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1.  Attitudes Towards Non-Invasive Prenatal Testing for Aneuploidy Among United States Adults of Reproductive Age 
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 Design
A 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.
There 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.
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.
PMCID: PMC4399855  PMID: 24603453
Prenatal Testing; Prenatal Diagnosis; Ethics
2.  Safe, accurate, prenatal diagnosis of thanatophoric dysplasia using ultrasound and free fetal DNA 
Prenatal Diagnosis  2014;33(5):416-423.
To improve the prenatal diagnosis of thanatophoric dysplasia by defining the change in fetal size across gestation and the frequency of sonographic features, and developing non-invasive molecular genetic diagnosis based on cell-free fetal DNA (cffDNA) in maternal plasma.
Fetuses with a confirmed diagnosis of thanatophoric dysplasia were ascertained, records reviewed, sonographic features and measurements determined. Charts of fetal size were then constructed using the LMS (lambda-mu-sigma) method and compared with charts used in normal pregnancies and those complicated by achondroplasia. Cases in this cohort referred to our Regional Genetics Laboratory for molecular diagnosis using cffDNA were identified and results reviewed.
Forty-two cases were scanned in our units. Commonly reported sonographic features were very short and sometimes bowed femora, frontal bossing, cloverleaf skull, short fingers, a small chest and polyhydramnios. Limb shortening was obvious from as early as 13 weeks' gestation, with minimal growth after 20 weeks. Analysis of cffDNA in three of these pregnancies confirmed the presence of the c.742C>CT (p.Arg248Cys) or the c.1948A>AG (p.Lys650Glu) mutation in the fibroblast growth factor receptor 3 gene.
These data should improve the accuracy of the sonographic diagnosis of thanatophoric dysplasia and have implications for reliable and safe targeted molecular confirmation using cffDNA. © 2013 The Authors. Prenatal Diagnosis published by John Wiley & Sons Ltd.
PMCID: PMC4166694  PMID: 23408600
3.  Pre-Analytical Conditions in Non-Invasive Prenatal Testing of Cell-Free Fetal RHD 
PLoS ONE  2013;8(10):e76990.
Non-invasive prenatal testing of cell-free fetal DNA (cffDNA) in maternal plasma can predict the fetal RhD type in D negative pregnant women. In Denmark, routine antenatal screening for the fetal RhD gene (RHD) directs the administration of antenatal anti-D prophylaxis only to women who carry an RhD positive fetus. Prophylaxis reduces the risk of immunization that may lead to hemolytic disease of the fetus and the newborn. The reliability of predicting the fetal RhD type depends on pre-analytical factors and assay sensitivity. We evaluated the testing setup in the Capital Region of Denmark, based on data from routine antenatal RHD screening.
Blood samples were drawn at gestational age 25 weeks. DNA extracted from 1 mL of plasma was analyzed for fetal RHD using a duplex method for exon 7/10. We investigated the effect of blood sample transportation time (n = 110) and ambient outdoor temperatures (n = 1539) on the levels of cffDNA and total DNA. We compared two different quantification methods, the delta Ct method and a universal standard curve. PCR pipetting was compared on two systems (n = 104).
The cffDNA level was unaffected by blood sample transportation for up to 9 days and by ambient outdoor temperatures ranging from -10°C to 28°C during transport. The universal standard curve was applicable for cffDNA quantification. Identical levels of cffDNA were observed using the two automated PCR pipetting systems. We detected a mean of 100 fetal DNA copies/mL at a median gestational age of 25 weeks (range 10–39, n = 1317).
The setup for real-time PCR-based, non-invasive prenatal testing of cffDNA in the Capital Region of Denmark is very robust. Our findings regarding the transportation of blood samples demonstrate the high stability of cffDNA. The applicability of a universal standard curve facilitates easy cffDNA quantification.
PMCID: PMC3800077  PMID: 24204719
4.  Evaluation of a Novel Assay for Detection of the Fetal Marker RASSF1A: Facilitating Improved Diagnostic Reliability of Noninvasive Prenatal Diagnosis 
PLoS ONE  2012;7(9):e45073.
Analysis of cell free fetal (cff) DNA in maternal plasma is used routinely for non invasive prenatal diagnosis (NIPD) of fetal sex determination, fetal rhesus D status and some single gene disorders. True positive results rely on detection of the fetal target being analysed. No amplification of the target may be interpreted either as a true negative result or a false negative result due to the absence or very low levels of cffDNA. The hypermethylated RASSF1A promoter has been reported as a universal fetal marker to confirm the presence of cffDNA. Using methylation-sensitive restriction enzymes hypomethylated maternal sequences are digested leaving hypermethylated fetal sequences detectable. Complete digestion of maternal sequences is required to eliminate false positive results.
cfDNA was extracted from maternal plasma (n = 90) and digested with methylation-sensitive and insensitive restriction enzymes. Analysis of RASSF1A, SRY and DYS14 was performed by real-time PCR.
