A statistical investigation of parameters associated with low cell-free fetal DNA fraction in maternal plasma for noninvasive prenatal testing.

BACKGROUND: Noninvasive prenatal testing (NIPT) is the most common method for prenatal aneuploidy screening. Low fetal fraction (LFF) is the primary reason for NIPT failure. Consequently, factors associated with LFF should be elucidated for optimal clinical implementation of NIPT. METHODS: In this study, NIPT data from January 2019 to December 2022 from the laboratory records and obstetrical and neonatal data from the electronic medical records were collected and analyzed. Subjects with FF >3.50% were assigned to the control group, subjects with FF <3.50% once were assigned to the LFF group, and subjects with FF <3.50% twice were assigned to the repetitive low fetal fraction (RLFF) group. Factors, including body mass index (BMI), gestational age, maternal age, twin pregnancy, and in vitro fertilization (IVF) known to be associated with LFF were assessed by Kruskal-Wallis H test and logistic regression. Clinical data on first trimester pregnancy-associated plasma protein-A (PAPP-A), beta-human chorionic gonadotropin (ß-hCG), gestational age at delivery, birth weight at delivery, and maternal diseases were obtained from the hospital's prenatal and neonatal screening systems (twin pregnancy was not included in the data on gestational age at delivery and the control group did not include data on maternal diseases.), and were analyzed using Kruskal-Wallis H test and Chi-square test. RESULTS: Among the total of 63,883 subjects, 63,605 subjects were assigned to the control group, 197 subjects were assigned to the LFF group, and 81 subjects were assigned to the RLFF group. The median of BMI in the three groups was 22.43 kg/m2 (control), 25.71 kg/m2 (LFF), and 24.54 kg/m2 (RLFF). The median gestational age in the three groups was 130 days (control), 126 days (LFF), and 122/133 days (RLFF). The median maternal age in the three groups was 29 (control), 29 (LFF), and 33-years-old (RLFF). The proportion of twin pregnancies in the three groups was 3.3% (control), 10.7% (LFF), and 11.7% (RLFF). The proportion of IVF in the three groups was 4.7% (control), 11.7% (LFF), and 21.3% (RLFF). The factors significantly associated with LFF included BMI [2.18, (1.94, 2.45), p < 0.0001], gestational age [0.76, (0.67, 0.87), p < 0.0001], twin pregnancy [1.62, (1.02, 2.52), p = 0.0353], and IVF [2.68, (1.82, 3.86), p < 0.0001]. The factors associated with RLFF included maternal age [1.54, (1.17, 2.05), p = 0.0023] and IVF [2.55, (1.19, 5.54), p = 0.016]. Multiples of the median (MOM) value of ß-hCG and pregnant persons' gestational age at delivery were significantly decreased in the LFF and RLFF groups compared to the control group. CONCLUSION: According to our findings based on the OR value, factors associated strongly with LFF include a high BMI and the use of IVF. Factors associated less strongly with LFF include early gestational age and twin pregnancy, while advanced maternal age and IVF were independent risk factors for a second LFF result.

Body mass index, gestational age, maternal age, twin pregnancy, and in vitro fertilization are associated with fetal fraction. We added the repetitive low fetal fraction population and used a large normal population as a control to identify the main factors associated with low fetal fraction.

Malformations of cortical development: Fetal imaging and genetics.

BACKGROUND: Malformations of cortical development (MCD) are a group of congenital disorders characterized by structural abnormalities in the brain cortex. The clinical manifestations include refractory epilepsy, mental retardation, and cognitive impairment. Genetic factors play a key role in the etiology of MCD. Currently, there is no curative treatment for MCD. Phenotypes such as epilepsy and cerebral palsy cannot be observed in the fetus. Therefore, the diagnosis of MCD is typically based on fetal brain magnetic resonance imaging (MRI), ultrasound, or genetic testing. The recent advances in neuroimaging have enabled the in-utero diagnosis of MCD using fetal ultrasound or MRI. METHODS: The present study retrospectively reviewed 32 cases of fetal MCD diagnosed by ultrasound or MRI. Then, the chromosome karyotype analysis, single nucleotide polymorphism array or copy number variation sequencing, and whole-exome sequencing (WES) findings were presented. RESULTS: Pathogenic copy number variants (CNVs) or single-nucleotide variants (SNVs) were detected in 22 fetuses (three pathogenic CNVs [9.4%, 3/32] and 19 SNVs [59.4%, 19/32]), corresponding to a total detection rate of 68.8% (22/32). CONCLUSION: The results suggest that genetic testing, especially WES, should be performed for fetal MCD, in order to evaluate the outcomes and prognosis, and predict the risk of recurrence in future pregnancies.

