Clinical implementation of cell-free dna analysis of maternal blood

  1. Quezada Rojas, María Soledad
Supervised by:
  1. Kypros H. Nicolaides Director
  2. Jesús Florido Navío Director

Defence university: Universidad de Granada

Fecha de defensa: 23 November 2015

Committee:
  1. Alberto Salamanca Ballesteros Chair
  2. Cristina Campoy Folgoso Secretary
  3. Francisca S. Molina García Committee member
  4. Karina Krajden Haratz Committee member
  5. Catalina De Paco Matallana Committee member

Type: Thesis

Abstract

CLINICAL IMPLEMENTATION OF CELL-FREE DNA ANALYSIS OF MATERNAL BLOOD INTRODUCTION Screening for trisomies 21, 18 and 13 in singleton and twin pregnancies Chromosomal abnormalities are major causes of perinatal death and childhood handicap. In the last 45 years a series of different methods have been used to identify the pregnancies at high-risk of fetal trisomy 21 that could be offered invasive diagnostic testing (Nicolaides 2011). During the 1970s and the early 1980s, advanced maternal age, defined in most countries as over 35 years, was the method of screening. At that time about 5% of the pregnant women were >35 years old and this population contained about 30% of the affected pregnancies. Since most of the screen positive group were actually normal the word screen positive became synonymous with false positive. Therefore, in screening by maternal age the detection rate (DR) was 30% and the false positive rate (FPR) was 5%. In recent years the trend to delayed childbearing has resulted in a significant increase in the number of pregnant women ¿ 35 years (20%). If all these women were to undergo invasive testing, the DR would be 50% and FPR 20%. In the late 1980s and early 1990s, it was realised that in pregnancies with fetal trisomy 21 there are altered maternal serum concentrations of various feto-placental products, including increased free ß-hCG and Inhibin A and decreased AFP and unconjugated estriol (uE3) (Merkatz et al., 1984; Canick et al., 1988; Macri et al., 1990; Van Lith et al., 1993; Brambati et al., 1993; Aitken et al., 1996). These biochemical changes were combined with maternal age to develop the double test (hCG and AFP), triple test (hCG, AFP and uE3) and the quad test (hCG, inhibin A, AFP and uE3). Screening by this approach was superior to that of maternal age alone with DR of 50-70% at FPR of 5% (Cuckle et al., 2005). In the 1990s, aneuploidy screening shifted to the first trimester with the `Combined¿ test which uses ultrasound measurement of fetal nuchal translucency (NT) together with maternal serum concentration of the placental proteins, free ß-human chorionic gonadotropin (free ß-hCG) and pregnancy-associated plasma protein (PAPP-A) (Nicolaides et al., 1992; Snijders et al., 1998; Wald et al., 2003a; Nicolaides, 2004; Malone et al., 2005; Spencer et al., 2003a; Kagan et al., 2008a; Wright et al., 2010 Brizot et al., 1994, 1995; Noble et al., 1995; Spencer et al., 1999; Bindra et al., 2002; Spencer et al., 2003c; Wapner et al., 2003; Nicolaides et al., 2005; Ekelund et al., 2008; Kagan et al., 2009a). This combined test has a DR of 90% with FPR of 5%. Research suggests that screening using a combination of NT with other ultrasound markers (nasal bone, tricuspid regurgitation and ductus venosus) and serum biochemistry with free ß-hCG, PAPP-A and placental growth factor (PLGF) can have a DR of 97% at FPR of 3% (Matias et al., 1998; Cicero et al., 2001, 2006; Huggon et al., 2003; Nicolaides, 2004; Faiola et al., 2005; Falcon et al., 2006; Kagan et al., 2009b, 2009c; 2010; Maiz et al., 2009). However, in most hospitals these additional ultrasound and biochemical markers are not used. Lo et al. in 1997, based on the previous knowledge that DNA of cancer patients can be detected in plasma, performed a study where he demonstrated the presence of fetal DNA in maternal blood. This discovery has revolutionized the field of fetal medicine. They used a rapid-boiling method to extract DNA from plasma and serum. DNA from plasma, serum, and nucleated blood cells from 43 pregnant women underwent a sensitive Y-PCR assay to detect circulating male fetal DNA from women bearing male fetuses. Fetus-derived Y sequences were detected in 24 (80%) of the 30 maternal plasma samples, and in 21 (70%) of the 30 maternal serum samples, from