2% mosaicism for trisomy 15 by FISH in buccal mucosal cells. The aCGH analysis revealed trisomy 15 in placenta and no genomic imbalance in cord blood. QF-PCR assays determined a maternal origin of trisomy 15 in placenta. ConclusionCytogenetic discrepancy may occur between uncultured and cultured amniocytes in mosaic trisomy 15 at amniocentesis. The cells of trisomy 15 cell line in prenatally detected mosaic trisomy 15 may decrease in number as the fetus grows. Whenever NIPT suspects trisomy 15, a confirmatory amniocentesis should include genetic analysis on both uncultured and cultured amniocytes to exclude mosaic trisomy 15 and maternal UPD 15, especially when the cultured amniocytes have a normal karyotype.

Non-invasive prenatal testing has altered positive predictive value following transfer of a euploid blastocyst

The positive predictive value (PPV) of non-invasive prenatal testing (NIPT) has been reported to range from 91.3-97.2% in the general population (1,2). However, PPV is dependent upon the prevalence of the disease in the population being tested. Patients who undergo in vitro fertilization (IVF) with preimplantation genetic testing for aneuploidy (PGT-A) and transfer a euploid embryo are presumably a lower risk population when compared to the general population. The objective of this study is to explore the PPV for NIPT following transfer of a euploid blastocyst. Retrospective cohort study. All patients at a single IVF center between 2014 and 2018 who pursued pre-implantation genetic testing for aneuploidy (PGT-A) and underwent transfer of a euploid blastocyst between 2014 and 2018 were contacted to request completion of a medical record release form authorizing release of antenatal records. Records were reviewed, and patients who had documentation of an abnormal NIPT were included in this study. Results of any subsequent prenatal or postnatal diagnostic testing were used to classify each positive NIPT as a “true positive” or a “false positive”. The PPV of NIPT was calculated. A total of 1,202 patients eligible for inclusion were contacted for completion of the medical record release form. Five patients with abnormal NIPT following transfer of a euploid blastocyst were identified. Four of these patients (80%) had subsequent definitive prenatal diagnostic testing which revealed a euploid karyotype concordant with their PGT-A results. One patient, who had a PGT-A result indicating 46,XX but a NIPT positive for Turner syndrome, underwent amniocentesis which confirmed Turner mosaicism (45,X karyotype in 80% of cells). Therefore, the PPV of NIPT in this patient cohort was 20%. The PPV of NIPT for patients undergoing transfer of a euploid blastocyst is lower than that for the general population. PGT-A may be more likely to yield inaccurate results in the presence of embryonic mosaicism, as illustrated by the true positive NIPT case in this study cohort. PGT-A is an imperfect screening tool and follow-up antenatal screening is advisable; however, clinicians and patients should recognize that patients undergoing transfer of a euploid blastocyst are at a relatively lower risk for fetal aneuploidy when compared to the general population and, as a result, the PPV of NIPT is altered in this setting.

Experience of chromosomal microarray applied in prenatal and postnatal settings in Hong Kong.

Chromosomal microarray (CMA) is recommended as a first tier investigation for patients with developmental delay (DD), intellectual disability (ID), autistic spectrum disorder (ASD), and multiple congenital anomalies (MCA). It is widely used in the prenatal and postnatal settings for detection of chromosomal aberrations. This is a retrospective review of all array comparative genomic hybridization (aCGH/ array CGH) findings ascertained in two major prenatal and postnatal genetic diagnostic centers in Hong Kong from June 2012 to December 2017. Medical records were reviewed for cases with pathogenic and variants of uncertain clinical significance (VUS). Classification of copy number variants (CNVs) was based on current knowledge and experience by August 2018. The aims of this review are to study the diagnostic yield of array CGH application in prenatal and postnatal settings in Hong Kong and to describe the spectrum of abnormalities found. Prenatal indications included abnormal ultrasound findings, positive Down syndrome screening, abnormal noninvasive prenatal test results, advanced maternal age and family history of chromosomal or genetic abnormalities. Postnatal indications included unexplained DD, ID, ASD, and MCA. A total of 1,261 prenatal subjects and 3,096 postnatal patients were reviewed. The prenatal diagnostic yield of pathogenic CNV and VUS (excluding those detectable by karyotype) was 3.5%. The postnatal diagnostic yield of pathogenic CNV was 15.2%. The detection rates for well-defined microdeletion and microduplication syndromes were 4.6% in prenatal and 6.1% (1 in 16 index patients) in postnatal cases, respectively. Chromosomes 15, 16, and 22 accounted for over 21 and 25% of pathogenic CNVs detected in prenatal and postnatal cohorts, respectively. This review provides the first large scale overview of genomic imbalance of mostly Chinese patients in prenatal and postnatal settings.

