Search

To evaluate the performance of cell-free DNA (cfDNA) screening for common fetal aneuploidies, choice of prenatal procedure, and chromosome conditions identified during pregnancy after low-risk cfDNA screening.A single-centre prenatal cfDNA screening test was employed to detect trisomies 21, 18 and 13 (T21, T18, T13) and sex chromosome aneuploidies (SCAs). Test performance, choice of prenatal procedure, and cytogenetic results in pregnancies with low-risk cfDNA screening were reviewed.CfDNA screening of 38,289 consecutive samples identified 720 (1.9%) pregnancies at increased risk for aneuploidy. Positive predictive values (PPVs) for high-risk singleton pregnancies were 98.5% (T21), 92.5% (T18) and 55.2% (T13). PPVs for SCAs ranged from 30.6-95.2%. Most women elected chorionic villus sampling for prenatal diagnosis of T21, T18 and T13; amniocentesis and/or postnatal testing were commonly chosen for SCAs. Cytogenetic tests from 616 screen-negative pregnancies identified 64 cases (12.7%) with chromosome conditions not detected by cfDNA screening, including triploidy (n=30) and pathogenic and likely pathogenic copy number variants (n=34). Fifteen (0.04%) false-negative common aneuploidy results were identified.CfDNA screening was highly accurate for detecting fetal aneuploidy in this general-risk obstetric population. Fetal ultrasound and prenatal diagnostic testing were important in identifying chromosome conditions in pregnancies screened as low-risk. This article is protected by copyright. All rights reserved.

Circulating cell-free DNA (cfDNA) is rapidly transitioning from discovery research to an important tool in clinical decision making. However, the lack of harmonization of preanalytic practices across institutions may compromise the reproducibility of cfDNA-derived data and hamper advancements in cfDNA testing in the clinic. Differences in cellular genomic contamination, cfDNA yield, integrity, and fragment length have been attributed to different collection tube types and anticoagulants, processing delays and temperatures, tube agitation, centrifugation protocols and speeds, plasma storage duration and temperature, the number of freeze-thaw events, and cfDNA extraction and quantification methods, all of which can also ultimately impact subsequent downstream analysis. Thus, there is a pressing need for widely applicable standards tailored for cfDNA analysis that include all preanalytic steps from blood draw to analysis. The NCI's Biorepositories and Biospecimen Research Branch has developed cfDNA-specific guidelines that are based upon published evidence and have been vetted by a panel of internationally recognized experts in the field. The guidelines include optimal procedures as well as acceptable alternatives to facilitate the generation of evidence-based protocols by individual laboratories and institutions. The aim of the document, which is entitled “Biospecimen Evidence-based Best Practices for Cell-free DNA: Biospecimen Collection and Processing,” is to improve the accuracy of cfDNA analysis in both basic research and the clinic by improving and harmonizing practices across institutions.

Guidelines discussed in article

Noninvasive prenatal testing (NIPT) is a screening test for fetal chromosomal aneuploidy using cell-free DNA derived from maternal blood. It has been rapidly accepted into obstetric practice because of its application from 10-weeks' gestation, and its high sensitivity and specificity. NIPT results can be influenced by several factors including placental or maternal mosaicism and co-twin demise; cell-free DNA from a maternal origin can also complicate interpretation, with evidence that NIPT can detect previously unsuspected malignancies. This study aimed to develop management guidelines for women with NIPT results suspicious of maternal malignancy. The Peter MacCallum Cancer Center's experience of seven cases where abnormal NIPT results led to investigation for maternal malignancy between 2016 and 2019 were reviewed, along with the published literature. Six of the seven women (86%) referred for investigation were diagnosed with advanced malignancies, including colorectal cancer, breast cancer, melanoma, and Hodgkin lymphoma. Based on our single-center experience, as well as the available literature, guidelines for the investigation of women with NIPT results suspicious of malignancy are proposed, including utilization of fluorodeoxyglucose positron emission tomography-computed tomography, which had a high concordance with other investigations and diagnoses. These guidelines include maternal and fetal investigations, as well as consideration of the complex medical, psychologic, social, and ethical needs of these patients and their families.

ispartof: PRENATAL DIAGNOSIS vol:41 issue:10 pages:1222-1232 ispartof: location:England status: published

Prenatal screening for sex chromosome aneuploidies (SCAs) is increasingly available through expanded non-invasive prenatal testing (NIPT). NIPT for SCAs raises complex ethical issues for clinical providers, prospective parents and future children. This paper discusses the ethical issues that arise around NIPT for SCAs and current guidelines and protocols for management. The first section outlines current practice and the limitations of NIPT for SCAs. It then outlines key guidelines before discussing the ethical issues raised by this use of NIPT. We conclude that while screening for SCAs should be made available for people seeking to use NIPT, its implementation requires careful consideration of what, when and how information is provided to users.

Balanced reciprocal translocation carriers are at increased risk of producing gametes with unbalanced forms of the translocation leading to miscarriage, fetal anomalies, and birth defects. We sought to determine if genome-wide cell-free DNA based noninvasive prenatal screening (gw-NIPS) could provide an alternative to prenatal diagnosis for carriers of these chromosomal rearrangements. This pilot series comprises a retrospective analysis of gw-NIPS and clinical outcome data from 42 singleton pregnancies where one parent carried a balanced reciprocal translocation. Gw-NIPS was performed between August 2015 and March 2018. Inclusion criteria required at least one translocation segment to be ≥15 Mb in size. Forty samples (95%) returned an informative result; 7 pregnancies (17.5%) were high risk for an unbalanced translocation and confirmed after diagnostic testing. The remaining 33 informative samples were low risk and confirmed after diagnostic testing or normal newborn physical exam. Test sensitivity of 100% (95% confidence interval [CI]: 64.6–100%) and specificity of 100% (95% CI: 89.6–100%) were observed for this pilot series. We demonstrate that gw-NIPS is a potential option for a majority of reciprocal translocation carriers. Further confirmation of this methodology could lead to adoption of this noninvasive alternative.