women bearing male fetuses. None of the 13 women bearing female fetuses,and none of the 10 non-pregnant control women, had positive results for plasma, serum or nucleated blood cells. There is evidence, from clinical validation and a few clinical implementation studies, that the performance of screening for trisomies by cfDNA analysis of maternal blood is superior to that of the combined test; the reported DR is 99% for trisomy 21, 96% for trisomy 18 and 91% trisomy 13, at combined FPR of 0.35%. However, the test may fail to give a result and the exact rate of such failure is difficult to quantify from the existing studies. Additionally, only four studies, on a limited number of cases and a wide gestational age range, reported on the clinical implementation of cfDNA testing in routine screening for trisomies in the general population In twin pregnancies, the performance of screening by the first-trimester combined test for trisomies is similar to that in singleton pregnancies but with a small increase in the FPR. Screening for trisomies in twins by cfDNA analysis of maternal blood is feasible. However, the number of cases reported is too small to allow accurate estimation of performance of screening. cfDNA analysis of maternal blood in screening for preterm birth There is some evidence that the cell free DNA in maternal blood is increased in pregnancy complications. Several studies have examined the potential value of cfDNA in maternal blood as a marker of pregnancy complications. Lo et al., (1998) described increasing concentrations of cf fetal DNA in maternal plasma from 3.4% to 6.2% between early and late gestation in a group of 12 women sampled sequentially throughout pregnancy. Ariga et al, (2001) studied serial blood samples from 20 healthy pregnant women carrying male fetuses in order to quantify the fetal DNA; they found a gradually increased fetal DNA during pregnancy, which peaked at term and in the postpartum period the levels rapidly fell to almost undetectable. Majer et al., (2007) examined cfDNA in the third trimester in 96 women carrying male foetuses and reported that the level was significantly correlated with placental weight and pregnancy-associated complications. Two studies examined women presenting with contractions and/or PPROM and reported that, in those with spontaneous preterm birth, maternal plasma fetal cfDNA was increased. Leung et al., (1998) using real-time quantitative polymerase chain reaction (PCR) for the detection of the SRY gene, demonstrated that the median fetal cfDNA was higher in 13 pregnancies with male fetuses, presenting with threatened preterm labor and subsequent delivery at 26¿34 weeks, when compared to 17 controls that delivered at term. Farina et al., (2005) assessed the DYS1 locus on the Y chromosome by real-time PCR to determine fetal cfDNA in 29 women with preterm labor delivering at <36 weeks, 21 with PPROM delivering at <36 weeks and 21 with preterm labor delivering at ¿36 weeks. They reported that cumulative rates of delivery at <30 weeks and delivery at <36 weeks were significantly higher for women with fetal cfDNA ¿1.82MoM than for those with fetal cfDNA concentrations below this cut-off (early preterm delivery: 45% vs 14%; preterm delivery: 73% vs 66%). Some contradictory evidence suggests that, in cases of spontaneous preterm delivery, the increase in cfDNA precedes the clinical event. A cohort study of 876 women undergoing routine fetal rhesus D (RhD) genotyping, at 23¿28 weeks¿ gestation, reported that if the fetal cfDNA level was above the 95th percentile there was a 6- and 16-fold increase in risk for spontaneous birth at <37 weeks (n=19) and at <34 weeks (n=8), respectively (Jakobsen et al., 2012). In contrast, Stein et al., (2013) reported that, in a cohort study of 611 women undergoing routine fetal RhD genotyping at 24¿25 weeks¿ gestation, the levels of fetal cfDNA were not altered in pregnancies complicated by preterm birth. A study examining 34 women with a short cervix and 22 women with normal cervical length at 22-24 weeks¿ gestation assessed the DYS14 locus on the Y chromosome by real-time PCR to determine fetal cfDNA levels and reported no significant difference in the level of fetal cfDNA between those that delivered before 37 weeks and those delivering at term (Illanes et