Non-invasive Prenatal Testing For Aneuploidy Research Articles

PREIMPLANTATION GENETIC TESTING: Non-invasive prenatal testing for aneuploidy, copy-number variants and single-gene disorders.

The discovery of cell-free fetal DNA (cffDNA) in maternal plasma has enabled a paradigm shift in prenatal testing, allowing for safer, earlier detection of genetic conditions of the fetus. Non-invasive prenatal testing (NIPT) for fetal aneuploidies has provided an alternative, highly efficient approach to first-trimester aneuploidy screening, and since its inception has been rapidly adopted worldwide. Due to the genome-wide nature of some NIPT protocols, the commercial sector has widened the scope of cell-free DNA (cfDNA) screening to include sex chromosome aneuploidies, rare autosomal trisomies and sub-microscopic copy-number variants. These developments may be marketed as 'expanded NIPT' or 'NIPT Plus' and bring with them a plethora of ethical and practical considerations. Concurrently, cfDNA tests for single-gene disorders, termed non-invasive prenatal diagnosis (NIPD), have been developed for an increasing array of conditions but are less widely available. Despite the fact that all these tests utilise the same biomarker, cfDNA, there is considerable variation in key parameters such as sensitivity, specificity and positive predictive value depending on what the test is for. The distinction between diagnostics and screening has become blurred, and there is a clear need for the education of physicians and patients regarding the technical capabilities and limitations of these different forms of testing. Furthermore, there is a requirement for consistent guidelines that apply across health sectors, both public and commercial, to ensure that tests are validated and robust and that careful and appropriate pre-test and post-test counselling is provided by professionals who understand the tests offered.

Noninvasive prenatal testing: from aneuploidy to single genes.

Noninvasive prenatal testing has undergone rapid advances in the last few years. Although researchers have long known about circulating pregnancy-based cell-free fragments of DNA in maternal plasma, it was the introduction of massively parallel sequencing that allowed noninvasive prenatal testing to become a widely used clinical test. This review will begin with an in-depth analysis of the use of noninvasive prenatal testing for aneuploidy, including common causes for inaccurate and/or discordant results. It will also review the ongoing expansion of noninvasive prenatal testing to include copy number variants and select single-gene disorders. Finally, integrated throughout the review is a comparison of noninvasive prenatal testing to more traditional screening methods along with some medical and ethical implications of the widespread use of this new technology.

The influence of fetal gender and maternal characteristics on fetal cell-free DNA in maternal plasma

ObjectiveTo examine the possible effects of fetal gender and maternal characteristics on concentration of fetal cell-free DNA (cfDNA). MethodsMaternal plasma that collected from 2638 singleton pregnancies women were analyzed using non-invasive prenatal testing for aneuploidy by next generation sequence technology. The effects of fetal gender and maternal BMI on fetal cfDNA was measured by Pearson correlation analysis. ResultsThe proportion of fetal cfDNA was positively correlated with gestational age (regression equation: Y=16.2483+6.8856X, r=0.1660, p<0.0001); and negatively correlated with BMI (Body Mass Index) (regression equation: Y=25.6342–19.0065X, r=−0.2146, p<0.0001); Concentration of female fetal cfDNA (mean fetal cfDNA is 13.07%, p<0.0001) is higher than male fetal cfDNA (mean fetal cfDNA is 8.37%, p<0.0001). ConclusionsFetal cfDNA increases stably between 12 and 20 weeks of gestation, and increases in a higher rate after 20 weeks. The maternal BMI is an important factor affecting fetal cfDNA, should be paid enough attention in clinical application.

Development of Reference Materials for Noninvasive Prenatal Aneuploidy Testing by Massively Parallel Sequencing: A Proof-of-Concept Study

