Objectives To evaluate the efficacy of non-invasive prenatal testing in detecting sex chromosome aneuploidies.Methods A total of 67,099 pregnant women were recruited in this retrospective observational study at Peking Union Medical College Hospital; 300 cases at high risk of sex chromosome aneuploidies were screened and 36 cases refused invasive prenatal diagnosis after detailed prenatal genetic counselling. The clinical data and prenatal diagnosis results were collected.Results 101 cases were in accordance with non-invasive prenatal testing results after invasive prenatal diagnosis: 95 cases had follow-up confirmation with abnormal sex aneuploidies (14 for monosomy X; 36 for 47, XXY; 22 for trisomy X and 23 for 47, XYY). The total positive predictive value was 38.26% (101/264). The accuracy of different Z scores was calculated, respectively, with positive predictive value ranging from 33.96% to 41.38% and a decreased sensitivity with rise of the Z score.Conclusions Non-invasive prenatal testing is an effective screening method for sex chromosome trisomy, rather than monosomy X, with relatively high accuracy. Z score = 3 is an optimum risk threshold in sex chromosome aneuploidies.

Optical genome mapping (OGM) is an emerging cytogenetic method for concurrently detecting structural variants (SVs) and copy number variants (CNVs). However, its clinical application in prenatal diagnosis remains underexplored. This study retrospectively evaluated the clinical validity of OGM in prenatal diagnosis by comparing with two routine genetic testing methods: karyotyping and chromosomal microarray analysis (CMA). Both positive and negative cases detected by routine genetic methods were enrolled to evaluate the technical concordance of OGM and its capability to improve diagnostic rate in negative cases. The exclusion criteria were balanced centromeric translocations, mosaic cases with cellular fractions < 20

OBJECTIVE:To develop a customized expanded carrier screening (ECS) solution for 8271 variants of 265 genes. Based on a microarray platform, this solution demonstrates comparable accuracy to next-generation sequencing (NGS) platform; however, it has a lower cost, a faster speed, and a simplified data-analysis process. METHOD:A dedicated panel composed of 8271 variants of 265 high-priority genes was developed for custom microarray fabrication. To test the performance of the custom microarray, we selected 1051 blood samples from an NGS-based ECS project to conduct a blind test. The evaluation indicators include sensitivity, specificity and positive predictive value. RESULTS:582 variants were identified in 69 genes. The concordance between the microarray platform and NGS platform results reaches 99.5%. The microarray demonstrated high sensitivity (99.8%) and specificity (99.8%), showing excellent performance in both positive and negative samples. The microarray platform reduces testing cycles to 3-4 days and lowers costs by 94%-96%. CONCLUSION:This study confirms that microarray-based carrier screening is a reliable, cost-effective, and timely alternative to NGS, and it is especially suitable for large-scale population screening.

INTRODUCTION:To evaluate the association between longitudinal changes in Observed-to-Expected Lung-to-Head Ratio (O/E LHR) measured by ultrasound and postnatal survival in fetuses with Congenital Diaphragmatic Hernia (CDH). METHODS:A total of 74 pregnant women carrying fetuses with CDH were retrospectively analyzed. The change in O/E LHR (ΔO/E LHR) was assessed as a continuous variable using univariate and multivariate logistic regression analyses. Hernia lateralization and liver herniation were combined into a single categorical variable to account for their clinical and statistical correlation. Receiver Operating Characteristic (ROC) analysis was performed to evaluate the discriminative ability of ΔO/E LHR for survival. RESULTS:The majority of patients (86% and 74% according to the first and last ultrasound examinations, respectively) in this cohort had mild CDH, with an overall survival rate of 73.0%. ΔO/E LHR differed significantly between survivors and non-survivors (13.32±22.09 vs. -13.93±17.59, p < 0.001). In multivariate analysis, ΔO/E LHR (OR = 1.089, 95% CI 1.024-1.158; p = 0.007), gestational age at diagnosis (OR = 1.462, 95% CI 1.041-2.053; p = 0.029), and the combined variable of hernia lateralization and liver herniation (p = 0.011) were associated with survival. ROC analysis of ΔO/E LHR yielded an AUC of 0.836 (95% CI 0.725-0.948). CONCLUSION:In this single-center cohort of fetuses with predominantly mild CDH, ΔO/E LHR was associated with postnatal survival and showed good discriminative performance. Gestational age at diagnosis and the combined variable of hernia lateralization and liver herniation were also associated with outcomes. However, estimates for individual anatomical subgroups were imprecise because of the limited number of cases and should not be used for individual risk prediction. These findings suggest that longitudinal assessment of O/E LHR may be more informative than a single measurement in this specific population. Further validation in larger cohorts is warranted.