al., 2011). More recently, a study used chromosome-selective sequencing of non-polymorphic and polymorphic loci, in which fetal alleles differ from maternal alleles, to determine the cfDNA counts of fetal and maternal origin in maternal plasma at 11¿13 weeks¿ gestation (Poon et al., 2013). Both fetal and maternal cfDNA counts were affected by maternal characteristics, but the corrected values in 20 cases of spontaneous preterm birth were not significantly different from those of 1805 unaffected pregnancies. PUBLISHED STUDIES Study 1: Screening for trisomies 21,18 and 13 by cell-free DNA analysis of maternal blood at 10-11 week¿s gestation and the combined test at 11-13 weeks. Ultrasound Obstet Gynecol 2015 Jan; 45: 36-41 The objective of this study was to report the results of clinical implementation of cfDNA testing for trisomies 21, 18 and 13 in the general population at 10-11 weeks¿ gestation and compare its performance to that of the first trimester combined test. In 2,905 singleton pregnancies prospective screening for trisomies was performed by chromosome-selective sequencing of cfDNA in maternal blood at 10-11 weeks and by the combined test at 11-13 weeks. The median maternal age was 36.9 (range 20.4-51.9) years. Results from cfDNA analysis were provided for 2,851 (98.1%) cases and these were available within 14 days from sampling in 2,848 (98.0%). The trisomic status of the pregnancies was determined by prenatal or postnatal karyotyping or clinical examination of the neonates. In the 2,785 pregnancies with cfDNA result and known trisomic status, cfDNA testing correctly identified all 32 cases with trisomy 21, nine of 10 with trisomy 18 and two of five with trisomy 13 with respective false positive rates of 0.04%, 0.19% and 0.07%. In cases with discordant results between cfDNA testing and fetal karyotype the median fetal fraction was lower than in those with concordant results (6% vs 11%). In the combined test, the estimated risk for trisomy 21 was >1:100 in all trisomic cases and in 4.4% of the non-trisomic pregnancies. It was concluded that the performance of first-trimester cfDNA testing for trisomies 21 and 18 in the general population is similar to that in high-risk pregnancies. Most false positive and negative results from cfDNA testing could be avoided if the a priori risk from the combined test is taken into account in the interpretation of individual risk. Study 2: Cell-free DNA analysis for trisomy risk assessment in first trimester twin pregnancies. Fetal Diagn Ther 2014; 35: 204-2011. The objective of this study was to examine the clinical implementation of chromosome-selective sequencing of cfDNA in maternal blood in the assessment of risk for trisomies in twin pregnancies. Risk for trisomies 21, 18 and 13 by cfDNA testing were estimated in stored plasma samples obtained at 11-13 weeks¿ gestation from 207 pregnancies with known outcome and prospectively in 68 twin pregnancies undergoing screening at 10-13 weeks. Cell-free DNA was extracted from the maternal plasma or blood samples and chromosome- selective sequencing was carried out as in singleton pregnancies. However, in the assessment of risk for trisomies the smallest fetal fraction contribution of the two fetuses was considered. Risk scores for trisomies were provided for 192 (92.8%) of stored plasma and for 63 (92.6%) of the prospective cases. In the retrospective study 10 of 11 trisomic pregnancies were correctly identified with no false positive results. In the prospective study three trisomic pregnancies were correctly identified with no false positive results. The median of the lower fetal fraction in the prospective study of twins was 7.4% (inter-quartile range 5.9-10.0%), which was lower than in our previous study in singletons (median 10.0%, IQR 7.8-13.0%). It was concluded that cfDNA testing in twins is feasible but the reporting rate of results is lower than in singletons due to a lower fetal fraction. The number of twin pregnancies undergoing cfDNA analysis in this and previous studies is too small to provide accurate assessment of the performance of the test. Nevertheless, it is likely that the sensitivity will be high and the false positive rate low. However, the high no reporting rate at first sampling is of major concern; this would shift the option of prenatal