OBJECTIVES: To perform individual record linkage of women undergoing screening with cell-free DNA (cfDNA), combined first-trimester screening (CFTS), second-trimester serum screening (STSS), and/or prenatal and postnatal cytogenetic testing with the aim to (1) obtain population-based estimates of utilization of prenatal screening and invasive diagnosis, (2) analyze the performance of different prenatal screening strategies, and (3) report the residual risk of any major chromosomal abnormality following a low-risk aneuploidy screening result. METHODS: This was a retrospective study of women residing in the state of Victoria, Australia, who underwent prenatal screening or invasive prenatal diagnosis in 2015. Patient-funded cfDNA referrals from multiple providers were merged with state-wide results for government-subsidized CFTS, STSS and invasive diagnostic procedures. Postnatal cytogenetic results from products of conception and infants up to 12 months of age were obtained to ascertain cases of false-negative screening results and atypical chromosomal abnormalities. Individual record linkage was performed using LinkageWizTM . RESULTS: During the study period, there were 79 140 births and 66 166 (83.6%) women underwent at least one form of aneuploidy screening. Linkage data were complete for 93.5% (n = 61 877) of women who underwent screening, and of these, 73.2% (n = 45 275) had CFTS alone, 20.2% (n = 12 486) had cfDNA alone; 5.3% (n = 3268) had STSS alone, 1.3% (n = 813) had both CFTS and cfDNA, and

Circulating cell-free DNA (cfDNA) is rapidly transitioning from discovery research to an important tool in clinical decision making. However, the lack of harmonization of preanalytic practices across institutions may compromise the reproducibility of cfDNA-derived data and hamper advancements in cfDNA testing in the clinic. Differences in cellular genomic contamination, cfDNA yield, integrity, and fragment length have been attributed to different collection tube types and anticoagulants, processing delays and temperatures, tube agitation, centrifugation protocols and speeds, plasma storage duration and temperature, the number of freeze-thaw events, and cfDNA extraction and quantification methods, all of which can also ultimately impact subsequent downstream analysis. Thus, there is a pressing need for widely applicable standards tailored for cfDNA analysis that include all preanalytic steps from blood draw to analysis. The NCI's Biorepositories and Biospecimen Research Branch has developed cfDNA-specific guidelines that are based upon published evidence and have been vetted by a panel of internationally recognized experts in the field. The guidelines include optimal procedures as well as acceptable alternatives to facilitate the generation of evidence-based protocols by individual laboratories and institutions. The aim of the document, which is entitled “Biospecimen Evidence-based Best Practices for Cell-free DNA: Biospecimen Collection and Processing,” is to improve the accuracy of cfDNA analysis in both basic research and the clinic by improving and harmonizing practices across institutions.

PurposeTo describe our experience of offering simultaneous genetic carrier screening for cystic fibrosis (CF), fragile X syndrome (FXS), and spinal muscular atrophy (SMA).MethodsCarrier screening is offered through general practice, obstetrics, fertility, and genetics settings before or in early pregnancy. Carriers are offered genetic counseling with prenatal/preimplantation genetic diagnosis available to those at increased risk.ResultsScreening of 12,000 individuals revealed 610 carriers (5.08%; 1 in 20): 342 CF, 35 FXS, 241 SMA (8 carriers of 2 conditions), approximately 88% of whom had no family history. At least 94% of CF and SMA carriers' partners were tested. Fifty couples (0.42%; 1 in 240) were at increased risk of having a child with one of the conditions (14 CF, 35 FXS, and 1 SMA) with 32 pregnant at the time of testing. Of these, 26 opted for prenatal diagnosis revealing 7 pregnancies affected (4 CF, 2 FXS, 1 SMA).ConclusionThe combined affected pregnancy rate is comparable to the population risk for Down syndrome, emphasizing the need to routinely offer carrier screening. The availability of appropriate genetic counseling support and a collaborative approach between laboratory teams, genetics services, health professionals offering screening, and support organizations is essential.

A complete hydatidiform mole (CHM) coexisting with a viable fetus is a rare finding in pregnancies. Accurate diagnosis often relies on ultrasonographic, histopathological and molecular techniques in the definite diagnosis. To the best of our knowledge, a liveborn fetus coexisting with CHM with trisomy 9 has not been described. The use of molecular genotyping and immunohistochemical laboratory investigations enabled the CHM to be fully characterized. Postzygotic diploidization of a triploid conception arising from dispermy is the proposed mechanism of its formation.

