Early detection of active Human CytomegaloVirus (hCMV) infection in pregnant women using data generated for noninvasive fetal aneuploidy testing.

BACKGROUND: Prenatal hCMV infections can lead to severe embryopathy and neurological sequelae in neonates. Screening during pregnancy is not recommended by global societies, as there is no effective therapy. Recently, several groups showed that maternal-fetal hCMV transmission can be strongly reduced by administering anti-viral agents early in pregnancy. This calls for a screening method to identify at risk pregnancies at an appropriate gestational age, with the possibility for large-scale enrolment. Non-Invasive Prenatal Testing (NIPT) for fetal aneuploidy screening early in pregnancy is already implemented in many countries and performed on a large-scale basis. We investigated the use of whole genome cell-free DNA (cfDNA) sequencing data, generated for the purpose of NIPT, as (pre-)screening tool to identify women with active hCMV-infections, eligible for therapy. METHODS: Coded raw sequencing NIPT data from 204,818 pregnant women from three testing laboratories were analyzed for the presence of hCMV-cfDNA. Samples were stratified by cfDNA-hCMV load. For validation and interpretation, diagnostic hCMV-qPCR and serology testing were performed on a subset of cfDNA-hCMV-positive (n = 112) and -negative (n = 127) samples. FINDINGS: In 1930 samples (0.94%) hCMV fragments were detected. Validation by hCMV-qPCR showed that samples with high cfDNA-hCMV load tested positive and cfDNA-hCMV-negative samples tested negative. In 32/112 cfDNA-hCMV-positive samples (28.6%) the serological profile suggested a recent primary infection: this was more likely in samples with high cfDNA-hCMV load (78.6%) than in samples with low cfDNA-hCMV load (11.0%). In none of the cfDNA-hCMV-negative samples serology was indicative of a recent primary infection. INTERPRETATION: Our study shows that large-scale (pre-)screening for both genetic fetal aberrations and active maternal hCMV infections during pregnancy can be combined in one cfDNA sequencing test, performed on a single blood sample, drawn in the first trimester of pregnancy. FUNDING: This work was partly funded by the Prenatal Screening Foundation Nijmegen, the Netherlands.

Evaluating the effectiveness of pre-operative diagnosis of ovarian cancer using minimally invasive liquid biopsies by combining serum human epididymis protein 4 and cell-free DNA in patients with an ovarian mass.

OBJECTIVE: To assess the feasibility of scalable, objective, and minimally invasive liquid biopsy-derived biomarkers such as cell-free DNA copy number profiles, human epididymis protein 4 (HE4), and cancer antigen 125 (CA125) for pre-operative risk assessment of early-stage ovarian cancer in a clinically representative and diagnostically challenging population and to compare the performance of these biomarkers with the Risk of Malignancy Index (RMI). METHODS: In this case-control study, we included 100 patients with an ovarian mass clinically suspected to be early-stage ovarian cancer. Of these 100 patients, 50 were confirmed to have a malignant mass (cases) and 50 had a benign mass (controls). Using WisecondorX, an algorithm used extensively in non-invasive prenatal testing, we calculated the benign-calibrated copy number profile abnormality score. This score represents how different a sample is from benign controls based on copy number profiles. We combined this score with HE4 serum concentration to separate cases and controls. RESULTS: Combining the benign-calibrated copy number profile abnormality score with HE4, we obtained a model with a significantly higher sensitivity (42% vs 0%; p<0.002) at 99% specificity as compared with the RMI that is currently employed in clinical practice. Investigating performance in subgroups, we observed especially large differences in the advanced stage and non-high-grade serous ovarian cancer groups. CONCLUSION: This study demonstrates that cell-free DNA can be successfully employed to perform pre-operative risk of malignancy assessment for ovarian masses; however, results warrant validation in a more extensive clinical study.

Current controversies in prenatal diagnosis: Noninvasive prenatal testing should replace other screening strategies for fetal trisomies 13, 18, 21.