Hypermethylated RASSF1A was amplified for 79 samples (88%) indicating the presence of cffDNA. SRY real time PCR results and fetal sex at delivery were 100% accurate. Eleven samples (12%) had no detectable hypermethylated RASSF1A and 10 of these (91%) had gestational ages less than 7 weeks 2 days. Six of these samples were male at delivery, five had inconclusive results for SRY analysis and one sample had no amplifiable SRY.
Use of this assay for the detection of hypermethylated RASSF1A as a universal fetal marker has the potential to improve the diagnostic reliability of NIPD for fetal sex determination and single gene disorders.
PMCID: PMC3443218  PMID: 23024794
5.  Fetal Sex Determination using Non-Invasive Method of Cell-free Fetal DNA in Maternal Plasma of Pregnant Women During 6th– 10th Weeks of Gestation 
In previous years, identification of fetal cells in maternal blood circulation has caused a new revolution in non-invasive method of prenatal diagnosis. Low number of fetal cells in maternal blood and long-term survival after pregnancy limited the use of fetal cells in diagnostic and clinical applications. With the discovery of cell-free fetal DNA (cffDNA) in plasma of pregnant women, access to genetic material of the fetus had become possible to determine early gender of a fetus in pregnancies at the risk of X-linked genetic conditions instead of applying invasive methods. Therefore in this study, the probability of detecting sequences on the Y chromosome in pregnant women has been evaluated to identify the gender of fetuses. Peripheral blood samples were obtained from 80 pregnant women at 6th to 10th weeks of gestation and then the fetal DNA was extracted from the plasma. Nested PCR was applied to detect the sequences of single copy SRY gene and multi copy DYS14 & DAZ genes on the Y chromosome of the male fetuses. At the end, all the obtained results were compared with the actual gender of the newborns. In 40 out of 42 born baby boys, the relevant gene sequences were identified and 95.2% sensitivity was obtained. Non-invasive early determination of fetal gender using cffDNA could be employed as a pre-test in the shortest possible time and with a high reliability to avoid applying invasive methods in cases where a fetus is at the risk of genetic diseases.
PMCID: PMC3558193  PMID: 23407464
Fetus; Genetic material; Prenatal diagnosis; Sex determination
6.  The use of cffDNA in fetal sex determination during the first trimester of pregnancy of female DMD carriers 
Chorionic villus sampling (CVS) or amniocentesis for fetal sex determination is generally the first step in the prenatal diagnosis of X-linked genetic disorders such as Duchenne muscular dystrophy (DMD). However, non-invasive prenatal diagnostic (NIPD) techniques such as measurement of cell-free fetal DNA (cffDNA) in maternal plasma are preferable given the procedure-related miscarriage rate of CVS. We determined fetal sex during the first trimester using a quantitative real-time polymerase chain reaction (PCR) assay of cffDNA in pregnant carriers of DMD. The fetal sex was confirmed by amniocentesis karyotype analysis and multiplex ligation-dependent probe amplification (MLPA) at 16 weeks. This procedure may avoid unnecessary CVS or amniocentesis of female fetuses.
PMCID: PMC4204568  PMID: 25343090
Cell-free fetal DNA (cffDNA); non-invasive prenatal diagnostic (NIPD); Duchenne muscular dystrophy (DMD); fetal sex determination
7.  Reliable Detection of Paternal SNPs within Deletion Breakpoints for Non-Invasive Prenatal Exclusion of Homozygous α0-Thalassemia in Maternal Plasma 
PLoS ONE  2011;6(9):e24779.