The American College of Obstetricians and Gynecologists and the Society for Maternal-Fetal Medicine recently recommended offering genetic counseling and diagnostic testing for enlarged nuchal translucency at ≥3.0 mm, regardless of previous negative screening with noninvasive prenatal testing.This study aimed to perform a population-based, individual record linkage study to determine the optimal definition of an enlarged nuchal translucency for the detection of atypical chromosome abnormalities.This was a retrospective study of women resident in Victoria, Australia, undergoing combined first-trimester screening during the 24-month period from January 2015 to December 2016. Linkages between statewide results for combined first-trimester screening, prenatal diagnostic procedures, and postnatal cytogenetic results from products of conception and infants up to 12 months of age were used to ascertain the frequency and type of chromosome abnormality by gestation and nuchal translucency measurement. An atypical chromosome abnormality was defined as any major chromosome abnormality other than whole chromosome aneuploidy involving chromosomes 21, 18, 13, X, and Y.Of the 81,244 singleton pregnancies undergoing combined first-trimester screening, 491 (0.60%) had a nuchal translucency of ≥3.5 mm, 534 (0.66%) had a nuchal translucency of 3.0 to 3.4 mm, and 80,219 (98.74%) had a nuchal translucency of3.0 mm. When grouped by nuchal translucency multiples of the median (MoM), 192 (0.24%) had a nuchal translucency of ≥3.0 MoM, 513 (0.63%) had a nuchal translucency of 1.9 to 2.9 MoM, and 80,539 (99.13%) had a nuchal translucency of1.9 MoM. A total of 1779 pregnancies underwent prenatal or postnatal diagnostic testing, of which 89.60% were performed by whole-genome single-nucleotide polymorphism chromosomal microarray. The frequency of total major chromosome abnormalities was significantly higher in the group with a nuchal translucency of ≥3.5 mm (147 of 491, 29.94%) than the group with a nuchal translucency of 3.0 to 3.4 mm (21 of 534, 3.93%) or a nuchal translucency of3.0 mm (71 of 80,219, 0.09%) (P.001). There were 93 atypical chromosome abnormalities in the total screened cohort. The frequency of an atypical chromosome abnormality was 4.07% (95% confidence interval, 2.51-6.22), 0.37% (95% confidence interval, 0.05-1.35), and 0.09% (95% confidence interval, 0.07-0.11) in the groups with a nuchal translucency of ≥3.5 mm, 3.0 to 3.4 mm, and3.0 mm, respectively. The frequency of atypical chromosome abnormalities was 4.69% (95% confidence interval, 2.17-8.71), 2.53% (95% confidence interval, 1.36-4.29), and 0.09% (95% confidence interval, 0.07-0.11) in the groups with a nuchal translucency of ≥3.0 MoM, 1.9 to 2.9 MoM, and1.9 MoM, respectively. When defining thresholds for offering diagnosis with chromosomal microarray at 11 to 13 weeks, both a nuchal translucency threshold of 1.9 MoM and a fixed threshold of 3.0 mm captured 22 of 93 fetuses (23.7%) with an atypical chromosome abnormality. Of these, 50.0% had a coexisting fetal abnormality on ultrasound. However, the gestation-specific threshold of 1.9 MoM had a better specificity than 3.0 mm. The positive predictive value of an enlarged nuchal translucency for any atypical chromosome abnormality was 1 in 47 for nuchal translucency of3.0 mm and 1 in 32 for nuchal translucency of1.9 MoM. Our nuchal translucency threshold of 1.9 MoM captured 0.87% of fetuses, thus approximating the 99th centile.A gestational age-adjusted nuchal translucency threshold of 1.9 MoM or 99th centile is superior to the fixed cutoff of 3.0 mm for the identification of atypical chromosome abnormalities. The risk of an atypical chromosome abnormality in a fetus with an enlarged nuchal translucency is more than tripled in the presence of an additional ultrasound abnormality.

OBJECTIVES: To explore the association between timing of diagnosis of common autosomal trisomies, maternal age, and socio-economic status (SES). DESIGN: Retrospective study of cytogenetic diagnoses of trisomy 21 (T21), trisomy 18 (T18), and trisomy 13 (T13) in Victoria, Australia, in 2015 to 2016, stratified by timing (prenatal less than 17 weeks gestation, prenatal including or greater than or 17 weeks gestation, and postnatal before 12 months of age), maternal age, and SES region. Utilisation of prenatal testing following a live-born T21 infant was ascertained via record linkage. RESULTS: Among 160 230 total births were 571 diagnoses of T21 and 246 of T18/T13. The overall and live birth prevalences of T21 were 3.56 and 0.47 per 1000 births, respectively. Compared with women from disadvantaged SES regions, women from high SES regions were more likely to have a prenatal diagnosis of a trisomy before 17 weeks than after (P

STUDY QUESTION What is the frequency of major chromosome abnormalities in a population-based diagnostic data set of genomic tests performed on miscarriage, fetal and infant samples in a state with >73 000 annual births? SUMMARY ANSWER The overall frequency of major chromosome abnormalities in the entire cohort was 28.2% (2493/8826), with a significant decrease in the detection of major chromosome abnormalities with later developmental stage, from 50.9% to 21.3% to 15.6% of tests in the miscarriage, prenatal and postnatal cohorts, respectively. WHAT IS KNOWN ALREADY Over the past decade, technological advances have revolutionized genomic testing at every stage of reproduction. Chromosomal microarrays (CMAs) are now the gold standard of chromosome assessment in prenatal diagnosis and pediatrics. STUDY DESIGN, SIZE, DURATION A population-based cohort study including all chromosome analysis was performed in the Australian state of Victoria during a 24-month period from January 2015 to December 2016. All samples obtained via invasive prenatal diagnosis and postnatal samples from pregnancy tissue and infants ≤12 months of age were included. PARTICIPANTS/MATERIALS, SETTING, METHODS A research collaboration of screening and diagnostic units in the Australian state of Victoria was formed (the Perinatal Record Linkage collaboration), capturing all instances of prenatal and postnatal chromosome testing performed in the state. Victoria has over 73 000 births per annum and a median maternal age of 31.5 years. We analyzed our population-based diagnostic data set for (i) chromosome assessment of miscarriage, prenatal diagnosis and postnatal samples; (ii) testing indications and diagnostic yields for each of these cohorts; (iii) and the combined prenatal/infant prevalence of 22q11.2 deletion syndrome (DS) as a proportion of all births ≥20 weeks gestation. MAIN RESULTS AND THE ROLE OF CHANCE During the 24-month study period, a total of 8826 chromosomal analyses were performed on prenatal and postnatal specimens in Victoria. The vast majority (91.2%) of all chromosome analyses were performed with CMA. The overall frequency of major chromosome abnormalities in the entire cohort was 28.2% (2493/8826). There was a significant decreasing trend in the percentage of chromosome abnormalities with later developmental stage from 50.9% to 21.3% to 15.6% in the miscarriage, prenatal and postnatal cohorts, respectively (χ2 trend = 790.0, P LIMITATIONS, REASONS FOR CAUTION Clinical information was missing on 11.6% of postnatal samples, and gestational age was rarely provided on the miscarriage specimens. We were unable to obtain rates of termination of pregnancy and stillbirth in our cohort due to incomplete data provided by clinical referrers. We therefore cannot make conclusions on pregnancy or infant outcome following diagnostic testing. Childhood and adult diagnoses of 22q11 DS were not collected. WIDER IMPLICATIONS OF THE FINDINGS Our study marks a complete transition in genomic testing from the G-banded karyotype era, with CMA now established as the first line investigation for pregnancy losses, fetal diagnosis and newborn/infant assessment in a high-income setting. Integration of prenatal and postnatal diagnostic data sets provides important opportunities for estimating the prevalence of clinically important congenital syndromes, such as 22q11 DS. STUDY FUNDING/COMPETING INTEREST(S) L.H. is funded by a National Health and Medical Research Council Early Career Fellowship (1105603); A.L. was funded by a Mercy Perinatal Research Fellowship; J.H. was funded by a National Health and Medical Research Council Senior Research Fellowship (10121252). The funding bodies had no role in the conduct of the research or the manuscript. Discretionary funding from the Murdoch Children’s Research Institute has supported the prenatal diagnosis data collection and reporting over the years. Dr Ricardo Palma-Dias reports a commercial relationship with Roche Diagnostics, personal fees from Philips Ultrasound, outside the submitted work. Debbie Nisbet reports a commercial relationship with Roche Diagnostics, outside the submitted work. TRIAL REGISTRATION NUMBER NA