This is a written summary of the oral debate presented at the International Society for Prenatal Diagnosis annual conference in Edinburgh in 2023. The topic under debate is whether noninvasive prenatal testing (NIPT) using cell-free fetal DNA should replace other screening strategies for the detection of fetal trisomies 13, 18, 21. There is no disagreement that NIPT is far more sensitive and has better positive predictive values for identifying trisomies 13, 18, and 21 than traditional screening approaches using biochemical markers and measurement of nuchal translucency. The major issue lies in the potential adverse consequences associated with abandoning traditional screening methods. The source of disagreement stems primarily from whether you consider the role of ultrasound in the context of screening to be strictly for nuchal translucency measurement or whether it should be combined with a fetal anatomy scan. The debate featured two experts who presented evidence in favor of each argument.

Fetal fraction of cell-free DNA in noninvasive prenatal testing and adverse pregnancy outcomes: a nationwide retrospective cohort study of 56,110 pregnant women.

BACKGROUND: Noninvasive prenatal testing by cell-free DNA analysis is offered to pregnant women worldwide to screen for fetal aneuploidies. In noninvasive prenatal testing, the fetal fraction of cell-free DNA in the maternal circulation is measured as a quality control parameter. Given that fetal cell-free DNA originates from the placenta, the fetal fraction might also reflect placental health and maternal pregnancy adaptation. OBJECTIVE: This study aimed to assess the association between the fetal fraction and adverse pregnancy outcomes. STUDY DESIGN: We performed a retrospective cohort study of women with singleton pregnancies opting for noninvasive prenatal testing between June 2018 and June 2019 within the Dutch nationwide implementation study (Trial by Dutch Laboratories for Evaluation of Non-Invasive Prenatal Testing [TRIDENT]-2). Multivariable logistic regression analysis was used to assess associations between fetal fraction and adverse pregnancy outcomes. Fetal fraction was assessed as a continuous variable and as <10th percentile, corresponding to a fetal fraction <2.5%. RESULTS: The cohort comprised 56,110 pregnancies. In the analysis of fetal fraction as a continuous variable, a decrease in fetal fraction was associated with increased risk of hypertensive disorders of pregnancy (adjusted odds ratio, 2.27 [95% confidence interval, 1.89-2.78]), small for gestational age neonates <10th percentile (adjusted odds ratio, 1.37 [1.28-1.45]) and <2.3rd percentile (adjusted odds ratio, 2.63 [1.96-3.57]), and spontaneous preterm birth from 24 to 37 weeks of gestation (adjusted odds ratio, 1.02 [1.01-1.03]). No association was found for fetal congenital anomalies (adjusted odds ratio, 1.02 [1.00-1.04]), stillbirth (adjusted odds ratio, 1.02 [0.96-1.08]), or neonatal death (adjusted odds ratio, 1.02 [0.96-1.08]). Similar associations were found for adverse pregnancy outcomes when fetal fraction was <10th percentile. CONCLUSION: In early pregnancy, a low fetal fraction is associated with increased risk of adverse pregnancy outcomes. These findings can be used to expand the potential of noninvasive prenatal testing in the future, enabling the prediction of pregnancy complications and facilitating tailored pregnancy management through intensified monitoring or preventive measures.

Association between low fetal fraction in cell-free DNA screening and fetal chromosomal aberrations: A systematic review and meta-analysis.

OBJECTIVE: To perform a systematic review and meta-analysis of the available literature on low fetal fraction (LFF) in cell-free DNA (cfDNA) screening and the risk of fetal chromosomal aberrations. METHOD: We searched articles published between January 2010 and May 2021 in PubMed and EMBASE databases. Risk of bias was assessed using QUADAS-2. RESULTS: Twenty-seven studies met the inclusion criteria, comprising data of 243,700 singleton pregnancies. Compared to normal fetal fraction, LFF was associated with a higher risk of trisomy 13 (OR 5.99 [3.61-9.95], I 2 of heterogeneity = 0%, n = 22 studies), trisomy 18 (OR 4.46 [3.07-6.47], I 2  = 0%, n = 22 studies), monosomy X (OR 5.88 [2.34-14.78], I 2  = 18%, n = 10 studies), and triploidy (OR 36.39 [9.83-134.68], I 2  = 61%, n = 6 studies), but not trisomy 21 (OR 1.25 [0.76-2.03], I 2  = 36%, n = 23 studies). LFF was also associated with a higher risk of various other types of fetal chromosomal aberrations (OR 4.00 [1.78-9.00], I 2  = 2%, n = 11 studies). Meta-analysis of proportions showed that absolute rates of fetal chromosomal aberrations ranged between 1% and 2% in women with LFF. A limitation of this review is the potential risk of ascertainment bias because of differences in outcome assessment between pregnancies with LFF and those with normal fetal fraction. Heterogeneity in population characteristics or applied technologies across included studies may not have been fully addressed. CONCLUSION: An LFF test result in cfDNA screening is associated with an increased risk of fetal trisomy 13, trisomy 18, monosomy X, and triploidy, but not trisomy 21. Further research is needed to assess the association between LFF and other specific types of fetal chromosomal aberrations.