Noninvasive prenatal aneuploidy testing (NIPT) represents the first large-scale clinical application of massively parallel sequencing technology. However, no NIPT reference material (RM) has yet been widely adopted, impeding the development of quality management systems and standardization. Developing an NIPT RM from a biological sample is complicated by the low concentration of cell-free DNA (cfDNA), which implies pooling specimens and frequent resampling. We tested the feasibility of using DNA from immortalized cell lines of a woman and her aneuploid offspring to spike an artificial plasma matrix. Enzymatic fragmentation of extracted DNA was optimized to achieve fragment size profiles with a mode of 150 to 200 bp, similar to biological cfDNA. This synthetic material was compared with routine biological samples from pregnant women by a targeted NIPT assay in a multiplex sequencing run on a Proton platform. Sequencing statistics were similar between artificially prepared material and routine biological samples, as well as relative chromosomal representation, and no matrix effects could be detected. Estimate of fetal fraction (FF) was within the range of expected value, and aneuploidy detection statistic (z-score) was also comparable between both types of samples. Artificial plasma spiked with DNA from cell lines of mother and offspring is a promising strategy for developing NIPT RM. This type of material would offer the advantage of a constant and stable composition, allowing for greater standardization of NIPT assays. Moreover, it preserves the parental relatedness used by targeted assay to estimate FF by identification of paternal alleles in single-nucleotide polymorphisms or other variable regions.

Measurement of fetal fraction in cell-free DNA from maternal plasma using a panel of insertion/deletion polymorphisms.

ObjectiveCell-free DNA from maternal plasma can be used for non-invasive prenatal testing for aneuploidies and single gene disorders, and also has applications as a biomarker for monitoring high-risk pregnancies, such as those at risk of pre-eclampsia. On average, the fractional cell-free fetal DNA concentration in plasma is approximately 15%, but can vary from less than 4% to greater than 30%. Although quantification of cell-free fetal DNA is straightforward in the case of a male fetus, there is no universal fetal marker; in a female fetus measurement is more challenging. We have developed a panel of multiplexed insertion/deletion polymorphisms that can measure fetal fraction in all pregnancies in a simple, targeted sequencing reaction.MethodsA multiplex panel of primers was designed for 35 indels plus a ZFX/ZFY amplicon. cfDNA was extracted from plasma from 157 pregnant women, and maternal genomic DNA was extracted for 20 of these samples for panel validation. Sixty-one samples from pregnancies with a male fetus were subjected to whole genome sequencing on the Ion Proton sequencing platform, and fetal fraction derived from Y chromosome counts was compared to fetal fraction measured using the indel panel. A total of 157 cell-free DNA samples were sequenced using the indel panel, and informativity was assessed, along with the proportion of fetal DNA.ResultsUsing gDNA we optimised the indel panel, removing amplicons giving rise to PCR bias. Good correlation was found between fetal fraction using indels and using whole genome sequencing of the Y chromosome (Spearmans r = 0.69). A median of 12 indels were informative per sample. The indel panel was informative in 157/157 cases (mean fetal fraction 14.4% (±0.58%)).ConclusionsUsing our targeted next generation sequencing panel we can readily assess the fetal DNA percentage in male and female pregnancies.

Implementing non-invasive prenatal testing for aneuploidy in a national healthcare system: global challenges and national solutions

BackgroundSince the introduction of non-invasive prenatal testing (NIPT) in 2011, mainly by commercial companies, a growing demand for NIPT from the public and healthcare professionals has been putting pressure on the healthcare systems of various countries. This study identifies the challenges of establishing a responsible implementation of NIPT for aneuploidy in prenatal healthcare, by looking at the Netherlands.MethodsA mixed methods approach involving 13 stakeholder interviews, document analysis and (participatory) observations of the Dutch NIPT Consortium meetings were used. The Diffusion of Innovation Theory and a Network of Actors model were used to interpret the findings.ResultsImplementation of NIPT was facilitated by several factors. The set-up of a national NIPT Consortium enabled discussion and collaboration between stakeholders. Moreover, it led to the plan to offer NIPT through a nationwide research setting (TRIDENT studies), which created a learning phase for careful implementation. The Dutch legal context was perceived as a delaying factor, but eventually gave room for the parties involved to organise themselves and their practices.ConclusionsThis study shows that implementing advanced technologies with profound effects on prenatal care benefit from a learning phase that allows time to carefully evaluate the technical performance and women’s experiences and to enable public debate. Such a coordinated learning phase, involving all stakeholders, will stimulate the process of responsible and sustainable implementation.

Women's Experiences and Preferences for Service Delivery of Non-Invasive Prenatal Testing for Aneuploidy in a Public Health Setting: A Mixed Methods Study.