Introduction Human placental mesenchymal stem/stromal cells (MSC) are an attractive source of MSC with great therapeutic potential. However, primary MSC are difficult to study in vitro due to their limited lifespan and patient-to-patient variation. Methods Fetal and maternal MSC were prepared from cells of the chorionic and basal plates of the placenta, respectively. Fetal and maternal MSC were transduced with the human telomerase reverse transcriptase (hTERT). Conventional stem cell assays assessed the MSC characteristics of the cell lines. Functional assays for cell proliferation, cell migration and ability to form colonies in soft agar were used to assess the whether transduced cells retained properties of primary MSC. Results Fetal chorionic and maternal MSC were successfully transduced with hTERT to create the cell lines CMSC29 and DMSC23 respectively. The lifespans of CMSC29 and DMSC23 were extended in cell culture. Both cell lines retained important MSC characteristics including cell surface marker expression and multipotent differentiation potential. Neither of the cell lines was tumourigenic in vitro. Gene expression differences were observed between CMSC29 and DMSC23 cells and their corresponding parent, primary MSC. Both cell lines show similar migration potential to their corresponding primary, parent MSC. Discussion The data show that transduced MSC retained important functional properties of the primary MSC. There were gene expression and functional differences between cell lines CMSC29 and DMSC23 that reflect their different tissue microenvironments of the parent, primary MSC. CMSC29 and DMSC23 cell lines could be useful tools for optimisation and functional studies of MSC.

The use of mesenchymal stem/stromal cells (MSC) in regenerative medicine often requires MSC to function in environments of high oxidative stress. Human pregnancy is a condition where the mother's tissues, and in particular her circulatory system, are exposed to increased levels of oxidative stress. MSC in the maternal decidua basalis (DMSC) are in a vascular niche, and thus would be exposed to oxidative stress products in the maternal circulation. Aldehyde dehydrogenases (ALDH) are a large family of enzymes which detoxify aldehydes and thereby protect stem cells against oxidative damage. A subpopulation of MSC express high levels of ALDH (ALDH(br)) and these are more potent in repairing and regenerating tissues. DMSC was compared with chorionic villous MSC (CMSC) derived from the human placenta. CMSC reside in vascular niche and are exposed to the fetal circulation, which is in lower oxidative state. We screened an ALDH isozyme cDNA array and determined that relative to CMSC, DMSC expressed high levels of ALDH1 family members, predominantly ALDH1A1. Immunocytochemistry gave qualitative confirmation at the protein level. Immunofluorescence detected ALDH1 immunoreactivity in the DMSC and CMSC vascular niche. The percentage of ALDH(br) cells was calculated by Aldefluor assay and DMSC showed a significantly higher percentage of ALDH(br) cells than CMSC. Finally, flow sorted ALDH(br) cells were functionally potent in colony forming unit assays. DMSC, which are derived from pregnancy tissues that are naturally exposed to high levels of oxidative stress, may be better candidates for regenerative therapies where MSC must function in high oxidative stress environments.

False-negative cell-free DNA (cfDNA) screening results involving Down syndrome are rare, but have high clinical impact on patients and their healthcare providers. Understanding the biology behind these results may allow for improved diagnostic follow-up and counseling. In 5 different centers offering cfDNA prenatal screening, 9 false-negative results were documented in 646 confirmed cases of trisomy 21; a false-negative rate of 1.4% (95% CI, 0.7–2.6). False-negative results included 4 cases of classical trisomy 21 and 5 cases with a de novo 21q;21q rearrangement. Two out of five rearrangements had molecular studies and were confirmed as isochromosomes. When combined with reports from the cfDNA screening literature, 8 out of 29 (28%) Down syndrome cases with a false-negative “non-invasive prenatal test” (NIPT) were associated with a 21q;21q rearrangement, compared with 2% reported in live born children with Down syndrome. In our laboratory series, evidence for placental or fetal mosaicism was present in 3 out of 3 true-positive cases involving a 21q;21q rearrangement and was confirmed in one false-negative case where placental material was available for study. Isochromosome 21q rearrangements are thus overrepresented among false-negative cfDNA screening results involving Down syndrome. Postzygotic isochromosome formation leading to placental mosaicism provides a biological cause for the increased prevalence of these rearrangements among false-negative cases. For clinical practice, a low trisomic fraction (z-score or equivalent measure) relative to the fetal fraction suggests placental mosaicism. Care should be taken as these cases may not reflect confined placental mosaicism, but rather full trisomy in the presence of a placenta containing normal cells.