This study aimed to resolve diagnostic discrepancies in which expanded carrier screening (ECS) in a healthy population suggested hemophilia A carrier status due to F8 intron 22 inversion (Inv22), while further analysis revealed a true distal Xq28 duplication. We evaluated the clinical utility of integrated genetic technologies in distinguishing between these two entities. A healthy pregnant woman underwent ECS, with long-distance polymerase chain reaction (LD-PCR) specifically employed for F8 Inv22 detection. Given the identified heterozygote of F8 inversion, prenatal diagnosis was conducted, integrating LD-PCR, single-nucleotide polymorphism array (SNP array), optical genome mapping (OGM), third-generation long-read sequencing, and X-chromosome inactivation (XCI) analysis to characterize the variant thoroughly. Initial LD-PCR suggested F8 Inv22 in both the fetus and the mother. SNP array revealed a 0.5-Mb Xq28 duplication spanning F8 exons 1-22. OGM and long-read sequencing confirmed the occurrence of duplication while excluding the F8 inversion, revealing int22h-1/int22h-2-mediated nonallelic homologous recombination as the mechanism. XCI analysis revealed skewed inactivation (18.1%) of the duplicated allele in the asymptomatic mother, whereas the fetus exhibited a random XCI pattern. This study highlights a critical diagnostic pitfall: even well-established techniques such as LD-PCR for F8 Inv22 may yield misleading results when applied to general population screening beyond their original diagnostic context. This underscores the importance of accurate interpretation of ECS results through appropriate analytic validation. We propose a tiered strategy: LD-PCR screening followed by OGM or long-read sequencing to differentiate F8 inversions from Xq28 duplications in the general population.

Optical genome mapping (OGM) has demonstrated significant potential in detecting structural variations (SVs) and has been comprehensively evaluated both retrospectively and prospectively in prenatal diagnosis. However, obtaining an adequate volume of amniotic fluid (AF) samples for OGM remains challenging due to the diverse detection techniques currently employed in prenatal diagnosis, which can limit the applicability of OGM in this setting. This study seeks to explore enhancements in cell culture techniques and quality control processes for prenatal samples when utilizing OGM in prenatal diagnosis. OGM successfully analyzed 188 AF samples with a minimum input of 0.225 million cells for ultra-high-molecular-weight DNA extraction. The study provides a comprehensive overview of the mass of chorionic villus samples, the volume of AF used for cell culture, the duration of culture, and the cell yields obtained for OGM. It was demonstrated that reducing the number of cells used for DNA isolation may not significantly decrease DNA quality for OGM with optimal cell viability and may even yield better results than those achieved with recommended cell amounts. This suggests that the current QC standards may be overly stringent, and that variant analysis remains feasible for some samples that do not meet these criteria. Based on the variant data analysis of these samples, standards appropriate for prenatal samples were summarized. The findings of this study indicate that a reduced volume of AF sample or a shortened cell culture duration can be achieved in prenatal OGM, thereby enhancing the feasibility of employing OGM in prenatal diagnosis and potentially benefiting patients. Furthermore, data QC metrics suitable for prenatal samples may be more tolerant than previously recommended, necessitating further investigation with larger cohorts to establish specific QC standards for prenatal samples.