Deep learning prediction of renal anomalies for prenatal ultrasound diagnosis.

Deep learning algorithms have demonstrated remarkable potential in clinical diagnostics, particularly in the field of medical imaging. In this study, we investigated the application of deep learning models in early detection of fetal kidney anomalies. To provide an enhanced interpretation of those models' predictions, we proposed an adapted two-class representation and developed a multi-class model interpretation approach for problems with more than two labels and variable hierarchical grouping of labels. Additionally, we employed the explainable AI (XAI) visualization tools Grad-CAM and HiResCAM, to gain insights into model predictions and identify reasons for misclassifications. The study dataset consisted of 969 ultrasound images from unique patients; 646 control images and 323 cases of kidney anomalies, including 259 cases of unilateral urinary tract dilation and 64 cases of unilateral multicystic dysplastic kidney. The best performing model achieved a cross-validated area under the ROC curve of 91.28% ± 0.52%, with an overall accuracy of 84.03% ± 0.76%, sensitivity of 77.39% ± 1.99%, and specificity of 87.35% ± 1.28%. Our findings emphasize the potential of deep learning models in predicting kidney anomalies from limited prenatal ultrasound imagery. The proposed adaptations in model representation and interpretation represent a novel solution to multi-class prediction problems.

Understanding genetic variability: exploring large-scale copy number variants through non-invasive prenatal testing in European populations.

Large-scale copy number variants (CNVs) are structural alterations in the genome that involve the duplication or deletion of DNA segments, contributing to genetic diversity and playing a crucial role in the evolution and development of various diseases and disorders, as they can lead to the dosage imbalance of one or more genes. Massively parallel sequencing (MPS) has revolutionized the field of genetic analysis and contributed significantly to routine clinical diagnosis and screening. It offers a precise method for detecting CNVs with exceptional accuracy. In this context, a non-invasive prenatal test (NIPT) based on the sequencing of cell-free DNA (cfDNA) from pregnant women's plasma using a low-coverage whole genome MPS (WGS) approach represents a valuable source for population studies. Here, we analyzed genomic data of 12,732 pregnant women from the Slovak (9,230), Czech (1,583), and Hungarian (1,919) populations. We identified 5,062 CNVs ranging from 200 kbp and described their basic characteristics and differences between the subject populations. Our results suggest that re-analysis of sequencing data from routine WGS assays has the potential to obtain large-scale CNV population frequencies, which are not well known and may provide valuable information to support the classification and interpretation of this type of genetic variation. Furthermore, this could contribute to expanding knowledge about the central European genome without investing in additional laboratory work, as NIPTs are a relatively widely used screening method.

Post-mortem rapid aneuploidy testing for holoprosencephaly.

BACKGROUND: Abortion and fetal death are common in fetuses with holoprosencephaly, so genetic examinations often have to be made in a post-mortem setting. The efficiency of the conventional karyotyping using cultured fibroblasts in these situations is limited due to frequent culture failure. In the current study, archived cases of holoprosencephaly, where post-mortem genetic evaluation was requested and sufficient frozen material was available, were reevaluated using the quantitative fluorescence polymerase chain reaction (QF-PCR) technique. METHODS: Testing for aneuploidies of chromosomes 13, 15, 16, 18, 21, 22, X, and Y with the QF-PCR technique was carried out on DNA isolated from archived frozen chorionic villi in seven cases of holoprosencephaly. RESULTS: QF-PCR was successful in all seven cases. Two cases of trisomy 13, two cases of triploidy, and one case of trisomy 18 was found meaning a 71% diagnostic yield. The success rate of QF-PCR (100%, 7/7) was superior compared to conventional karyotyping (43%, 3/7). CONCLUSIONS: Rapid aneuploidy testing using the QF-PCR technique is a simple, reliable, time- and cost-effective method sufficient to conclude the etiologic investigation in the majority of holoprosencephaly cases post-mortem.