Genome-wide sequencing of cell-free DNA (cfDNA) from maternal plasma enables noninvasive prenatal testing (NIPT) of all 24 chromosomes. A key advantage of the genome-wide approach is its potential to identify pathogenic copy number changes that would go unrecognized using standard methods of cfDNA-based NIPT, which screen only for aneuploidies of chromosomes 13, 18, 21, X and Y. An expanded cfDNA screening approach can identify rare autosomal trisomies associated with pregnancy complications that include miscarriage, true fetal mosaicism, uniparental disomy, fetal growth restriction, and fetal demise; while subchromosomal copy number imbalances such as deletions, duplications, and unbalanced chromosomal rearrangements, which have serious consequences for the normal development of the fetus, can also be identified. However, genome-wide screening for whole chromosome and segmental aneuploidies is not without its technical, biological, and interpretative challenges. This Chapter explores the rapidly expanding field of genome-wide cfDNA-based NIPT and highlights the applications, benefits, and limitations of this approach.

OBJECTIVES: Women's choices of prenatal screening and diagnostic pathways have increased in complexity since the introduction of cell-free DNA (cfDNA) screening and chromosomal microarrays. We performed individual record-linkage of women undergoing screening with cfDNA, combined first trimester screening (CFTS), second trimester serum screening (STSS), and/or prenatal and postnatal cytogenetic testing to (i) obtain population-based estimates on women's utilization of screening and diagnosis, (ii) analyse the performance of different screening strategies, and (iii) report the residual risks of any major chromosome abnormality following a low risk aneuploidy screen. METHODS: Retrospective study of women resident in Victoria, Australia, undergoing screening or prenatal diagnosis in 2015. Patient-funded cfDNA referrals from multiple providers were merged with state-wide results for government-subsidized CFTS, STSS and invasive diagnostic procedures. Postnatal cytogenetic results from products of conception and infants up to 12 months of age were obtained to ascertain cases of false negative screening results and atypical chromosome abnormalities. Individual record-linkage was performed with LinkageWizTM and statistical analyses with STATA v14.0. RESULTS: There were 79,140 births during the study period; 66,166 women (83.4%) underwent at least one form of aneuploidy screening. Linkage data were complete for 92.4% of women undergoing screening (n=61,911) and of these, 73.1% (n=45,275) used CFTS alone, 20.2% (n=12,520) used cfDNA alone; 5.3% (n=3268) used STSS alone, 1.3% (n=813) used both CFTS and cfDNA, and

The decidua basalis and placental chorionic villi are critical components of maternal-fetal interface, which plays a critical role in normal placental development. Failure to form a proper maternal-fetal interface is associated with clinically important placental pathologies including preeclampsia and fetal growth restriction. Placental trophoblast cells are well known for their critical roles in establishing the maternal-fetal interface; however accumulating evidence also implicates mesenchymal stem/stromal cells that envelop the maternal and fetal blood vessels as playing an important role in the formation and efficient functioning of the interface. Moreover, recent studies associate abnormal mesenchymal stem/stromal cell function in the development of preeclampsia. Further research is needed to fully understand the role that these cells play in this clinically important placental pathology.The intimate relationship between maternal and fetal tissues at the interface poses significant problems in the enrichment of decidua basalis and chorionic villous mesenchymal stem/stromal cells without significant cross-contamination. The protocols described below for the enrichment and characterization of mesenchymal stem/stromal cells from the maternal-fetal interface produce highly enriched cells that conform to international standards and show minimal cross-contamination.

The decidua basalis and placental chorionic villi are critical components of maternal-fetal interface, which plays a critical role in normal placental development. Failure to form a proper maternal-fetal interface is associated with clinically important placental pathologies including preeclampsia and fetal growth restriction. Placental trophoblast cells are well known for their critical roles in establishing the maternal-fetal interface; however accumulating evidence also implicates mesenchymal stem/stromal cells that envelop the maternal and fetal blood vessels as playing an important role in the formation and efficient functioning of the interface. Moreover, recent studies associate abnormal mesenchymal stem/stromal cell function in the development of preeclampsia. Further research is needed to fully understand the role that these cells play in this clinically important placental pathology.The intimate relationship between maternal and fetal tissues at the interface poses significant problems in the enrichment of decidua basalis and chorionic villous mesenchymal stem/stromal cells without significant cross-contamination. The protocols described below for the enrichment and characterization of mesenchymal stem/stromal cells from the maternal-fetal interface produce highly enriched cells that conform to international standards and show minimal cross-contamination.

This study aimed to examine the choice pregnant women make about the amount of fetal genetic information they want from chromosome microarray. Women having invasive prenatal testing in the absence of fetal structural abnormality were recruited in Victoria, Australia. A decision aid for women described ‘targeted’ analysis as reporting only copy number variants implicated in a highly penetrant and well-described phenotype and ‘extended’ as additionally reporting variants of uncertain or unknown significance. Participant’s choice and demographics were collected by survey before chorionic villus sampling or amniocentesis; psychological data were also collected then and again about 10 days after receiving results. High-resolution single-nucleotide polymorphism array analysis was performed, and a clinical review committee assessed variants for reporting before returning results to participants. Sixty-six participants (59.5%) chose extended analysis and 45 (40.5%) targeted. Choosing extended information was associated with (1) indication for prenatal diagnosis: maternal age alone (adjusted odds ratio (adjOR) 9.6, 95% confidence interval (CI): 1.4–66.0, p= 0.02), or ‘other’ indication (adjOR 7.1, 95% CI: 1.5–33.1, p= 0.01)); (2) >12 months to conceive (adjOR 4.1, 95% CI: 1.0–17.7, p= 0.05); and (3) Asian background (adjOR 4.67, 95% CI: 1.0–21.0, p= 0.04). No adverse psychological impact occurred in either group. We conclude that offering pregnant women different levels of fetal genetic analysis is warranted, alongside decision support.