diagnosis and selective fetocide from the first to the second trimester with consequent increase in the rate of miscarriage. Study 3: Fetal fraction of cell-free DNA in maternal plasma in the prediction of spontaneous preterm delivery Ultrasound Obstet Gynecol 2015 Jan; 45: 101-105. The objective of this study was explore whether in pregnancies that are complicated by spontaneous preterm delivery fetal fraction of cfDNA in maternal plasma at 11-13 weeks¿ gestation is altered and if this measurement is useful in the prediction of preterm birth. Fetal fraction of cfDNA was measured at 10+0-13+6 weeks¿ gestation in 3,066 (94.7%) pregnancies that delivered at >37 weeks¿ gestation and 103 (3.2%) with spontaneous delivery at <37 weeks, including 21 that delived at <34 weeks and 82 that delivered at 34-36 weeks. Fetal fraction measured was converted to multiples of median (MoM), corrected for maternal characteristics and gestational age. Mann-Whitney U test was used to determine the significance of differences in the median values in the spontaneous preterm delivery groups to that in the term delivery group. In the spontaneous preterm delivery groups (<34 weeks, 34-37 weeks, <37 weeks), compared to the term delivery group, there was no significant difference in the median fetal fraction MoM (1.004, 0.922 and 0.946, respectively, vs. 1.015). It was concluded that measurement of fetal fraction in maternal plasma at 11-13 weeks is not predictive of spontaneous preterm birth. This was a cross-sectional study and the conclusions in relation to the inability of fetal fraction to distinguish between pregnancies that subsequently deliver preterm and those delivering at term are confined to this early gestational age. Longitudinal studies are needed to examine whether a change in fetal fraction precedes the onset of labor and the interval between the two events. DISCUSSION Implementation of cfDNA testing for trisomies in singleton pregnancies This prospective study in 2,905 singleton pregnancies having routine first trimester screening for the major trisomies by cfDNA analysis of maternal blood and by the combined test, provided outcome data for nearly 98% of cases, which makes it possible to assess accurately the performance of screening. The combined test, at a risk cut-off of 1:100, identified all cases of trisomies 21, 18 and 13 with FPR of 4.4%. Screening by cfDNA analysis of maternal blood provided results in 98% of pregnancies and these were available within two weeks of sampling in 98% of cases. In the pregnancies with a cfDNA result all cases of fetal trisomy 21 were detected at FPR of 0.04%. The test also detected nine of the 10 cases with fetal trisomy 18 at FPR of 0.19%; in the one false negative case, QF PCR of chorionic villi reported disomy 18. The performance of screening for trisomy 13 was poorer with only two of five affected cases being detected. The study has also highlighted that in cases of discordant results between cfDNA testing and fetal karyotype the fetal fraction was lower than in those with concordant normal or abnormal results. A potential limitation of the study is that the patients were self-selected and inevitably a high proportion of women was of advanced age and conceived by assisted reproduction techniques. Nevertheless, the women did not have prior screening for trisomies by other methods and their results are applicable to a general population. Another limitation of the study relates to the high performance of the combined test. The results of cfDNA analysis were commonly available at the time of the ultrasound scan for measurement of fetal NT and could have potentially biased the measurements. Our findings on the performance of maternal blood cfDNA analysis in screening for trisomies 21 and 18 in a general population are compatible with the results of previous studies in high-risk pregnancies, but also those which examined general populations. The detection rate of trisomy 13 was lower, but the number of cases we have examined is too small for valid conclusions to be drawn. In the three previous prospective screening studies in the general population the median gestational age at cfDNA testing was 14-17 weeks (Song et al., 2013; Bianchi et al., 2014; Comas et al., 2014). Our study has focused on the application of cfDNA