A comprehensive performance analysis of sequence-based within-sample testing NIPT methods.

BACKGROUND: Non-Invasive Prenatal Testing is often performed by utilizing read coverage-based profiles obtained from shallow whole genome sequencing to detect fetal copy number variations. Such screening typically operates on a discretized binned representation of the genome, where (ab)normality of bins of a set size is judged relative to a reference panel of healthy samples. In practice such approaches are too costly given that for each tested sample they require the resequencing of the reference panel to avoid technical bias. Within-sample testing methods utilize the observation that bins on one chromosome can be judged relative to the behavior of similarly behaving bins on other chromosomes, allowing the bins of a sample to be compared among themselves, avoiding technical bias. RESULTS: We present a comprehensive performance analysis of the within-sample testing method Wisecondor and its variants, using both experimental and simulated data. We introduced alterations to Wisecondor to explicitly address and exploit paired-end sequencing data. Wisecondor was found to yield the most stable results across different bin size scales while producing more robust calls by assigning higher Z-scores at all fetal fraction ranges. CONCLUSIONS: Our findings show that the most recent available version of Wisecondor performs best.

A cross-country comparison of pregnant women's decision-making and perspectives when opting for non-invasive prenatal testing in the Netherlands and Belgium.

BACKGROUND: The Netherlands and Belgium have been among the first countries to offer non-invasive prenatal testing (NIPT) as a first-tier screening test. Despite similarities, differences exist in counseling modalities and test uptake. This study explored decision-making and perspectives of pregnant women who opted for NIPT in both countries. METHODS: A questionnaire study was performed among pregnant women in the Netherlands (NL) (n = 587) and Belgium (BE) (n = 444) opting for NIPT, including measures on informed choice, personal and societal perspectives on trisomy 21, 18 and 13 and pregnancy termination. RESULTS: Differences between Dutch and Belgian women were shown in the level of informed choice (NL: 83% vs. BE: 59%, p < 0.001), intention to terminate the pregnancy in case of confirmed trisomy 21 (NL: 51% vs. BE: 62%, p = 0.003) and trisomy 13/18 (NL: 80% vs. BE: 73%, p = 0.020). More Belgian women considered trisomy 21 a severe condition (NL: 64% vs. BE: 81%, p < 0.001). Belgian women more frequently indicated that they believed parents are judged for having a child with trisomy 21 (BE: 42% vs. NL: 16%, p < 0.001) and were less positive about quality of care and support for children with trisomy 21 (BE: 23% vs. NL: 62%, p < 0.001). CONCLUSION: Differences in women's decision-making regarding NIPT and the conditions screened for may be influenced by counseling aspects and country-specific societal and cultural contexts.

In Vitro CRISPR-Cas12a-Based Detection of Cancer-Associated TP53 Hotspot Mutations Beyond the crRNA Seed Region.

Cost-effective and time-efficient detection of oncogenic mutations supports improved presymptomatic cancer diagnostics and post-treatment disease monitoring. Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas12a is an RNA-guided endonuclease that, upon protospacer adjacent motif (PAM)-dependent recognition of target DNA in cis, exhibits indiscriminate ssDNase activity in trans, which can be harnessed for diagnostics. TP53, one of the most frequently mutated tumor suppressor genes in cancer, displays recurring point mutations at so-called "hotspots." In this study, we optimized Cas12a-based assay conditions for in vitro detection of six TP53 hotspot mutations at the codon for p.R273, located outside the Cas12a seed region, and evaluated the specificities of four commercial Cas12a variants. We found that nonengineered LbCas12a significantly outperformed the other tested nucleases specifically in distinguishing mutant p.R273 codons in synthetic DNA, mock cell-free DNA, and tissue biopsies, despite the suboptimal PAM-distal positioning of the corresponding mutations. Future clinical Cas12a-based applications may include point-of-care tumor analysis, cost-effective mutation screening, and improved monitoring of individual cancer patients.