Clinical application value of pre-pregnancy carrier screening in Chinese Han childbearing population.

BACKGROUND: To explore the clinical application value of pre-conception expanded carrier screening (PECS) in the Chinese Han ethnicity population of childbearing age. METHODS: The results of genetic testing of infertile parents who underwent PECS in the Reproductive Medicine Center of the Second Affiliated Hospital of Zhengzhou University, China, from September 2019 to December 2021, were retrospectively analyzed. The carrier rate of single gene disease, the detection rate of high-risk parents, and the clinical outcome of high-risk parents were statistically analyzed. RESULTS: A total of 1372 Chinese Han ethnicity patients underwent PECS, among which 458 patients underwent the extended 108-gene test, their overall carrier rate was 31.7%, and the detection rate of high-risk parents was 0.3%. The highest carrier rates were SLC22A (2.4%), ATP7B (2.4%), MMACHC (2.2%), PAH (1.8%), GALC (1.8%), MLC1 (1.3%), UNC13D (1.1%), CAPN3 (1.1%), and PKHD1 (1.1%). There were 488 women with fragile X syndrome-FMR1 gene detection, and 6 patients (1.2%) had FMR1 gene mutation. A total of 426 patients were screened for spinal muscular atrophy-SMN1, and the carrier rate was 3.5%, and the detection rate of parents' co-carrier was 0.5%. CONCLUSION: Monogenic recessive hereditary diseases had a high carrier rate in the population. Pre-pregnancy screening could provide good prenatal and postnatal care guidance for patients and preimplantation genetic testing for monogenic/single gene disorders (PGT-M) and prenatal diagnosis could provide more precise reproductive choices for high-risk parents.

The yield of SNP microarray analysis for fetal ultrasound cardiac abnormalities.

BACKGROUND: Chromosomal microarray analysis (CMA) has emerged as a critical instrument in prenatal diagnostic procedures, notably in assessing congenital heart diseases (CHD). Nonetheless, current research focuses solely on CHD, overlooking the necessity for thorough comparative investigations encompassing fetuses with varied structural abnormalities or those without apparent structural anomalies. OBJECTIVE: This study sought to assess the relation of single nucleotide polymorphism-based chromosomal microarray analysis (SNP-based CMA) in identifying the underlying causes of fetal cardiac ultrasound abnormalities. METHODS: A total of 2092 pregnant women who underwent prenatal diagnosis from 2017 to 2022 were included in the study and divided into four groups based on the presence of ultrasound structural abnormalities and the specific type of abnormality. The results of the SNP-Array test conducted on amniotic fluid samples from these groups were analyzed. RESULTS: Findings from the study revealed that the non-isolated CHD group exhibited the highest incidence of aneuploidy, overall chromosomal abnormalities, and trisomy 18, demonstrating statistically significant differences from the other groups (p < 0.001). Regarding the distribution frequency of copy number variation (CNV) segment size, no statistically significant distinctions were observed between the isolated CHD group and the non-isolated CHD group (p > 0.05). The occurrence rates of 22q11.2 and 15q11.2 were also not statistically different between the isolated CHD group and the non-isolated congenital heart defect group (p > 0.05). CONCLUSION: SNP-based CMA enhances the capacity to detect abnormal CNVs in CHD fetuses, offering valuable insights for diagnosing chromosomal etiology and facilitating genetic counseling. This research contributes to the broader understanding of the utility of SNP-based CMA in the context of fetal cardiac ultrasound abnormalities.