testing at 10-11 weeks because first-trimester screening and diagnosis of aneuploidies lead to early reassurance of the majority of parents that their fetus is unlikely to be trisomic, and for the few with an affected fetus the parents have the option of an earlier and safer termination of pregnancy. An important clinical implication of the data is that in the interpretation of cfDNA results, particularly in cases with low fetal fraction, the a priori risk should be considered. The ability to detect the small increase in the amount of a given chromosome in maternal plasma in a trisomic compared to a disomic pregnancy is directly related to the relative proportion of the fetal to maternal origin of the cfDNA in maternal plasma. In our cases with false positive and false negative results the median fetal fraction was lower than in those with concordant results between the cfDNA test and fetal karyotype. Clinical implementation of cfDNA testing for trisomies in twin pregnancies This study examined two datasets of twin pregnancies, first, stored plasma samples obtained at 11-13 weeks¿ gestation from 207 pregnancies with known outcome and second, 68 prospectively examined twin pregnancies undergoing screening at 10-13 weeks. In both groups, the failure rate of cfDNA testing to provide a result was 7%, which is considerably higher than the 2% observed in singleton pregnancies using the same method of sequencing. The reason for the higher failure rate in twins was low fetal fraction and this is the inevitable consequence of selecting the lower fetal fraction of the two fetuses, rather than the total, in estimating the risk for aneuploidies (Struble et al., 2014). The rationale for this choice is to avoid a false negative result in a dizygotic twin pregnancy discordant for aneuploidy where the total fetal fraction is satisfactory but the contribution of the affected fetus could be less than 4%. In terms of results, there were 12 cases of trisomy 21 and a correct diagnosis by cfDNA testing was made in 11; in one case there was a false negative result. There were two cases of trisomy 18 and a correct diagnosis by cfDNA testing was made in one; in one case there was no result. There were three cases of trisomy 13 and a correct diagnosis by cfDNA testing was made in one; in two cases there was no result. The case of trisomy 21 with a negative cfDNA result had a fetal fraction of only 5.3% which illustrates the fact that a low fetal fraction the accuracy of cfDNA testing, particularly in twins may be limited. The number of twin pregnancies undergoing cfDNA analysis in this and previous studies is too small to provide accurate assessment of the performance of the test. Nevertheless, it is likely that the sensitivity will be high and the false positive rate low. However, the high no reporting rate at first sampling is of major concern; this would shift the option of prenatal diagnosis and selective fetocide from the first to the second trimester with consequent increase in the rate of miscarriage. In twin pregnancies, as in singletons, there are essentially two options in the introduction of cfDNA testing in such a way as to retain the advantages of the 11-13 weeks scan (Gil, et al 2013, Nicolaides et al 2014). One option is to carry cfDNA testing in all women at 10 weeks¿ gestation followed by a scan at 12 weeks; in patients with a high risk score from cfDNA testing, invasive diagnostic testing and selective fetocide can be carried out in the first trimester. In cases with no result from cfDNA testing, pregnancy management could be based on the results of the combined test. The alternative to universal screening by the cfDNA test is a strategy of cfDNA testing contingent on the results of first-line screening by ultrasound and biochemical testing. This approach retains the major advantages of cfDNA testing in increasing the detection rate of trisomies and decreasing the false positive rate, but at considerably lower cost than offering the test to the whole population. The disadvantage of this approach, arising from delay or failure to obtain a result is the resultant shift in diagnosis from the first to the second trimester. This could be partly ameliorated by offering invasive testing when the estimated risk from the combined test is very high and reserving cfDNA