Whole-genome sequencing (WGS) of maternal plasma cell-free DNA (cfDNA) can potentially evaluate all 24 chromosomes to identify abnormalities of the placenta, fetus, or pregnant woman. Current bioinformatics algorithms typically only report on chromosomes 21, 18, 13, X, and Y; sequencing results from other chromosomes may be masked. We hypothesized that by systematically analyzing WGS data from all chromosomes, we could identify rare autosomal trisomies (RATs) to improve understanding of feto-placental biology. We analyzed two independent cohorts from clinical laboratories, both of which used a similar quality control parameter, normalized chromosome denominator quality. The entire data set included 89,817 samples. Samples flagged for analysis and classified as abnormal were 328 of 72,932 (0.45%) and 71 of 16,885 (0.42%) in cohorts 1 and 2, respectively. Clinical outcome data were available for 57 of 71 (80%) of abnormal cases in cohort 2. Visual analysis of WGS data demonstrated RATs, copy number variants, and extensive genome-wide imbalances. Trisomies 7, 15, 16, and 22 were the most frequently observed RATs in both cohorts. Cytogenetic or pregnancy outcome data were available in 52 of 60 (87%) of cases with RATs in cohort 2. Cases with RATs detected were associated with miscarriage, true fetal mosaicism, and confirmed or suspected uniparental disomy. Comparing the trisomic fraction with the fetal fraction allowed estimation of possible mosaicism. Analysis and reporting of aneuploidies in all chromosomes can clarify cases in which cfDNA findings on selected “target” chromosomes (21, 18, and 13) are discordant with the fetal karyotype and may identify pregnancies at risk of miscarriage and other complications.

Background Cardiac rhythm abnormalities are a leading cause of morbidity and mortality in developed countries. Loss-of-function variants in the ANK2 gene can cause a variety of cardiac rhythm abnormalities including sinus node dysfunction, atrial fibrillation and ventricular arrhythmias (called the "ankyrin-B syndrome"). ANK2 encodes ankyrin-B, a molecule critical for the membrane targeting of key cardiac ion channels, transporters, and signalling proteins. Methods and Results Here, we describe a family with a reciprocal chromosomal translocation between chromosomes 4q25 and 9q26 that transects the ANK2 gene on chromosome 4 resulting in loss-of-function of ankyrin-B . Select family members with ankyrin-B haploinsufficiency due to the translocation displayed clinical features of ankyrin-B syndrome. Furthermore, evaluation of primary lymphoblasts from a carrier of the translocation showed altered levels of ankyrin-B as well as a reduced expression of downstream ankyrin-binding partners. Conclusions Thus, our data conclude that, similar to previously described ANK2 loss-of-function "point mutations", large chromosomal translocations resulting in ANK2 haploinsufficiency are sufficient to cause the human cardiac ankyrin-B syndrome. The unexpected ascertainment of ANK2 dysfunction via the discovery of a chromosomal translocation in this family, the determination of the familial phenotype, as well as the complexities in formulating screening and treatment strategies are discussed.

We describe a method for determining the parental HLA haplotypes of a single individual without recourse to conventional segregation genetics. Blood samples were cultured to identify and sort chromosome 6 by bivariate flow cytometry. Single chromosome 6 amplification products were confirmed with a single nucleotide polymorphism (SNP) array and verified by deep sequencing to enable assignment of both alleles at the HLA loci, defining the two haplotypes. This study exemplifies a rapid and efficient method of haplotyping that can be applied to any chromosome pair, or indeed all chromosome pairs, using a single sorting operation. The method represents a cost-effective approach to complete phasing of SNPs, which will facilitate a deeper understanding of the links between SNPs, gene regulation and protein function.

Chromosome microarrays are an essential tool for investigation of copy number changes in children with congenital anomalies and intellectual deficit. Attempts to standardise microarray testing have focused on establishing technical and clinical quality criteria, however external quality assessment programs are still needed. We report on a microarray proficiency testing program for Australasian laboratories. Quality metrics evaluated included analytical accuracy, result interpretation, report completeness, and laboratory performance data: sample numbers, success and abnormality rate and reporting times. Between 2009 and 2014 nine samples were dispatched with variable results for analytical accuracy (30-100%), correct interpretation (32-96%), and report completeness (30-92%). Laboratory performance data (2007-2014) showed an overall mean success rate of 99.2% and abnormality rate of 23.6%. Reporting times decreased from90 days to30 days for normal results and from102 days to35 days for abnormal results. Data trends showed a positive correlation with improvement for all these quality metrics, however only 'report completeness' and reporting times reached statistical significance. Whether the overall improvement in laboratory performance was due to participation in this program, or from accumulated laboratory experience over time, is not clear. Either way, the outcome is likely to assist referring clinicians and improve patient care.