The 15q11.2 BP1–BP2 microdeletion (Burnside-Butler syndrome) is a recurrent copy number variation associated with variable neurodevelopmental outcomes. Its incomplete penetrance and broad phenotypic spectrum challenge prenatal genetic counseling. This study aimed to characterize prenatal and postnatal clinical outcomes associated with the 15q11.2 BP1–BP2 microdeletion and to provide evidence to inform prenatal genetic counseling. Among 5246 high-risk pregnancies undergoing invasive prenatal diagnosis at a single center between January 2022 and February 2026, 25 fetuses with 15q11.2 BP1–BP2 microdeletion were identified by chromosomal microarray analysis. Of these, 9 fetuses showed soft ultrasound markers, 3 had fetal growth restriction, and 3 had major structural anomalies. Five pregnancies were electively terminated, whereas 20 were continued. Postnatal follow-up ranging from 3 months to 3 years and 8 months revealed no developmental or growth abnormalities among live-born children (penetrance 0

OBJECTIVE:To identify risk factors associated with noninvasive prenatal testing (NIPT) failures due to a low fetal fraction (LFF, < 4%) and to evaluate appropriate management strategies. METHODS:This was a single retrospective cohort study conducted on consecutive NIPT procedures performed at a national prenatal diagnosis center between April 2016 and June 2022. RESULTS:Among the 41,693 NIPT procedures conducted at Peking Union Medical College Hospital, 524 cases (1.3%) failed due to LFF. Testing failures were associated with an increased risk of rare fetal chromosomal abnormalities (OR 18.9). Independent risk factors for NIPT failure included antiphospholipid syndrome (OR 19.7), rare fetal chromosomal abnormalities (OR 17.9), body mass index ≥ 25 kg/m2 (OR 8.1), low molecular weight heparin administration (OR 7.7), systemic lupus erythematosus (OR 6.1), and dichorionic twins (OR 2.1). NIPT failure was also associated with a higher incidence of gestational hypertension (2.9% vs. 0.7%; OR 4.0), preeclampsia (9.1% vs. 1.0%; OR 10.4), gestational diabetes mellitus (26.0% vs. 11.8%; OR 2.6), fetal growth restriction (4.7% vs. 0.9%; OR 5.4), spontaneous abortion (2.4% vs. 0.6%; OR 4.1), and preterm birth (10.0% vs. 3.6%; OR 3.0). CONCLUSIONS:Pregnancies with NIPT failures are at a heightened risk for fetal chromosomal abnormalities and placenta-mediated complications, highlighting the need for enhanced monitoring and individualized management during perinatal care.

Purpose Secondary findings (SFs) identified through genomic sequencing are results unrelated to the primary indication but with potential clinical utility. Although genomic technologies are increasingly used in perinatal diagnosis, the clinical implications of SFs remain insufficiently explored. We evaluated the detection rates, phenotypic concordance, and clinical implications of SFs in a prenatal cohort undergoing trio exome sequencing (trio-ES). Methods This was a single-center cohort study of 424 consecutive families who underwent prenatal trio-ES at a tertiary maternal-fetal medicine center from January 2019 to December 2023. SFs were classified using a 3-category framework (medically actionable risks, childhood-onset diseases, and carrier status) and analyzed via a stepwise protocol, including validation by a specialized clinical review panel. Results Among 1272 individuals, SFs were identified in 2.9% (37/1272), including 2.1% of fetuses (9/424) and 3.3% of parents (28/848). SF-related phenotypes were observed in 1 fetus and 3 adults. SFs prompted medical management changes in 27.0% (10/37) of cases, including 1 pregnancy termination, 7 adults initiating medical evaluations, and 2 couples committing to prenatal diagnosis/neonatal screening in subsequent pregnancies. Conclusion Systematic SF reporting in prenatal diagnosis has demonstrated clinical value by facilitating pregnancy decisions, informing parental health risks, and enabling preventive reproductive strategies.

