BACKGROUND: Introduction of cell-free fetal DNA (cff-DNA) testing in maternal blood opened possibilities to improve the performance of combined first-trimester screening (cFTS) in terms of better detection of trisomies and lowering invasive testing rate. The use of new molecular methods, such as chromosomal microarray analysis (CMA) and next-generation sequencing (NGS), has shown benefits in prenatal diagnosis of chromosomal and genetic diseases, which are not detectable with cff-DNA screening, but require an invasive procedure.
METHODS: The objective of this study was to evaluate prospectively during two years performance of CMA and NGS in high-risk pregnancies. Initially, we investigated 14,566 singleton pregnancies with cFTS. A total of 334 high-risk pregnancies were selected for CMA diagnostic performance evaluation and 28 cases of highly dysmorphic fetuses for NGS analysis. CMA study group was divided into two groups based on the indications for testing; group A patients with high-risk for trisomies after cFTS, but normal ultrasound and group B patients who met criteria for CMA as a first-tier diagnostic test.
RESULTS: The diagnostic yield of CMA was overall 3.6% (1.6% in Group A and 6.0% in Group B). In NGS analysis group, we report diagnostic yield of 17.9%.
CONCLUSIONS: The use of CMA in high-risk pregnancies is justified and provides relevant clinical information in 3.6% of the cases. NGS analysis in fetuses with multiple anomalies shows promising results, but more investigations are needed for a better understanding of practical applications of this molecular diagnosis method in prenatal settings.

Recently, chromosomal microarray analysis (CMA) has been implemented as a first-tier test in pregnancies with ultrasound anomalies. However, its application for pregnancies with abnormal maternal serum screening (AMSS) only is not widespread. This study evaluated the value of CMA compared to traditional karyotyping in pregnancies with increased risk following first- or second-trimester maternal serum screening. Data from 3973 pregnancies with referral for invasive prenatal testing following AMSS were obtained from April 2016 to May 2020. Routine karyotyping was performed and single nucleotide polymorphism array was recommended. The foetuses were categorized according to the indications as AMSS only (group A) and AMSS with ultrasound anomalies (group B). CMA was performed on 713 prenatal samples. The proportion of women opting for CMA testing in both groups increased over the years. The incremental yield of clinically significant findings for pregnancies with high risk of screening results was similar to that for the foetuses with ultrasound soft markers (P > 0.05), but significantly lower than that for the foetuses with structural anomalies (P < 0.05). The total frequencies of variants of unknown significance in groups A and B showed no significant difference (P > 0.05). CMA should be performed for pregnant women undergoing prenatal invasive testing due to AMSS, especially with high-risk results, regardless of ultrasound findings.

OBJECTIVE: The present study aimed to determine the diagnostic value of prenatal chromosomal microarray analysis (CMA) for fetuses with several indications of being at high risk for various conditions.
METHODS: This retrospective analysis included 1256 pregnancies that were prenatally evaluated due to high-risk indications using invasive CMA. The indications for invasive prenatal diagnosis mainly included ultrasound anomalies, high-risk for maternal serum screening (MSS), high-risk for non-invasive prenatal tests (NIPT), family history of genetic disorders or birth defects, and advanced maternal age (AMA). The rate of clinically significant genomic imbalances between the different groups was compared.
RESULTS: The overall prenatal diagnostic yield was 98 (7.8%) of 1256 pregnancies. Clinically significant genomic aberrations were identified in 2 (1.5%) of 132 patients with non-structural ultrasound anomalies, 36 (12.7%) of 283 with structural ultrasound anomalies, 2 (4.5%) of 44 at high-risk for MSS, 38 (26.6%) of 143 at high-risk for NIPT, 11 (3.8%) of 288 with a family history, and 7 (2.1%) of 328 with AMA. Submicroscopic findings were identified in 29 fetuses, 19 of whom showed structural ultrasound anomalies.
CONCLUSIONS: The diagnostic yields of CMA for pregnancies with different indications greatly varied. CMA could serve as a first-tier test for structural anomalies, especially multiple anomalies, craniofacial dysplasia, urinary defects, and cardiac dysplasia. Our results have important implications for genetic counseling.

The aim of this retrospective cohort study was to determine the potential diagnostic yield of prenatal whole exome sequencing in fetuses with structural anomalies on expert ultrasound scans and normal chromosomal microarray results.
In the period 2013-2016, 391 pregnant women with fetal ultrasound anomalies who received normal chromosomal microarray results, were referred for additional genetic counseling and opted for additional molecular testing pre- and/or postnatally. Most of the couples received only a targeted molecular test and in 159 cases (40.7%) whole exome sequencing (broad gene panels or open exome) was performed. The results of these molecular tests were evaluated retrospectively, regardless of the time of the genetic diagnosis (prenatal or postnatal).
In 76 of 391 fetuses (19.4%, 95% CI 15.8%-23.6%) molecular testing provided a genetic diagnosis with identification of (likely) pathogenic variants. In the majority of cases (91.1%, 73/76) the (likely) pathogenic variant would be detected by prenatal whole exome sequencing analysis.
Our retrospective cohort study shows that prenatal whole exome sequencing, if offered by a clinical geneticist, in addition to chromosomal microarray, would notably increase the diagnostic yield in fetuses with ultrasound anomalies and would allow early diagnosis of a genetic disorder irrespective of the (incomplete) fetal phenotype.