Prenatal diagnosis of monochorionic-diamniotic twins discordant for 45,X/46,XX mosaicism

To explore the prenatal screening and diagnosis for a pair of monochorionic-diamniotic (MCDA) twins discordant for 45,X/46,XX mosaicism. Amniotic fluid samples were taken from both twins for whom non-invasive prenatal testing has signaled a high risk for sex chromosomal abnormality. Uncultured amniotic fluid was analyzed by fluorescence in situ hybridization (FISH) and single nucleotide polymorphism array (SNP-array). Conventional G-banded karyotyping analysis was performed on the cultured amniotic fluid. Metaphase chromosome analysis showed that one of the twins had a mos 45,X[11]/46,XX[26] karyotype, while the other had a normal karyotype. FISH and SNP-array applied on uncultured amniotic fluid revealed about 30% mosaicism in one of the twins. The twins were confirmed to be monozygotic by SNP-array analysis. To avoid confusion arising from discordant karyotypes in MCDA twins with abnormal non-invasive prenatal testing (NIPT) results, dual amniocentesis should be carried out to obtain amniotic fluid samples for chromosomal as well as molecular analysis. To determine the ratio of 45,X and 46,XX cells in Turner syndrome can provide valuable information for prenatal genetic counseling.

The value and role of non-invasive prenatal testing in a select South African population.

Concerns have been raised about the injudicious use of non-invasive prenatal testing (NIPT) using cell-free DNA (cfDNA), which often leads to inaccuracies in interpretation of the role and value of cfDNA in prenatal screening. To determine the value and role of NIPT in a select South African (SA) population. A retrospective review of patients who elected to have NIPT between 1 October 2013 and 30 June 2015 at the Morningside Mediclinic Maternal and Fetal Medicine Centre in Johannesburg, SA. Patients had NIPT after either combined first-trimester screening (CFTS) or a second-trimester ultrasound scan. Data were collected on details of the first- and/or second-trimester screening, results of the NIPT, invasive tests done, decisions made in the event of abnormal results, and pregnancy outcomes. Overall, 3473 first- and second-trimester fetal assessments were done at the centre during the study period, and 2.3% of patients (n=82) elected to have NIPT. The majority of these individuals elected to have NIPT on the basis of positive findings on CFTS, or markers of aneuploidy detected on a second-trimester ultrasound scan. Of the tests done, 97.6% produced results. Of those with no results, one did not meet quality metrics and the other had a low fetal fraction of cfDNA. There were two abnormal NIPT results, one indicating a high risk of trisomy 13 and the other a triploidy. Patients who screened negative elected not to have an invasive test. The value of NIPT in this study was that it made it possible to avoid a number of invasive tests. NIPT had a role in contingency screening.

Enlarged NT (≥3.5mm) in the first trimester - not all chromosome aberrations can be detected by NIPT.

BackgroundSince non-invasive prenatal testing (NIPT) in maternal blood became available, we evaluated which chromosome aberrations found in our cohort of fetuses with an enlarged NT in the first trimester of pregnancy (tested with SNP microarray) could be detected by NIPT as well.Method362 fetuses were referred for cytogenetic testing due to an enlarged NT (≥3.5 mm). Chromosome aberrations were investigated using QF-PCR, karyotyping and whole genome SNP array.ResultsAfter invasive testing a chromosomal abnormality was detected in 137/362 (38 %) fetuses. 100/362 (28 %) cases concerned trisomy 21, 18 or 13, 25/362 (7 %) an aneuploidy of sex chromosomes and 3/362 (0.8 %) triploidy. In 6/362 (1.6 %) a pathogenic structural unbalanced chromosome aberration was seen and in 3/362 (0.8 %) a susceptibility locus for neurodevelopmental disorders was found. We estimated that in 2–10 % of fetuses with enlarged NT a chromosome aberration would be missed by current NIPT approaches.ConclusionBased on our cohort of fetuses with enlarged NT we may conclude that NIPT, depending on the approach, will miss chromosome aberrations in a significant percentage of pregnancies. Moreover all abnormal NIPT results require confirmatory studies with invasive testing, which will delay definitive diagnosis in ca. 30 % of patients. These figures are important for pretest counseling enabling pregnant women to make informed choices on the prenatal test. Larger cohorts of fetuses with an enlarged NT should be investigated to assess the additional diagnostic value of high resolution array testing for this indication.

Cytogenetic confirmation of a positive NIPT result: evidence-based choice between chorionic villus sampling and amniocentesis depending on chromosome aberration

ABSTRACTIt has been shown that in non-invasive prenatal testing (NIPT) there is a small chance of a false-positive or false-negative result. This is partly due to the fact that the fetal cell-free DNA present in maternal plasma is derived from the cytotrophoblast of chorionic villi (CV), which is not always representative for the fetal karyotype due to chromosomal mosaicism. Therefore, a positive NIPT result should always be confirmed with invasive testing, preferably amniocentesis, in order to investigate the fetal karyotype. However, since this invasive test can only be safely performed after 15.5 weeks of gestation while NIPT can be done from the 10th week of gestation, this potentially means an unacceptable long waiting time for the prospective parents to receive a definitive result. Based on our experience with cytogenetic investigations in CV and the literature, we determined whether CV sampling may be appropriate for confirmation of an abnormal NIPT result.

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