testing for the intermediate risk group. Cell-free DNA in the prediction of spontaneous preterm delivery Preterm delivery is the leading cause of perinatal death and handicap in children. Consequently, a method of early prediction and prevention of such pregnancy complication could have a major impact on perinatal death and handicap. There is some contradictory evidence that in cases of spontaneous preterm birth fetal DNA in maternal blood may be increased before the clinical event. Our study of 3,169 singleton pregnancies, including 103 (3.2%) with spontaneous preterm birth, demonstrated that in pregnancies resulting in early and late spontaneous preterm delivery, compared to those delivering at term, the median fetal fraction of cfDNA in the maternal plasma at 11-13 weeks¿ gestation is not significantly different. A potential limitation of our study arises from its cross-sectional nature confined to 11-13 weeks. The conclusions in relation to the inability of fetal fraction to distinguish between pregnancies that subsequently deliver preterm and those delivering at term are confined to this early gestational age. Longitudinal studies are needed to examine whether a change in fetal fraction precedes the onset of labor and the interval between the two events. The reported high performance of cfDNA analysis of maternal blood in screening for fetal trisomies will inevitably lead to widespread uptake of this technique and an integral part of such aneuploidy screening is measurement of the fetal fraction. A beneficial consequence of such measurement of the fetal fraction would have been improved performance of early screening for spontaneous preterm delivery. However, as demonstrated by our study the use of cfDNA testing is unlikely to be useful for the prediction of this pregnancy complication. REFERENCES Aitken DA, Wallace EM, Crossley JA, Swanston IA, Van Pereren Y, Van Maarle M, Groome NP, Macri JN, Connor JM. Dimeric inhibin A as a marker for Down¿s syndrome in early pregnancy. N Engl J Med 1996; 334: 1231-1236. Anker P, Stroun M. Progress in the knowledge of circulating nucleic acids: plasma RNA is particle-associated. Can it become a general detection marker for a cancer blood test? Clin Chem 2002; 48: 1210-1211. Ariga H, Ohto H, Busch MP, et al. Kinetics of fetal cellular and cell-free DNA in the maternal circulation during and after pregnancy: implications for noninvasive prenatal diagnosis. Transfusion 2001; 41: 1524-1530. Ashoor G, Poon L, Syngelaki A, Mosimann B, Nicolaides KH: Fetal fraction in maternal plasma cell-free DNA at 11-13 weeks' gestation: Effect of maternal and fetal factors. Fetal Diagn Ther 2012; 31: 237-243. Ashoor G, Syngelaki A, Poon LC, Rezende JC, Nicolaides KH: Fetal fraction in maternal plasma cell-free DNA at 11-13 weeks' gestation: relation to maternal and fetal characteristics. Ultrasound Obstet Gynecol 2013; 41: 26-32. Bevilacqua E, Gil MM, Nicolaides KH, Ordonez E, Cirigliano V, Dierickx H, Willems PJ, Jani JC. Performance of screening for aneuploidies by cell-free DNA analysis of maternal blood in twin pregnancies. Ultrasound Obstet Gynecol 2015; 45: 61-66. Bianchi DW, Parker RL, Wentworth J, Madankumar R, Saffer C, Das AF, Craig JA, Chudova DI, Devers PL, Jones KW, Oliver K, Rava RP, Sehnert AJ; CARE Study Group. DNA sequencing versus standard prenatal aneuploidy screening. N Engl J Med 2014; 370: 799-808. Bianchi DW. Fetal DNA in maternal plasma: the plot thickens and the placental barrier thins. Am J Hum Genet 1998; 62: 763-764. Bindra R, Heath V, Liao A, Spencer K, Nicolaides KH. One stop clinic for assessment of risk for trisomy 21 at 11¿14 weeks: a prospective study of 15,030 pregnancies. Ultrasound Obstet Gynecol 2002; 20: 219-225. Bischoff FZ, Lewis DE, Simpson JL. Cell-free fetal DNA in maternal blood: kinetics, source and structure Hum Reprod Update 2005; 11: 59-67. Brambati B, Macintosh MCM, Teisner B, Maguiness S, Chrimarker K, Lanzani A, Bonacchi I, Tulul L, Chard T, Grudzinskas JG. Low maternal serum level of pregnancy associated plasma protein (PAPP-A) in the first trimester in association with abnormal fetal karyotype. BJOG 1993; 100: 324-326. Brizot ML, Snijders RJM, Bersinger NA, Kuhn P, Nicolaides KH. Maternal serum pregnancy associated placental protein A and fetal nuchal translucency thickness