Variability in Fetal Fraction Estimation: Comparing Fetal Fractions Reported by Noninvasive Prenatal Testing Providers Globally.

BACKGROUND: Fetal fraction (FF) measurement is considered important for reliable noninvasive prenatal testing (NIPT). Using minimal FF threshold as a quality parameter is under debate. We evaluated the variability in reported FFs of individual samples between providers and laboratories and within a single laboratory. METHODS: Genomic quality assessment and European Molecular Genetics Quality Network provide joint proficiency testing for NIPT. We compared reported FFs across all laboratories and stratified according to test methodologies. A single sample was sequenced repeatedly and FF estimated by 2 bioinformatics methods: Veriseq2 and SeqFF. Finally, we compared FFs by Veriseq and SeqFF in 87 351 NIPT samples. RESULTS: For each proficiency test sample we observed a large variability in reported FF, SDs and CVs ranging from 1.7 to 3.6 and 17.0 to 35.8, respectively. FF measurements reported by single nucleotide polymorphism-based methods had smaller SDs (0.5 to 2.4) compared to whole genome sequencing-based methods (1.8 to 2.9). In the internal quality assessment, SDs were similar between SeqFF (SD 1.0) and Veriseq v2 (SD 0.9), but mean FF by Veriseq v2 was higher compared to SeqFF (9.0 vs 6.4, P 0.001). In patient samples, reported FFs were on average 1.12-points higher in Veriseq than in SeqFF (P 0.001). CONCLUSIONS: Current methods do not allow for a reliable and consistent FF estimation. Our data show estimated FF should be regarded as a laboratory-specific range, rather than a precise number. Applying strict universal minimum thresholds might result in unnecessary test failures and should be used with caution.

Experiences of pregnant women with genome-wide non-invasive prenatal testing in a national screening program.

Pregnant women's perspectives should be included in the dialogue surrounding the expanding offers of non-invasive prenatal testing (NIPT), especially now that technological possibilities are rapidly increasing. This study evaluated women's experiences with the offer of genome-wide (GW) first-tier NIPT in a national screening program. A nationwide pre-and post-test questionnaire was completed by 473 pregnant women choosing between targeted NIPT (trisomies 21, 18 and 13 only) and GW-NIPT (also other findings) within the Dutch TRIDENT-2 study. Measures included satisfaction, reasons for or against choosing GW-NIPT, anxiety, and opinion on the future scope of NIPT. Most respondents (90.4%) were glad to have been offered the choice between GW-NIPT and targeted NIPT; 76.5% chose GW-NIPT. Main reasons to choose GW-NIPT were 'wanting as much information as possible regarding the child's health' (38.6%) and 'to be prepared for everything' (23.8%). Main reasons to choose targeted NIPT were 'avoiding uncertain results/outcomes' (33.7%) and 'not wanting to unnecessarily worry' (32.6%). Nearly all respondents received a low-risk NIPT result (98.7%). No differences were found in anxiety between women choosing GW-NIPT and targeted NIPT. Most respondents were favorable toward future prenatal screening for a range of conditions, including life-threatening disorders, mental disabilities, disorders treatable in pregnancy and severe physical disabilities, regardless of their choice for GW-NIPT or targeted NIPT. In conclusion, women who chose first-tier NIPT were satisfied with the choice between GW-NIPT and targeted NIPT, and most women were favorable toward a broader future screening offer. Our results contribute to the debate concerning the expansion of NIPT.

Clinically relevant DNA viruses in pregnancy.

Infections by DNA viruses during pregnancy are associated with increased health risks to both mother and fetus. Although not all DNA viruses are related to an increased risk of complications during pregnancy, several can directly infect the fetus and/or cause placental dysfunction. During Non-Invasive Prenatal Testing analysis, the presence of viral DNA can be detected, theoretically allowing screening early in pregnancy. Although treatment options are currently limited, this might rapidly change in the near future. It is therefore important to be aware of the potential impact of these viruses on feto-maternal health. In this manuscript we provide a brief introduction into the most commonly detected DNA viruses in human cell-free DNA sequencing experiments and their pathogenic potential during pregnancy.