Non-invasive prenatal testing (NIPT) for aneuploidy is currently only available in the UK through the private sector outside of the research arena. As part of an implementation study in the UK National Health Service we conducted a mixed methods study to assess women’s experience of being offered NIPT using validated measures of decisional conflict, decisional regret and anxiety. Clinical service preferences were also explored. Women with a Down syndrome screening risk >1:1000 were invited to take part in the study and offered NIPT, NIPT and invasive testing (for women with a risk above 1:150) or no further testing. A cross-sectional survey and semi-structured interviews were conducted at two time points; at the time of testing and one month following receipt of results (or equivalent for NIPT decliners). In total, 845 questionnaires and 81 interviews were analysed. The main motivation to accept NIPT was for reassurance (30.8%). Decisional conflict occurred in a minimal number of cases (3.8%), however, none of the participants experienced decisional regret. Around a third (29.9%) of women had elevated anxiety at the time of testing, including intermediate risk women who traditionally would not be offered further testing (54.4% high risk; 20.1% medium risk), a finding supported through the qualitative interviews where prolonged or additional anxiety was found to occur in some medium risk cases. Women were overwhelmingly positive about the opportunity to have a test that was procedurally safe, accurate, reduced the need for invasive testing and identified cases of Down syndrome that might otherwise have been missed. Reassurance was identified as the main motivator for accepting NIPT, particularly amongst medium risk women, with high risk women inclined to accept NIPT to inform decisions around invasive testing. The current turnaround time for test result was identified as a key limitation. All the women interviewed thought NIPT should be adopted as part of NHS clinical practice, with the majority favouring NIPT offered as a first-line test. Our study highlights the potential that NIPT has to positively impact women’s experience of prenatal testing for aneuploidy.

Non-Invasive Prenatal Testing for Aneuploidy: A Review of the Literature

Aneuploidy is one of the leading causes of perinatal deaths and childhood handicaps. An important issue that has led to heated debates in the agenda of maternal-fetal medicine and that has been widely investigated in recent years is ‘the isolation of fetal deoxyribonucleic acid (DNA) from maternal blood. It is agreed that fetal DNA can be used in fetal aneuploidy screening and in predicting some pregnancy complications that are considered to be of placenta-based such as preeclampsia and preterm labor. The fetal DNA testing of maternal blood is regarded as an advanced screening test and generally named as ‘noninvasive prenatal test’ (NIPT). In this meta-study, we will review the accumulated data on the increasing clinical use of NIPT in perinatology as well as the other presently used conventional noninvasive aneuploidy screening methods.

Development and validation of a measure of informed choice for women undergoing non-invasive prenatal testing for aneuploidy.

Non-invasive prenatal testing (NIPT) using cell-free DNA for aneuploidy is a highly accurate screening test; however, concerns exist around the potential for routinisation of testing. The multidimensional measure of informed choice (MMIC) is a quantitative instrument developed to assess informed choice for Down syndrome screening (DSS). We have validated a modified MMIC for NIPT and measured informed choice among women offered NIPT in a public health service. The measure was distributed to women recruited across eight maternity units in the United Kingdom who had accepted DSS. Construct validity was assessed by simultaneously conducting qualitative interviews. Five hundred and eighty-five questionnaires were completed and 45 interviews conducted after blood-draw (or equivalent for those that declined NIPT). The measure demonstrated good internal consistency and internal validity. Results indicate the vast majority of women (89%) made an informed choice; 95% were judged to have good knowledge, 88% had a positive attitude and 92% had deliberated. Of the 11% judged to have made an uninformed choice, 55% had not deliberated, 41% had insufficient knowledge, and 19% had a negative attitude. Ethnicity (OR=2.78, P=0.003) and accepting NIPT (OR=16.05, P=0.021) were found to be significant predictors of informed choice. The high rate of informed choice is likely to reflect the importance placed on the provision of pre-test counselling in this study. It will be vital to ensure that this is maintained once NIPT is offered in routine clinical practice.

Erratum: Non-invasive prenatal testing for aneuploidy and beyond: challenges of responsible innovation in prenatal screening

Erratum: Non-invasive prenatal testing for aneuploidy and beyond: challenges of responsible innovation in prenatal screening

Non-invasive prenatal testing for aneuploidy: a systematic review of Internet advertising to potential users by commercial companies and private health providers.