Reliable detection of large deletions from cell-free fetal DNA (cffDNA) in maternal plasma is challenging, especially when both parents have the same deletion owing to a lack of specific markers for fetal genotyping. In order to evaluate the efficacy of a non-invasive prenatal diagnosis (NIPD) test to exclude α-thalassemia major that uses SNPs linked to the normal paternal α-globin allele, we established a novel protocol to reliably detect paternal SNPs within the (−−SEA) breakpoints and performed evaluation of the diagnostic potential of the protocol in a total of 67 pregnancies, in whom plasma samples were collected prior to invasive obstetrics procedures in southern China. A group of nine SNPs identified within the deletion breakpoints were scanned to select the informative SNPs in each of the 67 couples DNA by multiplex PCR based mini-sequencing technique. The paternally inherited SNP allele from cffDNA was detected by allele specific real-time PCR. A protocol for reliable detection of paternal SNPs within the (−−SEA) breakpoints was established and evaluation of the diagnostic potential of the protocol was performed in a total of 67 pregnancies. In 97% of the couples one or more different SNPs within the deletion breakpoint occurred between paternal and maternal alleles. Homozygosity for the (−−SEA) deletion was accurately excluded in 33 out of 67 (49.3%, 95% CI, 25.4–78.6%) pregnancies through the implementation of the protocol. Protocol was completely concordant with the traditional reference methods, except for two cases that exhibited uncertain results due to sample hemolysis. This method could be used as a routine NIPD test to exclude gross fetal deletions in α-thalassemia major, and could further be employed to test for other diseases due to gene deletion.
PMCID: PMC3182989  PMID: 21980356
8.  Determination of Fetal Rhesus D Status by Maternal Plasma DNA Analysis 
In this study, we assessed the feasibility of fetal RhD genotyping by analysis of cell-free fetal DNA(cffDNA) extracted from plasma samples of Rhesus (Rh) D-negative pregnant women by using real-time polymerase chain reaction (PCR). Fetal genotyping was performed on 30 RhD-negative women between 9 and 39 weeks of gestation who were referred to us for invasive testing [amniocentesis/chorionic villi sampling (CVS)]. The fetal RHD genotype was determined based on real-time PCR method. Exons 7 and 10 of the RHD and SRY genes were targeted.
Among the pregnant women, 12 were carrying male and 17 were carrying female fetuses. Out of 29 pregnant women, 21 had RhD-positive and nine had RhD-negative fetuses. One sample (case 12, whose blood group was found to be AB Rh [+]) was excluded due to controversial results from repeated serological analyses. All prenatal results were in concordance with postnatal RhD status and fetal sex without false- positive or -negative results. Performing real-time PCR on cffDNA showed accurate, efficient and reliable results, allowing rapid and high throughput non invasive determination of fetal sex and RhD status in clinical samples.
PMCID: PMC4001413  PMID: 24778561
Rhesus D (RhD); Non invasive prenatal diagnosis
9.  Evaluation of a Modified DNA Extraction Method for Isolation of Cell-Free Fetal DNA from Maternal Serum 
Discovery of short cell free fetal DNA (cffDNA) fragments in maternal plasma has created major changes in the field of prenatal diagnosis. The use of cffDNA to set up noninvasive prenatal test is limited due to the low concentration of fetal DNA in maternal plasma therefore, employing a high efficiency extraction method leads to more accurate results. The aim of this study was to evaluate the efficiency of Triton/Heat/Phenol (THP) protocol in comparison with the QIAamp DNA Blood mini Kit for cffDNA purification.
In order to evaluate the efficiency of THP protocol, DNA of Rhesus D (RhD) negative pregnant women's plasma was collected, then real-time PCR for RHD exon 7 was performed. The Ct value data of real time PCR obtained by two different methods were compared and after delivery serology test on cord blood was done to validate the real time PCR results.
The results indicated significant differences between two extraction methods (p=0.001). The mean±SD of Ct-value using THP protocol was 33.8±1.6 and 36.1±2.47 using QIAamp DNA Blood mini Kit.
Our finding demonstrated that THP protocol was more effective than the QIAamp DNA Blood mini Kits for cffDNA extraction and lead to decrease the false negative results.
PMCID: PMC4483320  PMID: 26140187
Fetus; Prenatal diagnosis; Real time polymerase chain reaction; THP protocol
10.  Quantification of Maternal Serum Cell-Free Fetal DNA in Early-Onset Preeclampsia 
The aim of this study was to determine whether the increased serum cell-free fetal DNA (cffDNA) level of gravidas developed into early-onset preeclampsia (EOPE) subsequently in the early second trimesters is related to prenatal screening markers. Serum was collected from 1011 gravidas. The level of cffDNA and prenatal screening markers were analyzed in 20 cases with EOPE and 20 controls. All fetuses were male. The maternal serum cffDNA level was assessed by amplification of the Y chromosome specific gene. Correlations between the variables were examined. (Logged) cffDNA in EOPE (median, 3.08; interquartile range, 2.93–3.68) was higher than controls (median, 1.79; interquartile range, 1.46–2.53). The increased level of (logged) cffDNA was correlated significantly with the increased human chorionic gonadotropin (HCG) level (r = 0.628, p < 0.001). Significant reciprocal correlations between cffDNA and babies’ birth weight as well as gestation weeks at delivery were noted (r = −0.516, p = 0.001; r = −0.623, p < 0.001, respectively). The sensitivity and specificity of cffDNA to discriminate between the EOPE cases and the controls were 90% and 85%, respectively. CffDNA is a potential marker for EOPE, which had a significant reciprocal correlation with babies’ birth weight and gestation weeks at delivery. Moreover, it may help in indicating the underlying hypoxic condition in the placenta.