The cell-free DNA virome of 108,349 Dutch pregnant women.

OBJECTIVE: Viral infections during pregnancy are a major health concern to mother and fetus. By repurposing cell-free Non Invasive Prenatal Testing (NIPT) sequencing data, we investigated prevalence and abundance of viral DNA in a cohort of 108,349 pregnant women. METHOD: Cell-free DNA (cfDNA) sequencing reads that did not map to any of the human chromosomes or mitochondrial DNA of the human reference genome build GRCh38 were aligned to 224 DNA viruses selected from the NCBI refseq viral database. RESULTS: In total 443,665 reads of viral origin were detected across 42,273 samples representing 165 viral species. Several are known to be potentially harmful during pregnancy and/or childbirth, including Cytomegalovirus, Parvovirus B19 and Hepatitis B. Viral sequences were mostly detected at very low abundance. However, several cases had exceptionally high viral loads for Parvovirus B19, Hepatitis B and others. We found statistically significant associations between presence of viral DNA and gestational age, maternal age, fetal fraction, cfDNA concentration and others. CONCLUSION: We demonstrate the feasibility to detect viral DNA from typical genome-wide NIPT cfDNA sequencing and describe the main characteristics of the viral DNA in our cohort. Our dataset of detected viral sequence reads is made publicly available to guide future clinical implementations.

Additional value of advanced ultrasonography in pregnancies with two inconclusive cell-free DNA draws.

OBJECTIVE: We aimed to evaluate the additional value of advanced fetal anatomical assessment by ultrasound in pregnancies with twice inconclusive noninvasive testing (NIPT) due to low fetal fraction (FF). METHODS: We performed a multicenter-retrospective study between 2017 and 2020 including 311 pregnancies with twice inconclusive NIPT due to low FF ≤ 1%. Women were offered invasive testing and advanced fetal anatomical assessment at ≤18 weeks' gestation. Ultrasound findings, genetic testing, and pregnancy/postnatal outcomes were evaluated. RESULTS: Ninety-two/311 (29.6%) women underwent invasive testing. Structural anomalies were diagnosed in 13/311 (4.2%) pregnancies (nine at the first scan and four at follow-up). In 6/13 (46.2%) cases, genetic aberrations were confirmed (one case of Trisomy 13 (detectable by NIPT), two of Triploidy, one of 16q12-deletion, HCN4-mutation and UPD(16) (nondetectable by NIPT). Genetic aberrations were found in 4/298 (1.3%) structurallynormal pregnancies (one 47XYY, two microscopic aberrations, one monogenic disorder found postpartum). Structural anomalies in genetically normal fetuses (2.0%) were not more prevalent compared to the general pregnant population (OR 1.0 [0.4-2.2]). CONCLUSION: In pregnancies with twice inconclusive NIPT due to low FF, fetal structural anomalies are not more prevalent than in the general obstetric population. The detailed anatomical assessment has the added value to detect phenotypical features suggestive of chromosomal/genetic aberrations and identify pregnancies where advanced genetic testing may be indicated.

Osteogenic transdifferentiation of primary human fibroblasts to osteoblast-like cells with human platelet lysate.

Inherited bone disorders account for about 10% of documented Mendelian disorders and are associated with high financial burden. Their study requires osteoblasts which play a critical role in regulating the development and maintenance of bone tissue. However, bone tissue is not always available from patients. We developed a highly efficient platelet lysate-based approach to directly transdifferentiate skin-derived human fibroblasts to osteoblast-like cells. We extensively characterized our in vitro model by examining the expression of osteoblast-specific markers during the transdifferentiation process both at the mRNA and protein level. The transdifferentiated osteoblast-like cells showed significantly increased expression of a panel of osteogenic markers. Mineral deposition and ALP activity were also shown, confirming their osteogenic properties. RNA-seq analysis allowed the global study of changes in the transcriptome of the transdifferentiated cells. The transdifferentiated cells clustered separately from the primary fibroblasts with regard to the significantly upregulated genes indicating a distinct transcriptome profile; transdifferentiated osteoblasts also showed significant enrichment in gene expression related to skeletal development and bone mineralization. Our presented in vitro model may potentially contribute to the prospect of studying osteoblast-dependent disorders in patient-derived cells.