OBJECTIVE:Expanded carrier screening (ECS) is used to assess the reproductive probability of having an offspring affected by an autosomal recessive or X-linked recessive genetic disorder. Rarely, a presumably healthy individual may be identified to carry variants that could influence their health. This study aimed to investigate the incidence of potentially affected individuals, with the objective of improving counseling for ECS. METHODS:A retrospective study of a cohort of 3001 individuals who sought ECS during early pregnancy or preconception was conducted. Potentially affected individuals carrying biallelic pathogenic variants for autosomal recessive diseases, as well as females carrying a heterozygous pathogenic variant for X-linked disease, were identified. RESULTS:A total of 13 potentially affected individuals (0.43%) were identified; 5 patients were homozygous or compound heterozygous for autosomal recessive diseases, and 8 were heterozygous for X-linked diseases. The majority of the potentially affected individuals were asymptomatic (85%, 11/13) at the time of assessment. Only two patients exhibited mild clinical manifestations. CONCLUSION:People who intend to undergo ECS should be informed that they themselves may be identified as potentially affected individuals for whom clinical evaluation and surveillance are recommended.

Optical genome mapping (OGM) offers high consistency in simultaneously detecting structural and copy number variants. This study aimed to retrospectively evaluate the efficacy and potential applications of OGM in preconception genetic counseling. Herein, 74 samples from 37 families were included, and their results of OGM were compared to conventional methods, namely karyotyping (KT) and chromosomal microarray analysis (CMA), which identified 27 variants across 16 positive families. Notably, OGM achieved a concordance rate of 94.7% and 100% with KT and CMA, respectively, presenting an overall concordance of 96.3%, as it missed detecting a centromeric translocation. Additionally, OGM detected two cryptic balanced translocations and a small deletion in three families that were missed by conventional methods, improving the diagnostic rate by 5.4%, along with assisting in the diagnoses of six families (16.2%) by identifying complex rearrangements and confirming cryptic translocations. The combination of KT with OGM yielded the highest diagnostic rate in all families. Overall, the findings of this study present the notable potential of OGM for its application, combined with KT per requirement, in clinical settings to improve the efficiency and accuracy of diagnoses and rapid screening of individuals seeking preconception genetic counseling.

OBJECTIVE:To evaluate the diagnostic value of prenatal exome sequencing (ES) integrated with copy number variant (CNV) and single nucleotide variant (SNV) analysis (ES-CNV/SNV) in fetuses with structural anomalies following negative chromosomal microarray analysis (CMA) and karyotyping, and to delineate the practical challenges encountered during its clinical implementation in prenatal settings. METHODS:In this multicenter prospective cohort study (2018-2021), 275 fetuses with structural anomalies were categorized into three groups: sporadic single-system (n = 128), sporadic multisystem (n = 88), and recurrent anomalies (n = 59). Trio-based ES-CNV/SNV analysis was used to identify SNVs, CNVs, or compound heterozygous CNV/SNV combinations- the latter being defined as two distinct pathogenic variants (a CNV and an SNV) coexisting within the same gene, resulting in biallelic gene dysfunction, which is pivotal for autosomal recessive disorders. Incremental diagnostic yields across groups, the incidence of CNV/SNV combinations, and expanded prenatal phenotypic-genotypic correlations were analyzed. RESULTS:ES-CNV/SNV analysis improved the diagnostic yield to 29.45% (81/275) compared with conventional chromosome analysis combined with CMA in fetuses with malformations. The highest diagnostic yield was observed in the recurrent anomaly group (40.68%, 24/59), followed by the multisystem (28.41%, 25/88) and single-system anomaly groups (25.00%, 32/128). Notably, 1.45% of cases harbored compound CNV/SNV combinations, underscoring the diagnostic potential of ES-CNV/SNV in autosomal recessive disorders. In addition, this study expanded prenatal phenotypic-genotypic correlations for 36 variants and five genes. CONCLUSION:ES-CNV/SNV analysis enhances prenatal diagnostic precision by detecting compound CNV/SNV combinations that are undetectable by standard testing. Recurrent anomalies demonstrated a greater diagnostic benefit than sporadic single-system or multisystem anomalies, underscoring the utility of this method in high-risk cases. This study broadens the prenatal phenotypic spectrum and deepens our understanding of developmental mechanisms.