Insights into non-informative results from non-invasive prenatal screening through gestational age, maternal BMI, and age analyses.

The discovery of cell-free fetal DNA fragments in the maternal plasma initiated a novel testing method in prenatal care, called non-invasive prenatal screening (NIPS). One of the limitations of NIPS is the necessity for a sufficient proportion of fetal fragments in the analyzed circulating DNA mixture (fetal fraction), otherwise, the sample is uninterpretable. We present the effect of gestational age, maternal body mass index (BMI), and maternal age on the fetal fraction (FF) of the sample. We retrospectively analyzed data from 5543 pregnant women with a single male fetus who underwent NIPS from which 189 samples received a repeat testing due to an insufficient FF. We showed the relationship between the failure rate of the samples after the repeated analysis, the FF, and the gestational age at the first sampling. Next, we found that different maternal BMI categories affect the FF and thus the chance of an informative redraw. A better understanding of the factors affecting the FF will reduce the number of non-informative calls from repeated analyzes. In this study, we provide helpful information to clinicians on how to approach non-informative analyses.

A rapid PCR-free next-generation sequencing method for comprehensive diagnosis of chromosome disease syndromes in prenatal samples.

The aim of this study is to investigate the application performance of rapid copy number variation sequencing (rCNV-seq) technology for the detection of chromosomal abnormalities during prenatal diagnosis. Samples were collected from 424 pregnant women who were at high-risk for noninvasive prenatal screening in Kunming Maternal and Child Care Hospital from January 2018 to May 2022. rCNV-seq technique was used to detect fetal chromosome abnormalities and compare the results with that of chromosomal karyotype analysis. The Result showed that 330 (77.83%, 330/424) cases indicated chromosomal abnormalities among 424 high-risk pregnant women who underwent rCNV-seq. Moreover, 94 (22.17%, 94/424) cases were discovered to have copy number variations. Among the 330 fetuses with chromosomal abnormalities, common autosomal aneuploidy was observed in 203 cases (47.87%, 203/424) and sex chromosome aneuploidy was observed in 91 cases (21.46%, 91/424). Moreover, the abnormalities in multiple chromosomes were discovered in 33 cases (7.78%, 33/424), and the rare autosomal aneuploidy was observed in 3 cases (0.71%, 3/424). There were 63 fetuses (14.86%, 63/424) with pathogenic CNVs among the 94 fetuses with variable copy numbers. Of the 245 pregnant women who voluntarily selected G-band karyotyping, 1 fetus with copy number variation had normal karyotype results, and the remaining women were consistent with rCNV-seq. Our study revealed that rCNV-seq has higher accuracy in detecting common trisomy and can also detect chromosomal microdeletions or microduplications that cannot be detected by G-banding karyotype analysis. There is no effective treatment for chromosomal diseases, so it is particularly important to prevent chromosomal diseases through genetic counseling and prenatal diagnosis of chromosomal diseases.

Noninvasive single-cell-based prenatal genetic testing: A proof of concept clinical study.

OBJECTIVE: To clinically assess a cell-based noninvasive prenatal genetic test using sequence-based copy number analysis of single trophoblasts from maternal blood. METHODS: Blood was obtained from 401 (243 + 158) individuals (8-22 weeks) and shipped overnight. Red cells were lysed, and nucleated cells stained for cytokeratin (CK) and CD45 and enriched for positive CK staining. Automated scanning was used to identify and pick single CK+ /CD45- trophoblasts which were subjected to next-generation sequencing. RESULTS: Blood was obtained from 243 pregnancies scheduled for CVS or amniocentesis. Luna results were normal for 160 singletons while 15 cases were abnormal (14 aneuploidy and one monozygotic twin with Williams syndrome deletion). The deletion was confirmed in both fetuses. Placental mosaicism occurred in 7 of 236 (3.0%) Luna cases and in 3 of 188 (1.6%) CVS cases (total 4.6%). No scorable trophoblasts were recovered in 32 of 236 usable samples. Additionally, 158 low-risk pregnancies not undergoing CVS/amniocentesis showed normal results in 133 cases. Seven had aneuploidy results, and there were three likely pathogenic deletions/duplications, including one15q11-q13 deletion. CONCLUSION: Although the sample size is modest and statistically accurate measures of test performance are not possible, the Luna test detected aneuploidy and deletions/duplications based on concordance with CVS/amniocentesis.