PMCID: PMC3645703  PMID: 23567271
preeclampsia; cell-free fetal DNA; HCG
11.  Integrating stakeholder perspectives into the translation of cell-free fetal DNA testing for aneuploidy 
Genome Medicine  2012;4(6):49.
The translation of novel genomic technologies from bench to bedside enjoins the comprehensive consideration of the perspectives of all stakeholders who stand to influence, or be influenced by, the translational course. Non-invasive prenatal aneuploidy testing that utilizes cell-free fetal DNA (cffDNA) circulating in maternal blood is one example of an innovative technology that promises significant benefits for its intended end users; however, it is currently uncertain whether it will achieve widespread clinical implementation. We conducted qualitative interviews with 18 diverse stakeholders in this domain, including prospective users of the technology and healthcare personnel, researchers and developers, and experts in social, legal, and regulatory aspects of genetic technology, and a pilot survey of 62 obstetric healthcare providers. Analysis of interview and survey data was combined with a review of the proceedings of a full-day, multidisciplinary conference on the topic and published scientific and ethics literature surrounding this and other relevant technologies.
We constructed potential pathways for technological implementation, identified broad stakeholder classes party to these translational processes, and performed a preliminary assessment of the viewpoints and interrelations among these diverse stakeholders. Some of the stakeholders whose priorities are critical to understand and integrate into translation include pregnant women and their families; healthcare providers; scientists, their institutions or companies, and the funding agencies that support them; regulatory and judicial bodies; third-party payers; professional societies; educational systems; disability rights communities; and other representatives from civil society. Stakeholder interviews, survey findings, and conference proceedings add complexity to these envisioned pathways and also demonstrate a paramount need to incorporate an iterative stakeholder analysis early and throughout the translational endeavor. We believe that the translational framework that we have developed will help guide crucial future stakeholder mapping and engagement activities for cffDNA aneuploidy testing and inform novel methods of technology assessment for other developments in the growing field of genomic medicine.
Mapping potential pathways for implementation and exploring the attitudes and interrelations of diverse stakeholders may lead to more effective translation of a novel method of prenatal aneuploidy testing.
PMCID: PMC3738226  PMID: 22720727
12.  Understanding the Limitations of Circulating Cell Free Fetal DNA: An Example of Two Unique Cases 
Circulating cell free fetal DNA (cffDNA) is an effective screening modality for fetal aneuploidy. We report two cases of false positive results. The first case involves a female, with self-reported Down syndrome. CffDNA returned positive for trisomy 18 leading to a maternal diagnosis of mosaicism chromosome 18 with normal fetal karyotype. The second case involves a patient with an anomalous fetal ultrasound and cffDNA positive for trisomy 13. Amniocentesis demonstrated a chromosome 8p duplication/deletion. False positive cffDNA may arise in clinical scenarios where diagnostic testing is clearly indicated. Practitioners should recognize the limitations of cffDNA.
PMCID: PMC4185925  PMID: 25298847
Prenatal diagnosis; Cell free fetal DNA; Fetal anomalies
13.  Cell-free fetal DNA testing for fetal aneuploidy and beyond: clinical integration challenges in the US context 
Human Reproduction (Oxford, England)  2012;27(11):3123-3131.
The recent release of new, non-invasive prenatal tests for fetal aneuploidy using cell-free fetal DNA (cffDNA) has been hailed as a revolution in prenatal testing and has triggered significant commercial interest in the field. Ongoing research portends the arrival of a wide range of cffDNA tests. However, it is not yet clear how these tests will be integrated into well-established prenatal testing strategies in the USA, as the timing of such testing and the degree to which new non-invasive tests will supplement or replace existing screening and diagnostic tools remain uncertain. We argue that there is an urgent need for policy-makers, regulators and professional societies to provide guidance on the most efficient and ethical manner for such tests to be introduced into clinical practice in the USA.