The development of non-invasive prenatal testing has increased accessibility of fetal testing. Companies are now advertising prenatal testing for aneuploidy via the Internet. The aim of this systematic review of websites advertising non-invasive prenatal testing for aneuploidy was to explore the nature of the information being provided to potential users. We systematically searched two Internet search engines for relevant websites using the following terms: 'prenatal test', 'antenatal test', 'non-invasive test', 'noninvasive test', 'cell-free fetal DNA', 'cffDNA', 'Down syndrome test' or 'trisomy test'. We examined the first 200 websites identified through each search. Relevant web-based text was examined, and key topics were identified, tabulated and counted. To analyse the text further, we used thematic analysis. Forty websites were identified. Whilst a number of sites provided balanced, accurate information, in the majority supporting evidence was not provided to underpin the information and there was inadequate information on the need for an invasive test to definitely diagnose aneuploidy. The information provided on many websites does not comply with professional recommendations. Guidelines are needed to ensure that companies offering prenatal testing via the Internet provide accurate and comprehensible information.

Non-invasive prenatal testing for aneuploidy and beyond: challenges of responsible innovation in prenatal screening. Summary and recommendations.

Non-invasive prenatal testing for aneuploidy and beyond: challenges of responsible innovation in prenatal screening. Summary and recommendations

Early clinical experience of cell-free DNA-based aneuploidy screening: A survey of obstetric sonologists in Australia and New Zealand.

Cell-free DNA-based non-invasive prenatal testing for aneuploidy (NIPT) is now established as the most accurate screening test for trisomy 21. This test became clinically available on a patient-funded basis in Australia and New Zealand in 2013. To investigate the clinical implementation of NIPT use by members of the Australian Association of Obstetrical and Gynaecological Ultrasonologists (AAOGU) during its first year of local availability. Email invitations with an embedded link to an anonymous online survey were sent to all 140 members of the AAOGU in December 2013. We received 54 responses to the survey (39% response rate). Two thirds of respondents were subspecialists in obstetric and gynaecological ultrasound or maternal fetal medicine. The majority of respondents had already used NIPT in their practice (94%). There was no significant difference in the proportion of respondents offering NIPT to high-risk women in private versus public practice (95 versus 82%, P=0.14). However, inequity of access due to cost was the most common ethical issue encountered. The vast majority continued to offer an 11-13week ultrasound in addition to NIPT. Almost all respondents (96%) were also willing to offer NIPT to low-risk women in December 2013 after appropriate genetic counselling. Non-invasive prenatal testing was introduced into clinical care by obstetric sonologists with confidence and in accordance with the current recommended guidelines. These results may help inform future prenatal screening policy and cost-effectiveness analyses.

Non-invasive prenatal testing for aneuploidy and beyond: challenges of responsible innovation in prenatal screening.

This paper contains a joint ESHG/ASHG position document with recommendations regarding responsible innovation in prenatal screening with non-invasive prenatal testing (NIPT). By virtue of its greater accuracy and safety with respect to prenatal screening for common autosomal aneuploidies, NIPT has the potential of helping the practice better achieve its aim of facilitating autonomous reproductive choices, provided that balanced pretest information and non-directive counseling are available as part of the screening offer. Depending on the health-care setting, different scenarios for NIPT-based screening for common autosomal aneuploidies are possible. The trade-offs involved in these scenarios should be assessed in light of the aim of screening, the balance of benefits and burdens for pregnant women and their partners and considerations of cost-effectiveness and justice. With improving screening technologies and decreasing costs of sequencing and analysis, it will become possible in the near future to significantly expand the scope of prenatal screening beyond common autosomal aneuploidies. Commercial providers have already begun expanding their tests to include sex-chromosomal abnormalities and microdeletions. However, multiple false positives may undermine the main achievement of NIPT in the context of prenatal screening: the significant reduction of the invasive testing rate. This document argues for a cautious expansion of the scope of prenatal screening to serious congenital and childhood disorders, only following sound validation studies and a comprehensive evaluation of all relevant aspects. A further core message of this document is that in countries where prenatal screening is offered as a public health programme, governments and public health authorities should adopt an active role to ensure the responsible innovation of prenatal screening on the basis of ethical principles. Crucial elements are the quality of the screening process as a whole (including non-laboratory aspects such as information and counseling), education of professionals, systematic evaluation of all aspects of prenatal screening, development of better evaluation tools in the light of the aim of the practice, accountability to all stakeholders including children born from screened pregnancies and persons living with the conditions targeted in prenatal screening and promotion of equity of access.