for the prediction of fetal trisomies in early pregnancy. Obstet Gynecol 1994; 84: 918-922. Canick J, Knight GJ, Palomaki GE, Haddow JE, Cuckle HS, Wald NJ. Low second trimester maternal serum unconjugated oestriol in pregnancies with Down¿s syndrome. BJOG 1988; 95: 330-333. Chan KC, Lo YM. Circulating nucleic acids as a tumor marker. Histol Histopathol 2002; 17: 937-943. Chen XQ, Stroun M, Magnenat JL, Nicod LP, Kurt AM, Lyautey J, Lederrey C, Anker P. Microsatellite alterations in plasma DNA of small cell lung cancer patients. Nat Med 1996; 2: 1033-1035. Cicero S, Curcio P, Papageorghiou A, Sonek J, Nicolaides KH. Absence of nasal bone in fetuses with Trisomy 21 at 11¿14 weeks of gestation: an observational study. Lancet 2001; 358: 1665-1667. Comas C, Echevarria M, Rodr¿guez MA, Prats P, Rodr¿guez I, Serra B. Initial experience with non-invasive prenatal testing of cell-free DNA for major chromosomal anomalies in a clinical setting. J Matern Fetal Neonatal Med 2014; 12: 1-6. Cuckle H, Benn P, Wright D.Down syndrome screening in the first and/or second trimester: model predicted performance using meta-analysis parameters. Semin Perinatol 2005; 29: 252-257. Cuckle H, Maymon R. Down syndrome risk calculation for a twin fetus taking account of the nuchal translucency in the co-twin. Prenat Diagn 2010; 30: 827-833. D'Onofrio BM, Class QA, Rickert ME, Larsson H, Långström N, Lichtenstein P. Preterm birth and mortality and morbidity: a population-based quasi-experimental study. JAMA Psychiatry 2013; 70: 1231-1240. Down LJ. Observations on an ethnic classification of idiots. Clinical Lectures and Reports, London Hospital 1866; 3: 259-262. Ekelund CK, Jørgensen FS, Petersen OB, Sundberg K, Tabor A; Danish Fetal Medicine Research Group. Impact of a new national screening policy for Down¿s syndrome in Denmark: population based cohort study. BMJ 2008; 337: DOI:10.1136/bmj.a2547. Faas BH, de Ligt J, Janssen I, Eggink AJ, Wijnberger LD, van Vugt JM, Vissers L, Geurts van Kessel A: Non-invasive prenatal diagnosis of fetal aneuploidies using massively parallel sequencing-by-ligation and evidence that cell-free fetal DNA in the maternal plasma originates from cytotrophoblastic cells. Expert Opin Biol Ther 2012; 12: S19-26. Faiola S, Tsoi E, Huggon IC, Allan LD, Nicolaides KH. Likelihood ratio for trisomy 21 in fetuses with tricuspid regurgitation at the 11 to 13 + 6-week scan. Ultrasound Obstet Gynecol 2005; 26: 22-27. Falcon O, Faiola S, Huggon I, Allan L, Nicolaides KH.Fetal tricuspid regurgitation at the 11 +0 to 13+ 6-week scan: association with chromosomal defects and reproducibility of the method. Ultrasound Obstet Gynecol 2006; 27: 609-612. Farina A, LeShane ES, Romero R, Gomez R, Chaiworapongsa T, Rizzo N, Bianchi DW. High levels of fetal cell-free DNA in maternal serum: a risk factor for spontaneous preterm delivery. Am J Obstet Gynecol 2005; 193: 421-425. Gil MM, Quezada MS, Revello R, Akolekar R, Nicolaides K. Analysis of cell free DNA in maternal blood in screening for fetal aneuploidies: updated meta-analysis. Ultrasound Obstet Gynecol 2015; 45: 249-266. Gil MM, Quezada MS, Bregant B, Ferraro M, Nicoliades KH. Implementation of maternal blood cell-free DNA testing in early screening for aneuploidies. Ultrasound Obstet Gynecol 2013; 42: 34-40. Goldenberg RL, Culhane JF, Iams JD, Romero R. Epidemiology and causes of preterm birth. Lacent 2008; 371: 75-84. Haghiac M, Vora NL, Basu S, Johnson KL, Presley L, Bianchi DW, Mouzon SH: Increased death of adipose cells, a path to release cell-free DNA into systemic circulation of obese women. Obesity (Silver Spring) 2012; 20: 2213-2219. Huggon IC, DeFigueiredo DB, Allan LD. Tricuspid regurgitation in the diagnosis of chromosomal anomalies in the fetus at 11¿14 weeks of gestation. Heart 2003; 89: 1071-1073. Hyett JA, Noble PL, Snijders RJ, Montenegro N, Nicolaides KH. Fetal heart rate in trisomy 21 and other chromosomal abnormalities at 10¿14 weeks of gestation. Ultrasound Obstet Gynecol 1996; 7: 239¿244. Iams JD. Prevention of preterm parturation. N Engl J Med 2014; 370: 254-261. Illanes S, Gomez R, Fornes R, Figueroa-Diesel H, Schepeler M, Searovic P, Serra R, Perez A, Nien JK. Free fetal DNA levels in patients at risk of preterm labour. Prenat Diagn 2011; 31: 1082-1085. Kagan KO, Gazzoni A, Sepulveda-Gonzalez