PMCID: PMC3472618  PMID: 22863603
ethics; prenatal diagnosis; aneuploidy
14.  Non Invasive Prenatal Diagnosis of Aneuploidy: Next Generation Sequencing or Fetal DNA Enrichment? 
Current invasive procedures [amniocentesis and chorionic villus sampling (CVS)] pose a risk to mother and fetus and such diagnostic procedures are available only to high risk pregnancies limiting aneuploidy detection rate. This review seeks to highlight the necessity of investing in non invasive prenatal diagnosis (NIPD) and how NIPD would improve patient safety and detection rate as well as allowing detection earlier in pregnancy.
Non invasive prenatal diagnosis can take either a proteomics approach or nucleic acid-based approach; this review focuses on the latter. Since the discovery of cell free fetal DNA (cffDNA) and fetal RNA in maternal plasma, procedures have been developed for detection for monogenic traits and for some have become well established (e.g., RHD blood group status). However, NIPD of aneuploidies remains technically challenging. This review examines currently published literature evaluating techniques and approaches that have been suggested and developed for aneuploidy detection, highlighting their advantages and limitations and areas for further research.
PMCID: PMC3776677  PMID: 24052738
Aneuploidy; Cell free fetal DNA (cff DNA); Non invasive prenatal diagnosis (NIPD)
15.  Non-invasive prenatal diagnosis of fetal trisomy 21 using cell-free fetal DNA in maternal blood 
Since the existence of cell-free fetal DNA (cff-DNA) in maternal circulation was discovered, it has been identified as a promising source of fetal genetic material in the development of reliable methods for non-invasive prenatal diagnosis (NIPD) of fetal trisomy 21 (T21). Currently, a prenatal diagnosis of fetal T21 is achieved through invasive techniques, such as chorionic villus sampling or amniocentesis. However, such invasive diagnostic tests are expensive, require expert technicians, and have a miscarriage risk approximately 1%. Therefore, NIPD using cff-DNA in the detection of fetal T21 is significant in prenatal care. Recently, the application of new techniques using single-molecular counting methods and the development of fetal-specific epigenetic markers has opened up new possibilities in the NIPD of fetal T21 using cff-DNA. These new technologies will facilitate safer, more sensitive and accurate prenatal tests in the near future. In this review, we investigate the recent methods for the NIPD of fetal T21 and discuss their implications in future clinical practice.
PMCID: PMC3784100  PMID: 24327983
Cell-free fetal DNA; Noninvasive prenatal diagnosis; Trisomy 21
16.  Cell-free fetal DNA and pregnancy-related complications (Review) 
Molecular Medicine Reports  2014;11(4):2367-2372.
Cell-free fetal DNA (cff-DNA) is a novel promising biomarker that has been applied in various aspects of obstetrical research, notably in prenatal diagnosis and complicated pregnancies. It is easily detected by semi-quantitative PCR for the SRY target gene. It is well recognized that the levels of circulating cff-DNA play a role in various complications of pregnancy. In this review, we explore the implications of the detection of cff-DNA in a range of pregnancy-related complications, such as preeclampsia, intrauterine growth restriction (IUGR), preterm labor, placenta previa and hyperemesis gravidarum. cff-DNA is released due to apoptotic mechanisms occurring on trophoblastic cells, although recent in vivo studies support the existence of additional mechanisms. The increase in the levels of cff-DNA can be used to predict pregnancy-related complications and has great value in the field of prenatal diagnosis and in common pregnancy-related complications, as it precedes the clinical symptoms of the disease. Gestational age is a factor that determines the elevation in cff-DNA levels in response to pathological conditions. In conclusion, the detection of cff-DNA levels has a number of valuable applications in prenatal screening; however, the detection of cff-DNA levels has not yet been applied in clinical practice for the diagnosis of pregnancy-related disorders. Thus, studies are focusing on unraveling the etiology of alterations in its levels under pathological conditions during pregnancy, in order to determine the potenial predictive and diagnostic applications of this biomarker.
PMCID: PMC4337476  PMID: 25530428
cell-free fetal DNA; intrauterine growth restriction; non-invasive prenatal screening; obstetrics; preeclampsia; pregnancy complications; preterm labor
17.  Method for the extraction of high quantity and quality cell-free DNA from amniotic fluid 
Circulating cell-free fetal deoxyribonucleic acids (cffDNA) are promising biomarkers with various promising clinical applications. Second and third trimester amniotic fluid (AF) is a rich source of cffDNA. Further improvements to the original protocol for the extraction of cffDNA from AF supernatant resulted in statistically significant higher yields of high quality cffDNA, allowing for a substantial majority of samples to be analyzed with subsequent molecular methods (e.g. comparative genomic hybridization [CGH] micro arrays) to further assess for genetic abnormalities. Several advantages have been realized with the optimized protocol. In addition to an improved yield from a greater proportion of samples as compared to the original protocol, the current method, using large silico-membranes, allows for the extraction of cffDNA from up to ten samples in less than three hours. The replacement of the original lysis buffer eliminates the need for a heating bath during the lysis step, and fewer overall steps are involved in the protocol (e.g. to reduce potential contamination).
