Disparities in integrating non-invasive prenatal testing into antenatal healthcare in Australia: a survey of healthcare professionals.

BACKGROUND: Non-invasive prenatal testing (NIPT) has been clinically available in Australia on a user-pays basis since 2012. There are numerous providers, with available tests ranging from targeted NIPT (only trisomies 21, 18, and 13 +/- sex chromosome aneuploidy) to genome-wide NIPT. While NIPT is being implemented in the public health care systems of other countries, in Australia, the implementation of NIPT has proceeded without public funding. The aim of this study was to investigate how NIPT has been integrated into antenatal care across Australia and reveal the successes and challenges in its implementation in this context. METHODS: An anonymous online survey was conducted from September to October 2022. Invitations to participate were sent to healthcare professionals (HCPs) involved in the provision of NIPT in Australia through professional society mailing lists and networks. Participants were asked questions on their knowledge of NIPT, delivery of NIPT, and post-test management of results. RESULTS: A total of 475 HCPs responded, comprising 232 (48.8%) obstetricians, 167 (35.2%) general practitioners, 32 (6.7%) midwives, and 44 (9.3%) genetic specialists. NIPT was most commonly offered as a first-tier test, with most HCPs (n = 279; 60.3%) offering it to patients as a choice between NIPT and combined first-trimester screening. Fifty-three percent (n = 245) of respondents always offered patients a choice between NIPT for the common autosomal trisomies and expanded (including genome-wide) NIPT. This choice was understood as supporting patient autonomy and informed consent. Cost was seen as a major barrier to access to NIPT, for both targeted and expanded tests. Equitable access, increasing time demands on HCPs, and staying up to date with advances were frequently reported as major challenges in delivering NIPT. CONCLUSIONS: Our findings demonstrate substantial variation in the clinical implementation of NIPT in Australia, including in the offers of expanded screening options. After a decade of clinical use, Australian clinicians still report ongoing challenges in the clinical and equitable provision of NIPT.

Performance of a cell-free DNA prenatal screening test, choice of prenatal procedure, and chromosome conditions identified during pregnancy after low-risk cell-free DNA screening.

OBJECTIVES: 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. METHOD: A single-center 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. RESULTS: 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% to 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). A further 15 (0.04%) false-negative common aneuploidy results were identified. CONCLUSIONS: 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.

Ethical issues associated with prenatal screening using non-invasive prenatal testing for sex chromosome aneuploidy.

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.

Performance of a Paired-End Sequencing-Based Noninvasive Prenatal Screening Test in the Detection of Genome-Wide Fetal Chromosomal Anomalies.

BACKGROUND: Noninvasive prenatal tests (NIPTs) detect fetal chromosomal anomalies with high clinical sensitivity and specificity. We examined the performance of a paired-end sequencing-based NIPT in the detection of genome-wide fetal chromosomal anomalies including common trisomies, sex chromosomal aneuploidies (SCA), rare autosomal aneuploidies (RAAs), and partial deletions/duplications ≥7 Mb. METHODS: Frozen plasma samples from pregnant women were tested using the VeriSeq NIPT Solution v2 assay. All samples were previously tested with a laboratory-developed NIPT and had known clinical outcomes. Individuals performing the sequencing were blinded to clinical outcome data. Clinical sensitivity and specificity were determined for basic (chromosomes 21, 18, 13, X, and Y) and genome-wide screening modes. RESULTS: Of 2335 samples that underwent genome-wide analysis, 28 did not meet QC requirements, resulting in a first-pass assay failure rate of 1.2%. Basic screening analysis, excluding known mosaics, correctly classified 130/130 trisomy 21 samples (sensitivity >99.9%, 95% confidence interval [CI] 97.1%-100%), 41/41 trisomy 18 samples (sensitivity >99.9%, 95% CI 91.4%-100%), and 26/26 trisomy 13 samples (sensitivity >99.9%, 95% CI 87.1%-100%) with 6 false-positive results; specificities ≥99.90% were reported for all 3 trisomies. Concordance for SCAs ranged from 90.5%-100%. Genome-wide screening analysis including known mosaics correctly classified 27/28 RAAs and 20/27 partial deletions/duplications with a specificity of 99.80% for both anomalies, and an overall genome-wide specificity for all anomalies of 99.34%. CONCLUSIONS: The VeriSeq NIPT Solution v2 assay enables accurate identification of fetal aneuploidy, allowing detection of genome-wide fetal chromosomal anomalies with high clinical sensitivities and specificities and a low assay failure rate.Clinical Trial Notification [CTN] identification number [ID]: CT-2018-CTN-01585-1 v1, Protocol: NIPT T05 002.