Using Noninvasive Prenatal Testing for Aneuploidies in a Developing Country: Lessons Learnt

AbstractThe objective of the present study was to analyze the utility of noninvasive prenatal testing (NIPT) for aneuploidies in a developing country like India. NIPT was offered to 500 pregnant women, after review of data of the ongoing pregnancy. Pre-test counseling included the different methodologies of testing, their benefits, limitations, turnaround time and interpretation of the result and was offered to all. Different vendors were used for the test. The results were explained in a post-test counseling session. The indications of NIPT were positive second (36.6 %) or first (22 %) trimester screen, advanced maternal age with or without positive biochemical screen (24.6 %), ultrasound soft markers (10.8 %), previous history of Down syndrome (4.6 %) and anxious couple (0.1 %). No aneuploidy was detected in 484 samples. In 1 case, no result was available. Fifteen (3 %) cases showed positive results. These included 8 cases of trisomy 21, 3 cases of trisomy 18, 3 cases of monosomy X, and 1 case of triploidy. Confirmatory testing revealed 6 cases (40 %) to be false positive—1 case of trisomy 21, 1 case of trisomy 18, 3 cases of monosomy X, and 1 case of triploidy. Of 484 cases, 230 have delivered healthy neonates, while the rest have yet to deliver. Four cases had to discontinue pregnancy due to complications in later pregnancy but unrelated to the NIPT results. Noninvasive prenatal test qualifies as an ‘advanced’ screening test, and requires invasive diagnostic tests for confirmation of the positive results. The pre- and post-test counseling is essential to appropriately explain the limitations, benefits, and the results to the couple. Recommendations are made for the appropriate deployment of this new technology in developing countries.

Noninvasive Prenatal Testing: The Indian Perspective

AbstractThis article summarizes the Professor Kamal Buckshee Oration given by the author at the annual conference of the Society of Fetal Medicine in Kochi, August 2014. The contributions of Professor Buckshee to fetal medicine are briefly described. The article traces the development of fetal medicine in India. It discusses the application of noninvasive prenatal testing (NIPT) in India. It briefly calculates the cost benefits of sexing of the fetus for X-linked disease. It also describes the results of screening 320 pregnant women using NIPT for aneuploidies. The benefits and limitations of NIPT are presented.

Clinical experience and follow-up with large scale single-nucleotide polymorphism–based noninvasive prenatal aneuploidy testing

We sought to report on laboratory and clinical experience following 6 months of clinical implementation of a single-nucleotide polymorphism-based noninvasive prenatal aneuploidy test in high- and low-risk women. All samples received from March through September 2013 and drawn ≥9 weeks' gestation were included. Samples that passed quality control were analyzed for trisomy 21, trisomy 18, trisomy 13, and monosomy X. Results were reported as high or low risk for fetal aneuploidy for each interrogated chromosome. Relationships between fetal fraction and gestational age and maternal weight were analyzed. Follow-up on outcome was sought for a subset of high-risk cases. False-negative results were reported voluntarily by providers. Positive predictive value (PPV) was calculated from cases with an available prenatal or postnatal karyotype or clinical evaluation at birth. Samples were received from 31,030 patients, 30,705 met study criteria, and 28,739 passed quality-control metrics and received a report detailing aneuploidy risk. Fetal fraction correlated positively with gestational age, and negatively with maternal weight. In all, 507 patients received a high-risk result for any of the 4 tested conditions (324 trisomy 21, 82 trisomy 18, 41 trisomy 13, 61 monosomy X; including 1 double aneuploidy case). Within the 17,885 cases included in follow-up analysis, 356 were high risk, and outcome information revealed 184 (51.7%) true positives, 38 (10.7%) false positives, 19 (5.3%) with ultrasound findings suggestive of aneuploidy, 36 (10.1%) spontaneous abortions without karyotype confirmation, 22 (6.2%) terminations without karyotype confirmation, and 57 (16.0%) lost to follow-up. This yielded an 82.9% PPV for all aneuploidies, and a 90.9% PPV for trisomy 21. The overall PPV for women aged ≥35 years was similar to the PPV for women aged <35 years. Two patients were reported as false negatives. The data from this large-scale report on clinical application of a commercially available noninvasive prenatal test suggest that the clinical performance of this single-nucleotide polymorphism-based noninvasive prenatal test in a mixed high- and low-risk population is consistent with performance in validation studies.