The improvements in the yield with the current protocol make it possible to augment current standard of care through the analysis of this previously unappreciated source of genetic material, and furthermore, will allow for exploration of widely unknown genetic, pathophysiological and kinetic issues of cell-free fetal DNA in amniotic fluid.
PMCID: PMC2679523  PMID: 18425491
Prenatal diagnosis; cell-free fetal DNA; prental screening; pregnancy
18.  Maternal Serum Screening Markers and Adverse Outcome: A New Perspective 
Journal of Clinical Medicine  2014;3(3):693-712.
There have been a number of studies evaluating the association of aneuploidy serum markers with adverse pregnancy outcome. More recently, the development of potential treatments for these adverse outcomes as well as the introduction of cell-free fetal DNA (cffDNA) screening for aneuploidy necessitates a re-evaluation of the benefit of serum markers in the identification of adverse outcomes. Analysis of the literature indicates that the serum markers tend to perform better in identifying pregnancies at risk for the more severe but less frequent form of individual pregnancy complications rather than the more frequent but milder forms of the condition. As a result, studies which evaluate the association of biomarkers with a broad definition of a given condition may underestimate the ability of such markers to identify pregnancies that are destined to develop the more severe form of the condition. Consideration of general population screening using cffDNA solely must be weighed against the fact that traditional screening using serum markers enables detection of severe pregnancy complications, not detectable with cffDNA, of which many may be amenable to treatment options.
PMCID: PMC4449652
aneuploidy screening; preeclampsia; IUGR; preterm birth; fetal loss; placenta accreta; open neural tube defects
19.  Routine administration of Anti-D: the ethical case for offering pregnant women fetal RHD genotyping and a review of policy and practice 
Since its introduction in the 1960s Anti-D immunoglobulin (Anti-D Ig) has been highly successful in reducing the incidence of haemolytic disease of the fetus and newborn (HDFN) and achieving improvements to maternal and fetal health. It has protected women from other invasive interventions during pregnancy and prevented deaths and damage amongst newborns and is a technology which has been adopted worldwide. Currently about one third of pregnant women with the blood group Rhesus D (RhD) negative in the UK (approximately 40,000 women per year in England and Wales), receive antenatal Anti-D Ig in pregnancy when they do not require it because they are carrying a RhD negative fetus. Since 1997, a test using cell free fetal DNA (cffDNA) in maternal blood has been developed to identify the genotype of the fetus and can be used to predict the fetal RhD blood group.
This paper considers whether it is ethically acceptable to continue administering antenatal Anti-D Ig to all RhD negative women when fetal RHD genotyping using maternal blood could identify those women who do not need this product.
The antenatal administration of Anti-D Ig to a third of RhD negative pregnant women who carry a RhD negative fetus and therefore do not need it raises important ethical issues. If fetal RHD genotyping using maternal blood was offered to all RhD negative pregnant women it would assist them to make an informed choice about whether or not to have antenatal Anti-D Ig.
PMCID: PMC3944436  PMID: 24568571
Anti-D immunoglobulin; Fetal RHD genotyping; RhD blood group; Ethics; Informed consent
20.  Impact of Cell-Free Fetal DNA Screening on Patients’ Choice of Invasive Procedures after a Positive California Prenatal Screen Result 
Journal of Clinical Medicine  2014;3(3):849-864.
Until recently, maternal serum analyte levels paired with sonographic fetal nuchal translucency measurement was the most accurate prenatal screen available for Trisomies 18 and 21, (91% and 94% detection and false positive rates of 0.31% and 4.5% respectively). Women with positive California Prenatal Screening Program (CPSP) results have the option of diagnostic testing to determine definitively if the fetus has a chromosomal abnormality. Cell-free fetal (cff-) DNA screening for Trisomies 13, 18, and 21 was first offered in 2012, allowing women with positive screens to choose additional screening before diagnostic testing. Cff-DNA sensitivity rates are as high as 99.9% and 99.1%, with false positive rates of 0.4% and 0.1%, for Trisomies 18 and 21, respectively. A retrospective chart review was performed in 2012 on 500 CPSP referrals at the University of California, San Diego Thornton Hospital. Data were collected prior to and after the introduction of cff-DNA. There was a significant increase in the number of participants who chose to pursue additional testing and a decrease in the number of invasive procedures performed after cff-DNA screening was available. We conclude that as fetal aneuploidy screening improves, the number of invasive procedures will continue to decrease.