Reexamining the optimal nuchal translucency cutoff for diagnostic testing in the cell-free DNA and microarray era: results from the Victorian Perinatal Record Linkage study.

BACKGROUND: 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. OBJECTIVE: 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. STUDY DESIGN: 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. RESULTS: 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 of < 3.0 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 of <1.9 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 of <3.0 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, and <3.0 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, and <1.9 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 of >3.0 mm and 1 in 32 for nuchal translucency of >1.9 MoM. Our nuchal translucency threshold of 1.9 MoM captured 0.87% of fetuses, thus approximating the 99th centile. CONCLUSION: 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.

Cancer Diagnoses Following Abnormal Noninvasive Prenatal Testing: A Case Series, Literature Review, and Proposed Management Model.

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.

A minimum estimate of the prevalence of 22q11 deletion syndrome and other chromosome abnormalities in a combined prenatal and postnatal cohort.

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 < 0.0001). The total frequency of abnormalities in the live infant subgroup was 13.4% (244/1816). The frequencies of pathogenic copy number variants (CNVs) detected via CMA for the miscarriage, prenatal and postnatal cohorts were 1.9% (50/2573), 2.2% (82/3661) and 4.9% (127/2592), respectively. There was a significant increasing trend in the frequency of pathogenic CNVs with later developmental stage (χ2 trend = 39.72, P < 0.0001). For the subgroup of live infants, the pathogenic CNV frequency on CMA analysis was 6.0% (109/1816). There were 38 diagnoses of 22q11.2 DS, including 1 miscarriage, 15 prenatal and 22 postnatal cases. After excluding the miscarriage case and accounting for duplicate testing, the estimated prevalence of 22q11 DS was 1 in 4558 Victorian births. 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.

Harmonizing Cell-Free DNA Collection and Processing Practices through Evidence-Based Guidance.

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.

Association between timing of diagnosis of trisomy 21, 18, and 13 and maternal socio-economic status in Victoria, Australia: A population-based cohort study from 2015 to 2016.

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 < .01) and less likely to have a live-born T21 infant than a prenatal diagnosis (P < .01). There was a significant trend to higher live birth rates of T21 with lower SES (P = .004). The majority (68.5%) of women who gave birth to a live infant with T21 did not utilise prenatal testing. CONCLUSION: There is a significant relationship between lower SES, later prenatal diagnosis of trisomy, and higher live birth rate of T21 in Victoria.

'Small cost to pay for peace of mind': Women's experiences with non-invasive prenatal testing.

BACKGROUND: Non-invasive prenatal testing (NIPT) has been available in Australia on a user-pays basis since 2012. Since its introduction, it has grown in popularity as a screening method for fetal aneuploidy and may become publicly funded. AIMS: To assess the motivations and experiences of women who have undergone NIPT in a user-pays system in Australia. MATERIALS AND METHODS: One thousand women who had undergone NIPT through the Victorian Clinical Genetics Services in Melbourne, Australia were contacted and asked to complete a mixed-methods survey. The number of eligible responses received was 235. Quantitative data analysis was performed in STATA IC 15.1, and qualitative data were examined using content analysis. RESULTS: Women reported generally positive experiences with NIPT and 95% of respondents indicated they would undergo NIPT in a future pregnancy. Most respondents received a low-risk result, with 2.2% receiving a high-risk result. Respondents viewed NIPT favourably compared to invasive testing and cited reassurance as a key reason they sought it. However, a small minority of women reported negative experiences with the testing process. Women were also supportive of NIPT becoming publicly funded, with 93% of respondents indicating support. Pre- and post-test counselling were identified as possible areas for improvement to ensure informed consent. CONCLUSION: In support of the existing literature, these results indicate that Australian women generally report positive experiences with NIPT. As NIPT becomes more common, with possible integration into public healthcare, further qualitative research would be valuable to examine the motivations and experiences of women undergoing NIPT.