The Y centromere sequence of house mouse, Mus musculus, remains unknown despite our otherwise significant knowledge of the genome sequence of this important mammalian model organism. Here, we report the complete molecular characterization of the C57BL/6J chromosome Y centromere, which comprises a highly diverged minor satellite-like sequence (designated Ymin) with higher-order repeat (HOR) sequence organization previously undescribed at mouse centromeres. The Ymin array is ∼90 kb in length and resides within a single BAC clone that provides sequence information spanning an endogenous animal centromere for the first time. By exploiting direct patrilineal inheritance of the Y chromosome, we demonstrate stability of the Y centromere DNA structure spanning at least 175 inbred generations to beyond the time of domestication of the East Asian M.m. molossinus “fancy” mouse through which the Y chromosome was first introduced into the classical inbred laboratory mouse strains. Despite this stability, at least three unequal genetic exchange events have altered Ymin HOR unit length and sequence structure since divergence of the ancestral Mus musculus subspecies around 900,000 yr ago, with major turnover of the HOR arrays driving rapid divergence of sequence and higher-order structure at the mouse Y centromere. A comparative sequence analysis between the human and chimpanzee centromeres indicates a similar rapid divergence of the primate Y centromere. Our data point to a unique DNA sequence and organizational architecture for the mouse Y centromere that has evolved independently of all other mouse centromeres.

We report an exceptional complex chromosomal rearrangement (CCR) found in three individuals in a family that involves 4 chromosomes with 5 breakpoints. The CCR was ascertained in a phenotypically abnormal newborn with additional chromosomal material on the short arm of chromosome 4. Maternal karyotyping indicated that the mother carried an apparently balanced CCR involving chromosomes 4, 6, 11, and 18. Maternal transmission of the derivative chromosome 4 resulted in partial trisomy for chromosomes 6q and 18q and a partial monosomy of chromosome 4p in the proband. Further family studies found that the maternal grandmother carried the same apparently balanced CCR as the proband’s mother, which was confirmed using the whole chromosome painting (WCP) FISH. High resolution whole genome microarray analysis of DNA from the proband’s mother found no evidence for copy number imbalance in the vicinity of the CCR translocation breakpoints, or elsewhere in the genome, providing evidence that the mother’s and grandmother’s CCRs were balanced at a molecular level. This structural rearrangement can be categorized as an exceptional CCR due to its complexity and is a rare example of an exceptional CCR being transmitted in balanced and/or unbalanced form across three generations.

Objective To determine the long-term health and development of a cohort of children in whom confined placental mosaicism (CPM) was diagnosed at prenatal diagnosis. Methods A retrospective cohort study was performed comparing 36 children in whom CPM had been diagnosed prenatally with 195 controls subjects in whom a normal karyotype had been detected prenatally. Data comprising birth information, health, health service utilisation, growth, development, behaviour, and the family were collected by a maternal questionnaire administered when the subjects were aged between 4 and 11 years. Results CPM cases did not differ from controls across a broad range of health measures and there were no major health problems or birth defects among the CPM group. No increase was detected in the incidence of intrauterine growth retardation (IUGR) among CPM cases; however, postnatal growth was reduced compared with controls (p = 0.047). Development and behaviour in CPM cases was similar to that of controls. Conclusions The prenatal diagnosis of CPM is not associated with an increased risk of birth defects or developmental problems, but may be associated with decreased growth. Copyright (C) 2006 John Wiley & Sons, Ltd.

Chromosome analysis of spontaneous miscarriages is clinically important but is hampered by frequent tissue culture failure and relatively low-resolution analysis. We have investigated replacement of conventional karyotype analysis with a quantitative subtelomere assay performed on uncultured tissue samples, which is based on Multiplex Ligation-Dependent Probe Amplification. This assay is suitable for this purpose as approximately 98% of all observed karyotype abnormalities in spontaneous miscarriages involve copy-number change to one or more subtelomere regions. A pilot study has compared karyotyping and subtelomere analysis on 78 samples. Extensive tissue necrosis accounted for failure of both karyotyping and subtelomere testing in four (5.1%) samples. Excluding these, there were no (0/74) subtelomere test failures compared to 9.5% (7/74) karyotype failures. Twenty-two (30%) whole chromosome aneuploidies and five (6.8%) structural abnormalities were detected using the subtelomere assay. With the exception of three cases of triploidy, all karyotype abnormalities were detected by the subtelomere assay. Following on from this study, a further 100 samples were tested using the subtelomere assay in conjunction with a simple ancillary FISH test using uncultured cells to exclude polyploidy in the event of a normal subtelomere assay result. Except for three necrotic samples, tests results were obtained for all cases revealing 18 abnormalities including one case of triploidy. Taking into consideration the high success rate for the combined MLPA and FISH test results, and the very significant additional advantages of cost-effective, high-throughput batching, and automated, objective analysis, this approach greatly facilitates routine investigation of chromosome abnormalities in spontaneous miscarriage.

Introduction Maternal decidua basalis tissue attached to the placenta following delivery is a source of decidual mesenchymal stem cells (DMSCs). The in vitro characteristics of DMSCs have been partly defined but their in vivo function(s) are poorly understood. The anatomic location, or niche, provides clues regarding potential in vivo function(s) of DMSCs, but the niche has not been described. Methods Cells were isolated from the decidua basalis and flow cytometric analyses showed the expected phenotypic profile for MSC cell surface markers. In vitro , the cells differentiated into adipocytes, osteocytes, and chondrocytes. DMSCs were then stained with antibodies by immunofluorescence detection. Results Immunocytochemistry revealed that DMSCs were positive for FZD-9, STRO-1, 3G5, and α-SMA as expected and lacked expression of vWF and Ck7. Fluorescence in situ hybridization analysis showed the cultured cells were of maternal origin. Immunofluorescence was carried out on placental bed biopsies using the FZD-9, STRO-1, 3G5, and α-SMA antibodies. DMSCs were located in the vascular niche in decidua basalis . Immunofluorescence with antibodies to FZD-9, Ck7 and vWF revealed DMSCs in the vascular niche surrounding intact non-transformed spiral arterioles but DMSCs were absent in fully transformed spiral arterioles. Discussion Spiral arteriole remodelling is a critical feature of human pregnancy. The DMSC niche was investigated in fully transformed and non-transformed spiral arterioles. DMSCs have not been previously implicated in spiral arteriole remodelling. The absence of DMSCs around fully transformed spiral arterioles suggests they are a target for replacement or destruction by invading placental extravillous trophoblast cells, which carry out spiral arteriole remodelling.