PMCID: PMC4449655
prenatal; cell-free fetal DNA screening; non-invasive prenatal testing
21.  Tracking fetal development through molecular analysis of maternal biofluids☆ 
Biochimica et biophysica acta  2012;1822(12):1970-1980.
Current monitoring of fetal development includes fetal ultrasonography, chorionic villus sampling or amniocentesis for chromosome analysis, and maternal serum biochemical screening for analytes associated with aneuploidy and open neural tube defects. Over the last 15 years, significant advances in noninvasive prenatal diagnosis (NIPD) via cell-free fetal (cff) nucleic acids in maternal plasma have resulted in the ability to determine fetal sex, RhD genotype, and aneuploidy. Cff nucleic acids in the maternal circulation originate primarily from the placenta. This contrasts with cff nucleic acids in amniotic fluid, which derive from the fetus, and are present in significantly higher concentrations than in maternal blood. The fetal origin of cff nucleic acids in the amniotic fluid permits the acquisition of real-time information about fetal development and gene expression. This review seeks to provide a comprehensive summary of the molecular analysis of cff nucleic acids in maternal biofluids to elucidate mechanisms of fetal development, physiology, and pathology. This article is part of a Special Issue entitled: Molecular Genetics of Human Reproductive Failure.
PMCID: PMC4378902  PMID: 22542507
Fetal DNA; Fetal mRNA; Fetal development; Amniotic fluid transcriptome; Pregnancy; Noninvasive prenatal diagnosis
22.  Haplotype-assisted accurate non-invasive fetal whole genome recovery through maternal plasma sequencing 
Genome Medicine  2013;5(2):18.
The applications of massively parallel sequencing technology to fetal cell-free DNA (cff-DNA) have brought new insight to non-invasive prenatal diagnosis. However, most previous research based on maternal plasma sequencing has been restricted to fetal aneuploidies. To detect specific parentally inherited mutations, invasive approaches to obtain fetal DNA are the current standard in the clinic because of the experimental complexity and resource consumption of previously reported non-invasive approaches.
Here, we present a simple and effective non-invasive method for accurate fetal genome recovery-assisted with parental haplotypes. The parental haplotype were firstly inferred using a combination strategy of trio and unrelated individuals. Assisted with the parental haplotype, we then employed a hidden Markov model to non-invasively recover the fetal genome through maternal plasma sequencing.
Using a sequence depth of approximately 44X against a an approximate 5.69% cff-DNA concentration, we non-invasively inferred fetal genotype and haplotype under different situations of parental heterozygosity. Our data show that 98.57%, 95.37%, and 98.45% of paternal autosome alleles, maternal autosome alleles, and maternal chromosome X in the fetal haplotypes, respectively, were recovered accurately. Additionally, we obtained efficient coverage or strong linkage of 96.65% of reported Mendelian-disorder genes and 98.90% of complex disease-associated markers.
Our method provides a useful strategy for non-invasive whole fetal genome recovery.
PMCID: PMC3706925  PMID: 23445748
23.  Circulating cell-free DNA levels increase variably following chorionic villus sampling 
Prenatal diagnosis  2010;30(4):325-328.
Cell-free fetal DNA (cffDNA) in maternal plasma results from degradation of fetal and/or placental cells. Our objective was to determine if chorionic villus sampling (CVS) causes increased release of fetal and/or maternal DNA.
Fifty-two pregnant women were recruited prior to CVS, performed for clinical indications, at 10 5/7 to 13 2/7 weeks. Maternal blood was collected before and within 15 minutes after CVS. cffDNA was extracted from plasma. Real-time polymerase chain reaction (PCR) amplification of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and the Y chromosome sequence DYS1 were used as measures of total and fetal DNA, respectively. All samples were analyzed in triplicate without knowledge of fetal gender.
Sensitivity of DYS1 detection in male fetuses was 100% (n=30); specificity in female fetuses was 100% (n=22). While a majority of women had >50% post-procedure increases in both fetal and total DNA, some showed post-procedure decreases. However, overall median proportional increases were not statistically significant. Gestational age (GA), placental location, and individual CVS operator did not correlate with changes in DNA levels.
While there were no statistically significant overall changes in DNA levels after CVS, as-yet undiscovered variables may influence the extent of post-procedure release of cell-free DNA in the circulation of pregnant women.