Review of the Evaluation of Pulmonary Hypoplasia as an Important Determinant of Clinical Outcomes in Infants with Congenital Diaphragmatic Hernia.

Pulmonary hypoplasia is one of main causes of neonatal mortality and morbidity in patients with congenital diaphragmatic hernia. With most cases diagnosed prenatally, the emphasis is put on prediction of the severity of this defect. Several attempts are made to reduce the mortality and provide optimal prenatal and postnatal care. Appropriate estimation of risk of pulmonary hypoplasia also provides an important inclusion criterion for prenatal intervention. The main tool used for the detection and prediction of pulmonary hypoplasia is ultrasound, with an increasing number of available formulas to estimate the risk of occurrence of this phenomenon and complication associated with it. For most of the formulas used in this measurement method, the main limitations are either gestational-age dependency or limited research. Other imaging methods used to assess the risk of pulmonary hypoplasia involve magnetic resonance imaging and vascular assessment of affected lungs. The limitation in these remains the limited accessibility. Currently, the most widely used indexes are observed-to-expected lungs-to-head ratio and presence of liver herniation. These are the 2 most commonly used measurement methods, as they are the basis for patient qualification for fetoscopic endoluminal tracheal occlusion. This article aims to review the evaluation of pulmonary hypoplasia or hypoplastic lung disease as an important determinant of clinical outcomes in infants with congenital diaphragmatic hernia. In this review, we emphasize the importance of early prenatal diagnosis of congenital diaphragmatic hernia and present a summary of different methods of prenatal risk assessment of lung hypoplasia in congenital diaphragmatic hernia.

[Genetic diagnosis and analysis of eight cases with central 22q11.2 deletion syndrome].

OBJECTIVE: To explore the pregnancy outcome and postpartum clinical phenotype of LCR22B/C~D central 22q11.2 deletion syndrome. METHODS: For fetuses diagnosed with central 22q11.2 deletion by chromosomal microarray analysis (CMA) at the Prenatal Diagnosis Center of the Third Affiliated Hospital of Zhengzhou University from January 2019 to April 2022, their prenatal imaging, parental CMA verification, pregnancy outcomes and postpartum clinical phenotype were analyzed. RESULTS: Eight cases of central 22q11.2 deletion syndrome were included, including six cases with LCR22B~D 22q11.2 deletions and two with LCR22C~D 22q11.2 deletions. Among the six cases with LCR22B~D type 22q11.2 deletions, three had shown cardiovascular malformations (right aortic arch, ventricular septal defect, mild tricuspid regurgitation), one had shown urinary defect (right kidney heterotopia). Two cases with LCR22C~D 22q11.2 deletions showed nonspecific ultrasonographic findings, including oligohydramnios with growth restriction and nuchal skin thickening. The CMA verification showed that six cases were inherited from their parents, and five couples had chosen to continue with the pregnancy. Postpartum follow-up showed that the physical and intellectual development of all children were normal. One couple had opted to terminate the pregnancy considering the ectopic fetal right kidney. Two remaining cases had decided to terminate their pregnancies without parental verification. CONCLUSION: The central 22q11.2 deletion syndrome of the LCR22B/C~D type is different from the classical types. Its genetic information mainly comes from parents. Prenatal imaging has mainly shown cardiovascular and urinary abnormalities. Postnatal growth and intellectual development have been normal. Therefore, the couples should be provided with suffice prenatal genetic counseling.

[Prenatal diagnosis and phenotypic analysis of two fetuses harboring heterozygous deletions of SHOX gene].