'Is it better not to know certain things?': views of women who have undergone non-invasive prenatal testing on its possible future applications.

Non-invasive prenatal testing (NIPT) is at the forefront of prenatal screening. Current uses for NIPT include fetal sex determination and screening for chromosomal disorders such as trisomy 21 (Down syndrome). However, NIPT may be expanded to many different future applications. There are a potential host of ethical concerns around the expanding use of NIPT, as examined by the recent Nuffield Council report on the topic. It is important to examine what NIPT might be used for before these possibilities become consumer reality. There is limited research exploring views of women on possible future uses of NIPT, particularly those of women who have undergone NIPT. In this study, we examined the views of women who undertook NIPT previously on the acceptability of and interest levels in using NIPT for a number of current and possible future applications. These included several medical conditions encompassing psychiatric, neurodevelopmental and adult-onset conditions as well as non-medical traits such as intelligence. One thousand women were invited to participate and 235 eligible surveys were received. Women generally reported an interest in using NIPT for medical conditions that severely impacted quality of life and with an onset earlier in life and stressed the importance of the accuracy of the test. Concerns were raised about the use of NIPT for non-medical traits. Respondents indicated that termination of pregnancy was not their only reason for testing, particularly in the case of sex. These results can further inform the ethical debate around the increasing integration of NIPT into healthcare systems.

Isochromosome 21q is overrepresented among false-negative cell-free DNA prenatal screening results involving Down syndrome.

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.

A fetus coexisting with a complete hydatidiform mole with trisomy 9 of maternal origin.

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.

Isolation and Characterization of Mesenchymal Stem/Stromal Cells Derived from Human Third Trimester Placental Chorionic Villi and Decidua Basalis.

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.

Rare autosomal trisomies, revealed by maternal plasma DNA sequencing, suggest increased risk of feto-placental disease.

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.

Abnormal plasma DNA profiles in early ovarian cancer using a non-invasive prenatal testing platform: implications for cancer screening.

BACKGROUND: Non-invasive prenatal testing (NIPT) identifies fetal aneuploidy by sequencing cell-free DNA in the maternal plasma. Pre-symptomatic maternal malignancies have been incidentally detected during NIPT based on abnormal genomic profiles. This low coverage sequencing approach could have potential for ovarian cancer screening in the non-pregnant population. Our objective was to investigate whether plasma DNA sequencing with a clinical whole genome NIPT platform can detect early- and late-stage high-grade serous ovarian carcinomas (HGSOC). METHODS: This is a case control study of prospectively-collected biobank samples comprising preoperative plasma from 32 women with HGSOC (16 'early cancer' (FIGO I-II) and 16 'advanced cancer' (FIGO III-IV)) and 32 benign controls. Plasma DNA from cases and controls were sequenced using a commercial NIPT platform and chromosome dosage measured. Sequencing data were blindly analyzed with two methods: (1) Subchromosomal changes were called using an open source algorithm WISECONDOR (WIthin-SamplE COpy Number aberration DetectOR). Genomic gains or losses ≥ 15 Mb were prespecified as "screen positive" calls, and mapped to recurrent copy number variations reported in an ovarian cancer genome atlas. (2) Selected whole chromosome gains or losses were reported using the routine NIPT pipeline for fetal aneuploidy. RESULTS: We detected 13/32 cancer cases using the subchromosomal analysis (sensitivity 40.6 %, 95 % CI, 23.7-59.4 %), including 6/16 early and 7/16 advanced HGSOC cases. Two of 32 benign controls had subchromosomal gains ≥ 15 Mb (specificity 93.8 %, 95 % CI, 79.2-99.2 %). Twelve of the 13 true positive cancer cases exhibited specific recurrent changes reported in HGSOC tumors. The NIPT pipeline resulted in one "monosomy 18" call from the cancer group, and two "monosomy X" calls in the controls. CONCLUSIONS: Low coverage plasma DNA sequencing used for prenatal testing detected 40.6 % of all HGSOC, including 38 % of early stage cases. Our findings demonstrate the potential of a high throughput sequencing platform to screen for early HGSOC in plasma based on characteristic multiple segmental chromosome gains and losses. The performance of this approach may be further improved by refining bioinformatics algorithms and targeting selected cancer copy number variations.

Haplotyping the human leukocyte antigen system from single chromosomes.

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.