PMCID: PMC2966017  PMID: 20127897
CVS; cell-free DNA; prenatal diagnosis
24.  Inhibitory Control Test, Critical Flicker Frequency, and Psychometric Tests in the Diagnosis of Minimal Hepatic Encephalopathy in Cirrhosis 
Minimal hepatic encephalopathy (MHE) impairs health-related quality of life and driving ability of the patient.
We assessed the utility of the inhibitory control test (ICT), critical flicker frequency (CFF), and psychometry in the diagnosis of MHE.
Patients and Methods:
Consecutive patients with cirrhosis underwent number connection tests A and B (NCT-A, B), digit symbol test (DST), line tracing test (LTT), serial dot test (SDT), CFF, and ICT at baseline and after four hours. Fifty healthy subjects served as controls for the ICT test.
Fifty patients with cirrhosis (43.4 ± 10.2 yrs, M: F 42:8) underwent psychometric tests [NCT-A (48.3 ± 17.7 vs. 42.6 ± 17.3 sec, P = 0.001), NCT-B (85.7 ± 40.1 vs. 90.2 ± 37.0 sec, P = 0.18), DST (23.5 ± 9.3 vs. 23.0 ± 8.7, P = 0.45), LTT (96.6 ± 48.2 vs. 96.8 ± 46.8 sec, P = 0.92), SDT (88.0 ± 39.5 vs. 83.4 ± 37.2 sec, P = 0.02)] at baseline and after four hours. Target accuracy of ICT was lower in patients with cirrhosis compared with controls (88.4 ± 5.6 vs. 95.6 ± 2.1, P = 0.01), whereas ICT lures were higher (18.3 ± 4.2 vs 10.2 ± 2.8, P = 0.01). Patients with cirrhosis showed a reduction in lures in the second evaluation compared with the first (18.3 ± 4.2 vs. 17.1 ± 4.3, P = 0.003) but no change in target accuracy (88.4 ± 5.6 vs. 88.4 ± 5.3, P = 0.97). Control subjects did not show any change either in lures (10.2 ± 2.8 vs. 10.3 ± 2.1, P = 0.65) or target accuracy (95.6 ± 2.1 vs. 95.5 ± 2.2, P = 0.82). The sensitivity and specificity of ICT test for the diagnosis of MHE at lure rate >16.5 was 88.5 and 56%, respectively. CFF in patients with MHE (38.4 ± 1.8 vs. 38.6 ± 1.5, P = 0.3) and non MHE (40.6 ± 2.2 vs. 40.8 ± 2.2, P = 0.6) did not show any difference after four hours as in controls (41.9 ± 2.4 vs. 42.1 ± 2.0, P = 0.3). Thirty one (31%) patients preferred psychometric tests, 57 (57%) preferred CFF and only 12 (12%) preferred ICT (P = 0.001).
ICT, CFF, and psychometric tests are useful tools to assess MHE, and CFF was preferred by this study cohort.
PMCID: PMC3603489  PMID: 23319037
Critical flicker frequency; inhibitory control test; psychometric tests
25.  Noninvasive Prenatal Detection for Pathogenic CNVs: The Application in α-Thalassemia 
PLoS ONE  2013;8(6):e67464.
The discovery of cell free fetal DNA (cff-DNA) in maternal plasma has brought new insight for noninvasive prenatal diagnosis. Combining with the rapidly developed massively parallel sequencing technology, noninvasive prenatal detection of chromosome aneuploidy and single base variation has been successfully validated. However, few studies discussed the possibility of noninvasive pathogenic CNVs detection.
Methodology/Principal Findings
A novel algorithm for noninvasive prenatal detection of fetal pathogenic CNVs was firstly tested in 5 pairs of parents with heterozygote α-thalassemia of Southeast Asian (SEA) deletion using target region capture sequencing for maternal plasma. Capture probes were designed for α-globin (HBA) and β-globin (HBB) gene, as well as 4,525 SNPs selected from 22 automatic chromosomes. Mixed adaptors with 384 different barcodes were employed to construct maternal plasma DNA library for massively parallel sequencing. The signal of fetal CNVs was calculated using the relative copy ratio (RCR) of maternal plasma combined with the analysis of R-score and L-score by comparing with normal control. With mean of 101.93× maternal plasma sequencing depth for the target region, the RCR value combined with further R-score and L-score analysis showed a possible homozygous deletion in the HBA gene region for one fetus, heterozygous deletion for two fetus and normal for the other two fetus, which was consistent with that of invasive prenatal diagnosis.
Our study showed the feasibility to detect pathogenic CNVs using target region capture sequencing, which might greatly extend the scope of noninvasive prenatal diagnosis.
PMCID: PMC3696090  PMID: 23840709

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