OBJECTIVE: To explore the clinical manifestations of two fetuses harboring heterozygous deletions of the SHOX gene. METHODS: Two pregnant women who had presented at the Prenatal Diagnosis Center of Nanjing Drum Tower Hospital respectively on June 24, 2022 and July 27, 2022 were selected as the study subjects. In case 1, prenatal ultrasonography had shown short femur and intrauterine growth retardation of the fetus. Case 2 had a history of spontaneous abortions due to structural chromosomal aberrations. Fetus 1 had undergone a test for the FGFR3 gene, and both fetuses were subjected to single nucleotide polymorphism-based microarray (SNP array) analysis. RESULTS: After excluding the influence of FGFR3 gene variant, fetus 1 was found to harbor a heterozygous 883 kb deletion at Xpter or Ypter, whilst fetus 2 was found to harbor a 5.75 Mb deletion in the Xpter region. Both deletions have encompassed the SHOX gene. The origin of the deletion in fetus 1 was unknown, whilst that in fetus 2 was inherited from its mother. Fetus 1 has been delivered at term with a normal phenotype, and fetus 2 was not born yet. CONCLUSION: The intrauterine and postnatal phenotypes of fetuses may be predicted by combining the ultrasound finding, parental phenotype and results of CMA, and the results can facilitate genetic counseling and decision making over the pregnancy.

Identification of copy number variations among fetuses with isolated ultrasound soft markers in pregnant women not of advanced maternal age.

BACKGROUND: Pathogenic (P) copy number variants (CNVs) may be associated with second-trimester ultrasound soft markers (USMs), and noninvasive prenatal screening (NIPS) can enable interrogate the entire fetal genome to screening of fetal CNVs. This study evaluated the clinical application of NIPS for detecting CNVs among fetuses with USMs in pregnant women not of advanced maternal age (AMA). RESULTS: Fetal aneuploidies and CNVs were identified in 6647 pregnant women using the Berry Genomics NIPS algorithm.Those with positive NIPS results underwent amniocentesis for prenatal diagnosis. The NIPS and prenatal diagnosis results were analyzed and compared among different USMs. A total of 96 pregnancies were scored positive for fetal chromosome anomalies, comprising 37 aneuploidies and 59 CNVs. Positive predictive values (PPVs) for trisomy 21, trisomy 18, trisomy 13, and sex chromosome aneuploidies were 66.67%, 80.00%, 0%, and 30.43%, respectively. NIPS sensitivity for aneuploidies was 100%. For CNVs, the PPVs were calculated as 35.59% and false positive rate of 0.57%. There were six P CNVs, two successfully identified by NIPS and four missed, of which three were below the NIPS resolution limit and one false negative. The incidence of aneuploidies was significantly higher in fetuses with absent or hypoplastic nasal bone, while that of P CNVs was significantly higher in fetuses with aberrant right subclavian artery (ARSA), compared with other groups. CONCLUSIONS: NIPS yielded a moderate PPV for CNVs in non-AMA pregnant women with fetal USM. However, NIPS showed limited ability in identifying P CNVs. Positive NIPS results for CNVs emphasize the need for further prenatal diagnosis. We do not recommend the use of NIPS for CNVs screening in non-AMA pregnant women with fetal USM, especially in fetuses with ARSA.

The Evaluation of Invasive Prenatal Diagnostic Tests in North Cyprus: A Retrospective Study.