What does next-generation sequencing mean for prenatal diagnosis?

The ability to gain genetic information from the fetus in the mother's blood during pregnancy has been a long desired goal of research in prenatal medicine. The detection of fetal DNA in maternal blood, coupled with the development of the powerful techniques of next-generation sequencing finally transferred this analysis into clinical practice. Following the commercial introduction of noninvasive prenatal testing for aneuploidies, there has been a very strong demand, which has fostered an extreme rapid development and improvement of technology. Publications in this field are so numerous so that it is challenging to keep up with the latest state of the art. Here, we describe the current basic concepts of cell-free DNA-based noninvasive prenatal testing, give an overview of the currently commercially available tests and the chromosomal aberrations that can be identified. We also present current and future concepts for the implementation of cell-free DNA testing into clinical care.

A single center’s experience with noninvasive prenatal testing

Massively parallel sequencing to detect fetal aneuploidy has high sensitivity and specificity for the detection of trisomies 21, 18, and 13 in high-risk populations. The purpose of our study was to review our institution's experience with the use of noninvasive prenatal testing for aneuploidy screening. This was a descriptive study of patients who had undergone noninvasive prenatal testing between January and September 2012 at the UNC Prenatal Diagnosis unit. Two hundred and eight women had undergone noninvasive prenatal testing during the study period. The majority of patients were white (62.9%) and of advanced maternal age (71.2%). The fetal fraction was below the threshold in three obese patients (1.4%). An abnormal noninvasive prenatal test (aneuploidy detected or "unclassified" result) was reported in 6.3% (13/208) of the patients. Noninvasive prenatal testing had a combined sensitivity of 87.5% and specificity of 99.5% for detection of trisomies 21, 18, and 13. There were "unclassified" results in 11.1% (5/45) of the patients. Over the study period, the number of patients requesting noninvasive prenatal testing increased monthly. The rate of amniocenteses significantly declined (8.1% before vs. 5.3% after noninvasive prenatal testing, P < 0.01). An increase in uptake of noninvasive prenatal testing and a significant decline in amniocentesis procedures were observed. The rates of "unclassified," false-positive, and false-negative results were higher than anticipated based on published preclinical trials.

Attitudes towards non-invasive prenatal testing for aneuploidy among US adults of reproductive age

Objective(s)To determine how adults in the United States (US) view non-invasive prenatal testing using cell-free fetal DNA (cffDNA testing) in order to help estimate uptake.Study DesignA national sample of 1,861 US-based adults was surveyed using a validated online survey instrument. The survey was administered by a commercial survey research company. Respondents were randomized to receive a survey about prenatal testing for trisomy 13 and 18 or trisomy 21. Participants were asked to select among testing modalities, including cffDNA testing, and rank the features of testing that they considered most important to decision making.ResultsThere was substantive interest in the use of cffDNA testing rather than traditional screening mechanisms with a minority of respondents reporting that they would support the use of both methods in combination. The lower rates of false negative and false positive test results and the ability to use the test earlier in the pregnancy were the most highly rated benefits of cffDNA testing. Participants expressed strong support for diagnostic confirmation via invasive testing after a positive result from either screening or cffDNA testing. However, almost one-third of participants reported that they would not endorse the use of either invasive or non-invasive prenatal testing.Conclusion(s)There appears to be support for uptake of non-invasive prenatal tests. Clinical guidelines should therefor go forward in providing guidance on how to integrate non-invasive methods into current standard of care. However, our findings indicate that even when accuracy, which is rated by patients as the most important aspect of prenatal testing, is significantly improved over existing screening methods and testing is offered non-invasively, the number of individuals who reported that they would decline any testing remained the same. Attention should therefor be directed at ensuring that the right of informed refusal of prenatal testing is not impacted by new, non-invasive methods.