DNA-based Chromosome MicroArrays (CMAs) are now well established as diagnostic tools in clinical genetics laboratories. Over the last decade, the primary application of CMAs has been the genome-wide detection of a particular class of mutation known as copy number variants (CNVs). Since 2010, CMA testing has been recommended as a first-tier test for detection of CNVs associated with intellectual disability, autism spectrum disorders, and/or multiple congenital anomalies…in the post-natal setting. CNVs are now regarded as pathogenic in 14-18 % of patients referred for these (and related) disorders.Through consideration of clinical examples, and several microarray platforms, we attempt to provide an appreciation of microarray diagnostics, from the initial inspection of the microarray data, to the composing of the patient report. In CMA data interpretation, a major challenge comes from the high frequency of clinically irrelevant CNVs observed within "patient" and "normal" populations. As might be predicted, the more common and clinically insignificant CNVs tend to be the smaller ones100 kb in length, involving few or no known genes. However, this relationship is not at all straightforward: CNV length and gene content are only very imperfect indicators of CNV pathogenicity. Presently, there are no reliable means of separating, a priori, the benign from the pathological CNV classes.This chapter also considers sources of technical "noise" within CMA data sets. Some level of noise is inevitable in diagnostic genomics, given the very large number of data points generated in any one test. Noise further limits CMA resolution, and some miscalling of CNVs is unavoidable. In this, there is no ideal solution, but various strategies for handling noise are available. Even without solutions, consideration of these diagnostic problems per se is informative, as they afford critical insights into the biological and technical underpinnings of CNV discovery. These are indispensable to any clinician or scientist practising within the field of genome diagnostics.

Human chorionic mesenchymal stem/stromal cells (CMSCs) derived from the placenta are similar to adult tissue-derived MSCs. The aim of this study was to investigate the role of these cells in normal placental development. Transcription factors, particularly members of the homeobox gene family, play crucial roles in maintaining stem cell proliferation and lineage specification in embryonic tissues. In adult tissues and organs, stem cells proliferate at low levels in their niche until they receive cues from the microenvironment to differentiate. The homeobox genes that are expressed in the CMSC niche in placental tissues have not been identified. We used the novel strategy of laser capture microdissection to isolate the stromal component of first trimester villi and excluded the cytotrophoblast and syncytiotrophoblast layers that comprise the outer layer of the chorionic villi. Microarray analysis was then used to screen for homeobox genes in the microdissected tissue. Candidate homeobox genes were selected for further RNA analysis. Immunohistochemistry of candidate genes in first trimester placental villous stromal tissue revealed homeobox genes Meis1, myeloid ectropic viral integration site 1 homolog 2 (MEIS2), H2.0-like Drosophila (HLX), transforming growth factor β-induced factor (TGIF), and distal-less homeobox 5 (DLX5) were expressed in the vascular niche where CMSCs have been shown to reside. Expression of MEIS2, HLX, TGIF, and DLX5 was also detected in scattered stromal cells. Real-time polymerase chain reaction and immunocytochemistry verified expression of MEIS2, HLX, TGIF, and DLX5 homeobox genes in first trimester and term CMSCs. These data suggest a combination of regulatory homeobox genes is expressed in CMSCs from early placental development to term, which may be required for stem cell proliferation and differentiation.

DNA-based Chromosome MicroArrays (CMAs) are now well established as diagnostic tools in clinical genetics laboratories. Over the last decade, the primary application of CMAs has been the genome-wide detection of a particular class of mutation known as copy number variants (CNVs). Since 2010, CMA testing has been recommended as a first-tier test for detection of CNVs associated with intellectual disability, autism spectrum disorders, and/or multiple congenital anomalies…in the post-natal setting. CNVs are now regarded as pathogenic in 14-18 % of patients referred for these (and related) disorders.Through consideration of clinical examples, and several microarray platforms, we attempt to provide an appreciation of microarray diagnostics, from the initial inspection of the microarray data, to the composing of the patient report. In CMA data interpretation, a major challenge comes from the high frequency of clinically irrelevant CNVs observed within "patient" and "normal" populations. As might be predicted, the more common and clinically insignificant CNVs tend to be the smaller ones

We report a case of a neonate who was shown with routine chromosome analysis on peripheral blood lymphocytes to have full monosomy 21. Further investigation on fibroblast cells using conventional chromosome and FISH analysis revealed two additional mosaic cell lines; one is containing a ring chromosome 21 and the other a double ring chromosome 21. In addition, chromosome microarray analysis (CMA) on fibroblasts showed a mosaic duplication of chromosome region 21q11.2q22.13 with approximately 45% of cells showing three copies of the proximal long arm segment, consistent with the presence of a mosaic ring chromosome 21 with ring instability. The CMA also showed complete monosomy for an 8.8 Mb terminal segment (21q22.13q22.3). Whilst this patient had a provisional clinical diagnosis of trisomy 21, the patient also had phenotypic features consistent with monosomy 21, such as prominent epicanthic folds, broad nasal bridge, anteverted nares, simple ears, and bilateral overlapping fifth fingers, features which can also be present in individuals with Down syndrome. The patient died at 4.5 months of age. This case highlights the need for additional studies using multiple tissue types and molecular testing methodologies in patients provisionally diagnosed with monosomy 21, in particular if detected in the neonatal period.

We describe six cases of trisomy 13 mosaicism detected at prenatal diagnosis. Most level I and level II trisomy 13 mosaicism detected at prenatal diagnosis is pseudomosaicism or confined placental mosaicism. Rarely, low-level mosaicism at chorionic villus sampling or amniocentesis reflects a true fetal mosaicism. In this case, a normal phenotype is a possible, but not a certain, outcome. Genetic counselling is not straightforward.