BACKGROUND: Congenital diseases are still an important medical, social, and economic problem all over the world. In North Cyprus, in addition to other reasons, early prenatal diagnostic measures are undertaken to prevent births with thalassemia major, a locally widespread genetic disease. AIM: This study aims to evaluate the results of prenatal invasive diagnostic tests performed in a private obstetrics clinic in Northern Cyprus and show the diagnosis process of thalassemia and chromosomal anomalies. MATERIALS AND METHODS: This study is a retrospective, descriptive study. Chorionic villus sampling (CVS) results and the amniocentesis tests performed between 1990 and 2022 are evaluated. Thalassemia and chromosome analysis of samples obtained by CVS and amniocentesis tests were performed. To diagnose alpha or beta thalassemia and sickle cell, 239 CVS was performed. And to diagnose chromosomal anomalies, 396 CVS and amniocentesis were performed. RESULTS: The mean age of the 480 pregnant women included in the study was 31.12 years (18-46) and 30% of them were older than 34 years. The most common indications for invasive prenatal diagnostic test (IPDT) were; mother/father thalassemia minor/major, advanced maternal age, high risk of ultrasonography erase findings, and the noninvasive screening test. The result of IPDT detected 7.3% chromosomal anomaly and 69.5% thalassemia and sickle cell anemia. Of the 239 CVS performed to diagnose alpha or beta thalasemia and sickle cell, 23.4% beta major, 42.3% beta minor, and 2.1% alpha minor were diagnosed. Of the 396 CVS and amniocentesis performed to diagnose chromosomal anormalies; 2.8% of Down syndrome and 4.54% of other chromosomal anomalies were diagnosed. CONCLUSION: IPDT is important in correctly diagnosing fetal anomalies at the prenatal stage to help families decide at the right time.

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.

Patient experiences with prenatal cell-free DNA screening in a safety net setting.

OBJECTIVES: Thirty-five states, including Florida, now cover cell-free DNA (cfDNA) screening of fetuses for all pregnant patients enrolled in state public insurance programs. We interviewed Black and Hispanic obstetric patients at a safety net clinic in Florida shortly after the state rolled out cfDNA as a first-tier screening method for publicly insured patients. METHODS: Black and Hispanic patients receiving prenatal care from a prenatal or maternal fetal medicine clinic at a federally qualified health center in Jacksonville, FL were invited to participate in a qualitative interview in English or Spanish to explore experiences and perceptions of prenatal cfDNA screening. Participants were recruited following their first prenatal visit when cfDNA is typically introduced. Interview transcripts were qualitatively analyzed for iterative themes based on principles of grounded theory. RESULTS: One hundred Black and Hispanic patients (n = 51 non-Hispanic Black, n = 43 Hispanic, n = 3 Hispanic Black, n = 3 Not Reported/Other) completed an interview. Participants described minimal opportunity for pre-screening counseling and limited health literacy about cfDNA or its uses. Some believed that cfDNA could positively impact pregnancy health. Many were unsure if they had received cfDNA even though they were aware of the information provided by it. Most participants expressed an interest in cfDNA as a means for early detection of fetal sex and as an additional indication of general fetal health. CONCLUSIONS: Patient experiences indicate limited informed consent and decision-making for cfDNA, discordant with professional guidelines on pre-screen counseling. Our findings suggest that there should be additional investment in implementing cfDNA in safety net settings to ensure that patients and providers receive the support necessary for effective patient counseling and follow-on care for the ethical implementation of cfDNA.

Usefulness of early morphological ultrasound in association with cell-free DNA testing in case of atypical serum markers in first trimester of pregnancy: A retrospective study over 5 years.

BACKGROUND: Early morphologic ultrasound, generally carried out in case of atypical first trimester serum markers (PAPP-A and/or free hCGß <0.30 MoM), has not been re-evaluated since the possibility of performing a cell-free fetal DNA analysis in this indication. Our objective was to evaluate the usefulness of early morphological ultrasound in case of atypical profile of serum markers performed in association with Non-Invasive Prenatal Testing (NIPT). METHODS: This was a single-center retrospective study in a tertiary maternity. Between January 2017 and December 2021, women with an atypical first trimester serum markers and low/intermediate risk for trisomy 21 (<1/50) were included. The clinical data, results of first trimester serum markers, NIPT, early morphological ultrasound and subsequent ultrasounds and other investigations (amniocentesis, pregnancy outcomes) were analyzed. RESULTS: After exclusion of women with high-risk of trisomy 21 and lost to follow-up, 163 women were included. In 72 % of cases (117/163), women had a low risk of trisomy 21, and 39 % (59/163) had an early morphological ultrasound. Early morphological ultrasound was useful to detect severe IUGR leading to the suspicion of triploidy (3/163, 1.8 %). In all other situations, it did not allow earlier management. After analysis of the 3 triploidy cases, a collapsed profile for both serum markers was demonstrated (<0.25 MoM). CONCLUSIONS: Systematic early morphological ultrasound in case of an atypical serum marker profile seems useless considering the performance of NIPT. An ultrasound restricted to women with both markers below 0.25 MoM would allow the early detection of triploidy.