Clinical impact of additional findings detected by genome-wide non-invasive prenatal testing: Follow-up results of the TRIDENT-2 study.

In the TRIDENT-2 study, all pregnant women in the Netherlands are offered genome-wide non-invasive prenatal testing (GW-NIPT) with a choice of receiving either full screening or screening solely for common trisomies. Previous data showed that GW-NIPT can reliably detect common trisomies in the general obstetric population and that this test can also detect other chromosomal abnormalities (additional findings). However, evidence regarding the clinical impact of screening for additional findings is lacking. Therefore, we present follow-up results of the TRIDENT-2 study to determine this clinical impact based on the laboratory and perinatal outcomes of cases with additional findings. Between April 2017 and April 2019, additional findings were detected in 402/110,739 pregnancies (0.36%). For 358 cases, the origin was proven to be either fetal (n = 79; 22.1%), (assumed) confined placental mosaicism (CPM) (n = 189; 52.8%), or maternal (n = 90; 25.1%). For the remaining 44 (10.9%), the origin of the aberration could not be determined. Most fetal chromosomal aberrations were pathogenic and associated with severe clinical phenotypes (61/79; 77.2%). For CPM cases, occurrence of pre-eclampsia (8.5% [16/189] vs 0.5% [754/159,924]; RR 18.5), and birth weight <2.3rd percentile (13.6% [24/177] vs 2.5% [3,892/155,491]; RR 5.5) were significantly increased compared to the general obstetric population. Of the 90 maternal findings, 12 (13.3%) were malignancies and 32 (35.6%) (mosaic) pathogenic copy number variants, mostly associated with mild or no clinical phenotypes. Data from this large cohort study provide crucial information for deciding if and how to implement GW-NIPT in screening programs. Additionally, these data can inform the challenging interpretation, counseling, and follow-up of additional findings.

Noninvasive Prenatal Test Results Indicative of Maternal Malignancies: A Nationwide Genetic and Clinical Follow-Up Study.

PURPOSE: Noninvasive prenatal testing (NIPT) for fetal aneuploidy screening using cell-free DNA derived from maternal plasma can incidentally raise suspicion for cancer. Diagnostic routing after malignancy suspicious-NIPT faces many challenges. Here, we detail malignancy suspicious-NIPT cases, and describe the clinical characteristics, chromosomal aberrations, and diagnostic routing of the patients with a confirmed malignancy. Clinical lessons can be learned from our experience. METHODS: Patients with NIPT results indicative of a malignancy referred for tumor screening between April 2017 and April 2020 were retrospectively included from a Dutch nationwide NIPT implementation study, TRIDENT-2. NIPT profiles from patients with confirmed malignancies were reviewed, and the pattern of chromosomal aberrations related to tumor type was analyzed. We evaluated the diagnostic contribution of clinical and genetic examinations. RESULTS: Malignancy suspicious-NIPT results were reported in 0.03% after genome-wide NIPT, and malignancies confirmed in 16 patients (16/48, 33.3%). Multiple chromosomal aberrations were seen in 23 of 48 patients with genome-wide NIPT, and a malignancy was confirmed in 16 patients (16/23, 69.6%). After targeted NIPT, 0.005% malignancy suspicious-NIPT results were reported, in 2/3 patients a malignancy was confirmed. Different tumor types and stages were diagnosed, predominantly hematologic malignancies (12/18). NIPT data showed recurrent gains and losses in primary mediastinal B-cell lymphomas and classic Hodgkin lymphomas. Magnetic resonance imaging and computed tomography were most informative in diagnosing the malignancy. CONCLUSION: In 231,896 pregnant women, a low percentage (0.02%) of NIPT results were assessed as indicative of a maternal malignancy. However, when multiple chromosomal aberrations were found, the risk of a confirmed malignancy was considerably high. Referral for extensive oncologic examination is recommended, and may be guided by tumor-specific hallmarks in the NIPT profile.