The aim of this study was to investigate the detection capability of medium-coverage whole-genome sequencing for chromosomal mosaicism in prenatal diagnosis, and to evaluate the pregnancy outcome of mosaicism. Thirty-four prenatal samples with chromosomal mosaicism diagnosed by chromosomal microarray analysis with single nucleotide polymorphism (CMA/SNP) were included and subsequently subjected to chromosome analysis by medium-coverage whole-genome sequencing (CMA-seq) for back-to-back comparison. The results of karyotyping or fluorescence in situ hybridization (FISH) were reviewed for additional validation. Data on pregnancy outcomes were collected to analyze the effect of mosaicism on fetuses. In total, 13 mosaic autosomal trisomies, 9 sex chromosome aneuploidies (SCAs), and 12 mosaic copy number variants (CNVs) (11 pathogenic or likely pathogenic) were detected by CMA-seq. Twenty-six cases were classified as having high-level (≥ 30

Fetal skeletal dysplasia (FSD) encompasses diverse clinical features and complicates prenatal diagnosis and perinatal care. In this retrospective study, we integrate prenatal deep phenotyping with exome or genome sequencing (ES/GS) to elucidate comprehensive genotype and phenotype landscapes, diagnostic outcomes, genotype-phenotype correlations, and postnatal follow-up findings and to refine genetic counseling and clinical decision-making. The study includes a cohort of 152 fetuses with FSD in China. All fetuses undergo prenatal deep phenotyping followed by ES/GS analysis. Prenatal deep phenotyping enables classification into isolated and non-isolated FSD groups and identifies previously unrecognized prenatal features associated with KBG syndrome and Segawa syndrome. Among skeletal anomalies, limb bone anomalies are the most common (72.4%). Genetic testing yields positive diagnoses in 88 fetuses (57.9%). Notably, fetuses with cranial and limb bone abnormalities demonstrate a higher diagnostic yield. Comparative analysis of prenatal and postnatal genotypes and phenotypes in individuals harboring pathogenic variants in four hotspot genes provides a deeper understanding of skeletal dysplasia phenotypes. Genetic findings from this cohort directly inform reproductive decisions in 16 subsequent pregnancies. Our findings significantly enhance genotype-phenotype correlations and contribute to improved prenatal counseling, informed clinical decision-making, and optimized perinatal care, and advance precision medicine strategies for FSD-affected families.

Expanded carrier screening (ECS) is crucial in preconception and early pregnancy care for identifying couples at risk of having children with autosomal recessive or X-linked conditions. This study developed a custom ECS array using an array platform designed to detect single nucleotide variations (SNVs) and indels across 1,625 genes associated with more than 1,000 genetic disorders. To validate the performance of the custom array, we utilized 1,112 blood samples from which next-generation sequencing (NGS)-based ECS was performed. Among these samples, 225 distinct variants were identified in 100 genes. The array demonstrated high sensitivity (98.62%) and specificity (99.17%). This custom array offers a standardized and efficient alternative to NGS-based ECS, potentially enhancing clinical accessibility and selectivity.