 We report a case of two consecutive pregnancies in the same couple presenting with very low pregnancy-associated plasma protein A (PAPP-A), with both pregnancies affected by multiple anomalies of a similar phenotype identified during mid-trimester ultrasound, and eventual diagnosis of Peters-plus syndrome. This case is important in expanding the differential for very low PAPP-A. It also demonstrates the diagnostic value of whole-exome sequencing (WES) after prenatal diagnosis of recurrent fetal ultrasonographic findings. The importance and complexity of providing patient education to enable informed consent for next generation sequencing technologies is discussed.

 We report a case of two consecutive pregnancies in the same couple presenting with very low pregnancy-associated plasma protein A (PAPP-A), with both pregnancies affected by multiple anomalies of a similar phenotype identified during mid-trimester ultrasound, and eventual diagnosis of Peters-plus syndrome. This case is important in expanding the differential for very low PAPP-A. It also demonstrates the diagnostic value of whole-exome sequencing (WES) after prenatal diagnosis of recurrent fetal ultrasonographic findings. The importance and complexity of providing patient education to enable informed consent for next generation sequencing technologies is discussed.

Prenatal diagnostics has been impacted by technological changes in the past decade, which have affected the diagnostic yield. The aim of this study was to evaluate the impact of SNP array and noninvasive prenatal testing (NIPT) on the diagnostic yield and the number of invasive tests in our center. The frequency of pathogenic fetal unbalanced chromosome aberrations was studied in 10,005 cases referred for prenatal testing in 2009-2015. Chromosomal SNP microarray analysis replaced karyotyping in all invasively tested pregnancies and since 2014 a choice between NIPT and diagnostic testing with microarray was offered to women with an increased risk for common aneuploidy. The introduction of microarray led to an additional yield of submicroscopic pathogenic chromosome aberrations: 3.6% in fetuses with ultrasound anomalies and 1.9% in fetuses without ultrasound anomalies. The introduction of NIPT led to a decrease of invasive tests and of diagnostic yield. Moreover, a diagnostic delay in about 1:350 cases was observed. Since 20%-33% of pathogenic fetal chromosome aberrations are different from the common aneuploidies and triploidy, whole-genome analysis should be offered after invasive sampling. Because NIPT (as a second screening) has led to a decreased diagnostic yield, it should be accompanied by an appropriate pretest counseling.

OBJECTIVE: To evaluate the application of non-invasive prenatal testing (NIPT) as an alternative to invasive diagnostic prenatal testing in pregnancies with abnormal ultrasound findings.
METHODS: This was a retrospective analysis of 251 singleton and multiple pregnancies at high risk for fetal chromosomal abnormality based on findings at sonographic examination, in which NIPT was performed as a first-tier genetic test. NIPT was performed by massively parallel sequencing of cell-free DNA in maternal plasma, allowing genome-wide detection of whole-chromosome, as well as partial, autosomal aneuploidy. Sex chromosomes were not analyzed, according to the current protocol in Dutch laboratories.
RESULTS: NIPT was performed at a median gestational age of 20 weeks, indicated by the presence of multiple congenital anomalies (n = 13), isolated structural anomalies (n = 57), increased nuchal translucency ≥ 3.5 mm (n = 58), soft markers (n = 73), growth restriction (n = 40) and other anomalies (n = 10). NIPT results were normal in 224 (89.2%) pregnancies, inconclusive in one (0.4%) and abnormal in 26 (10.4%). Most genetic aberrations detected by NIPT were common whole-chromosome aneuploidies: trisomy 21 (n = 13), trisomy 18 (n = 6) and trisomy 13 (n = 3). Four further NIPT results were abnormal; one was suspected of being confined placental mosaicism and one was of maternal origin. In those with normal NIPT results, sonographic follow-up or examination of the newborn indicated the need for diagnostic genetic testing in 33/224 (14.7%) pregnancies. Clinically relevant genetic aberrations were revealed in 7/224 (3.1%) cases, two of which were whole-chromosome aneuploidies: trisomy 13 and monosomy X. As sex chromosomal aberrations are not included in NIPT analysis, the latter cannot be considered a false-negative result. Other discordant findings were subchromosomal aberrations (< 20 megabases, n = 2) and monogenic aberrations (n = 3).
CONCLUSIONS: NIPT should not be recommended for genetic evaluation of the etiology of ultrasound anomalies, as both resolution and sensitivity, or negative predictive value, are inferior to those of conventional karyotyping and microarray analysis. Nonetheless, some pregnant women consider NIPT to be an acceptable alternative to invasive diagnostic testing. © 2016 The Authors. Ultrasound in Obstetrics & Gynecology published by John Wiley & Sons Ltd on behalf of the International Society of Ultrasound in Obstetrics and Gynecology.