Congenital Ichthyosis: Current Approaches to Prenatal Diagnoses.

INTRODUCTION: Congenital ichthyosis represents a wide spectrum of diseases. This article reviews prenatal testing for ichthyosis. METHODS: We used pubmed.ncbi.nlm.nih.gov to search for 38 types of congenital ichthyosis combined with 17 words related to prenatal testing. RESULTS: Search resulted in 408 publications covering 13 types of ichthyoses and four types of tests. DISCUSSION: Biochemical testing is diagnostic in trichothiodystrophy, but nonspecific in X-linked ichthyosis and Refsum syndrome. Except in X-linked ichthyosis, biochemical testing requires invasive procedures to obtain fetal skin biopsy, amniocytes, or chorionic villus samples. It is superior to histological and cytological examination of fetal skin biopsy or amniocytes because keratinization occurs later in pregnancy and microscopy cannot differentiate between ichthyosis types. Imaging is more acceptable due to noninvasiveness and routine use, although ultrasonography is operator-dependent, nonspecific, and captures abnormalities at late stage. Molecular tests are described in at-risk pregnancies but testing of free fetal DNA was not described.

[Prenatal diagnosis and outcome of pregnancy for women with high risks by screening of fetal free DNA from peripheral blood samples].

OBJECTIVE: To analyze the results of prenatal diagnosis and outcome of pregnancy for women with a high risk for fetal aneuploidies. METHODS: A total of 747 cases of prenatal diagnosis by amniocentesis due to high risks by non-invasive prenatal testing (NIPT) were selected from January 2015 to March 2022 in the Drum Tower Hospital Affiliated to Nanjing University Medical School. The amniotic fluid samples were subjected to chromosomal karyotyping and/or chromosomal microarray analysis. All cases were followed up by searching the birth information or telephone calls, and the results were recorded. 2 test or F test were used for comparing the difference between the groups. RESULTS: Among the 747 pregnant women with a high risk by NIPT, 387 were true positives, and the overall positive predictive value (PPV) was 51.81%. The PPVs for trisomy 21 (T21), trisomy 18 (T18), trisomy 13 (T13) and sex chromosome aneuploidies (SCA) were 80.24% (199/248), 60% (48/80), 14% (7/50) and 38.97% (106/272), respectively. The PPV for T21 was significantly higher than T18 and T13 (χ2 = 85.216, P < 0.0001). The PPV for other chromosomal aneuploidies and copy number variations (CNVs) were 11.11% (5/45) and 40.74% (22/52), respectively. The PPV for increased X chromosomes was significantly higher than X chromosome decreases (64.29% vs. 22.22%, χ2 = 5.530, P < 0.05). The overall PPV for elder women (≥ 35 years old) was significantly higher than younger women (69.35% vs. 42.39%, χ2 = 49.440, P < 0.0001). For T21 and T18, the PPV of Z ≥ 10 group was significantly higher than that for 3 ≤ Z < 5 group or 5 ≤ Z < 10 group (P < 0.05). Among 52 cases with a high risk for CNVs, the PPV for the ≤ 5 Mb group was significantly higher than the 5 Mb < CNVs < 10 Mb or > 10 Mb groups (60% vs. 30%60% vs. 23.53%, P < 0.05). Among the 387 true positive cases, 322 had opted for induced labor, 53 had delivered with no abnormal growth and development, and 12 were lost during the follow-up. CONCLUSION: The PPVs for common chromosomal aneuploidies are related to the age and Z value of the pregnant women, which were higher in the elder group and higher Z value group. In addition, the PPV is associated with high risk types. The PPV for T21 was higher than T18 and T13, and that for 45,X was lower than 47,XXX, 47,XYY or 47,XXY syndrome. NIPT therefore has relatively high PPVs for the identification of chromosomal CNVs.