Advances in next-generation sequencing (NGS) have revolutionized clinical and translational medicine. NGS is increasingly used in the diagnosis of genetic diseases, including newborn screening, prenatal screening and diagnosis, and preimplantation screening. While advancements in sequencing and bioinformatics have facilitated the discovery of novel genes and diseases, the identification of variants continues to outpace our ability to classify them accurately. Variants of uncertain significance (VUS), defined as genetic alterations with insufficient evidence for clinical interpretation, present significant diagnostic challenges. Clinically available functional studies, such as biochemical testing, offer valuable insights for interpreting VUS. This review synthesizes recent literature on amniotic fluid metabolite analysis for prenatal diagnosis and VUS interpretation in four organic acidurias. It aims to guide the application of biochemical testing for inherited metabolic disorders and enhance clinical decision-making regarding VUS classification.

OBJECTIVE:Congenital diaphragmatic hernia (CDH) is a rare abnormality with highly heterogeneous genetic causes. This study investigated chromosomal and monogenic abnormalities in fetal CDH patients and evaluated the efficacy of chromosomal microarray analysis (CMA) and whole-exome sequencing (WES) for genetic diagnosis. The clinical features of the patients were also evaluated. METHODS:We evaluated the genetic and clinical data of 51 prenatally diagnosed fetuses with CDH. CMA was performed for every patient. If CMA did not yield diagnostic results, the samples were subjected to WES. RESULTS:Compared with fetuses with isolated CDH (n = 42), those with non-isolated CDH (n = 9) presented a higher genetic diagnostic rate (22.2% vs. 2.4%). The overall diagnostic yield was 5.9%, comprising 3.9% from chromosomal microarray analysis (CMA) and an additional 2.0% from whole exome sequencing (WES). CMA identified (1) mosaic trisomy 18 in a patient with isolated CDH; and (2) 4q terminal deletion syndrome in a patient with non-isolated CDH. WES identified a novel missense mutation, PLS3 c.1763A > G, associated with X-linked CDH in a patient with non-isolated CDH and a family history of recurrent CDH. CONCLUSION:Genetic testing should be offered for all fetuses with CDH, regardless of whether the cases are isolated or non-isolated. WES should be considered if CMA fails to provide a diagnostic result, particularly in patients with non-isolated CDH and a family history of recurrent CDH.

Fetal growth restriction (FGR), a leading cause of perinatal morbidity and mortality, is caused by fetal, maternal, and placental factors. Uniparental disomy (UPD) is a rare condition that leads to imprinting effects, low-level mosaic aneuploidies and homozygosity for pathogenic variants. In the present study, UPD events were detected in 5 women with FGR by trio exome sequencing (trio-WES) of a cohort of 150 FGR cases. Furthermore, noninvasive prenatal testing results of the 5 patients revealed a high risk of rare autosomal trisomy. Trio-WES showed no copy-number variations (CNVs) or nondisease-causing mutations associated with FGR. Among the 5 women with FGR, two showed gene imprinting, and two exhibited confined placental mosaicism (CPM) by copy number variant sequencing (CNV-seq). The present study showed that in FGR patients with UPD, the detection of imprinted genes and CPM could enhance the genetic diagnosis of FGR.

Complex chromosomal rearrangements (CCRs) can result in spontaneous abortions, infertility, and malformations in newborns. In this study, we explored a familial CCR involving chromosome 6 by combining optical genomic mapping (OGM) and molecular cytogenetic methodologies. Within this family, the father and the paternal grandfather were both asymptomatic carriers of an identical balanced CCR, while the two offspring with an unbalanced paternal-origin CCR and two microdeletions presented with clinical manifestation. The first affected child, a 5-year-old boy, exhibited neurodevelopmental delay, while the second, a fetus, presented with hydrops fetalis. SNP-genotype analysis revealed a recombination event during gamete formation in the father that may have contributed to the deletion in his offspring. Meanwhile, the couple’s haplotypes will facilitate the selection of normal gametes in the setting of assisted reproduction. Our study demonstrated the potential of OGM in identifying CCRs and its ability to work with current methodologies to refine precise breakpoints and construct accurate haplotypes for couples with a CCR.