G, Sotiriadis A, Nicolaides KH. Discordance in nuchal translucency thickness in the prediction of severe twin-to-twin transfusion syndrome. Ultrasound Obstet Gynecol 2007; 29: 527-532. Kagan KO, Wright D, Spencer K, Molina FS, Nicolaides KH. First trimester screening for trisomy 21 by free beta-human chorionic gonadotropin and pregnancy-associated plasma protein-A: impact of maternal and pregnancy characteristics. Ultrasound Obstet Gynecol 2008a; 31: 493-502. Kagan KO, Wright D, Valencia C, Maiz N, Nicolaides KH. Screening for trisomies 21, 18 and 13 by maternal age, fetal nuchal translucency, fetal heart rate, free ß-hCG and pregnancy-associated plasma protein-A. Hum Reprod 2008b; 23: 1968-1975. Kagan KO, Etchegaray A, Zhou Y, Wright D, Nicolaides KH. Prospective validation of first-trimester combined screening for trisomy 21. Ultrasound Obstet Gynecol 2009a; 34: 14-18. Kagan KO, Cicero S, Staboulidou I, Wright D, Nicolaides KH. Fetal nasal bone in screening for trisomies 21, 18 and 13 and Turner syndrome at 11¿13 weeks of gestation. Ultrasound Obstet Gynecol 2009b; 33: 259¿264. Kagan KO, Valencia C, Livanos P, Wright D, Nicolaides KH. Tricuspid regurgitation in screening for trisomies 21, 18 and 13 and Turner syndrome at 11 + 0¿13 + 6 weeks of gestation. Ultrasound Obstet Gynecol 2009c; 33: 18-22. Kagan KO, Staboulidou I, Cruz J, Wright D, Nicolaides KH. Two stage first-trimester screening for trisomy 21 by ultrasound assessment and biochemical testing. Ultrasound Obstet Gynecol 2010; 36: 542-547. Kodjebacheva GD, Sabo T. Influence of premature birth on the health conditions, receipt of special education and sport participation of children aged 6-17 years in the USA. J Public Health (Oxf) 2015; pii: fdv098. Lejeune J, Turpin R, Gautier M. Mongolism; a chromosomal disease (trisomy) Bull Acad Nati Med 1959; 143: 256-265. Leon SA, Shapiro B, Sklaroff DM, Yaros MJ. Free DNA in the serum of cancer patients and the effect of therapy. Cancer Res 1977; 37: 646-650. Leung TN, Zhang J, Lau TK, Hjelm NM, Lo YM: Maternal plasma fetal DNA as a marker for preterm labour. Lancet 1998; 352: 1904-1905. Leung TY, Qu JZ, Liao GJ, Jiang P, Cheng YK, Chan KC, Chiu RW, Lo YM. Noninvasive twin zygosity assessment and aneuploidy detection by maternal plasma DNA sequencing. Prenat Diagn 2013; 33: 675-681. Liao AW, Snijders R, Geerts L, Spencer K, Nicolaides KH. Fetal heart rate in chromosomally abnormal fetuses. Ultrasound Obstet Gynecol 2000; 16: 610-613. Linskens IH, Spreeuwenberg MD, Blankenstein MA, van Vugt JM. Early first-trimester free beta-hCG and PAPP-A serum distributions in monochorionic and dichorionic twins. Prenat Diagn 2009; 29: 74-78. Lo YM, Corbetta N, Chamberlain PF, Rai V, Sargent IL, Redman CW, Wainscoat JS. Presence of fetal DNA in maternal plasma and serum. Lancet 1997; 350: 485-487. Lo YM, Tein MS, Lau TK, Haines C, Leung T, Poon P, Wainscoat J, Johnson P, Chang A, Hjelm M. Quantitative analysis of fetal DNA in maternal plasma and serum: implications for noninvasive prenatal diagnosis. Am J Hum Genet. 1998; 62 :768-775. Macri JN, Kasturi RV, Krantz DA, Cook EJ, Moore ND, Young JA, Romero K, Larsen JW Jr. Maternal serum Down syndrome screening: free beta protein is a more effective marker than human chorionic gonadotrophin. Am J Obstet Gynecol 1990; 163: 1248-1253. Madsen HN, Ball S, Wright D, Tørring N, Petersen OB, Nicolaides KH, Spencer K. A reassessment of biochemical marker distributions in trisomy 21-affected and unaffected twin pregnancies in the first trimester. Ultrasound Obstet Gynecol 2011; 37: 38-47. Maiz N, Valencia C, Kagan KO, Wright D, Nicolaides KH. Ductus venosus Doppler in screening for trisomies 21, 18 and 13 and Turner syndrome at 11¿13 weeks of gestation. Ultrasound Obstet Gynecol 2009; 33: 512-517. Majer S, Bauer M, Magnet E, Strele A, Giegerl E, Eder M, Lang U, Pertl B. Maternal urine for prenatal diagnosis¿an analysis of cell-free fetal DNA in maternal urine and plasma in the third trimester. Prenat Diagn 2007; 27: 1219-1223. Malone FD, Canick JA, Ball RH, Nyberg DA, Comstock CH, Bukowski R, Berkowitz RL, Gross SJ, Dugoff L, Craigo SD, Timor-Trisch IE, Carr SR, Wolfe HM, Dukes K, Bianchi DW, Rudnicka AR, Hackshaw AK, Lambert-Messerlian G, Wald NJ, D¿Alton ME. First- and Second-Trimester Evaluation of Risk (FASTER) Research Consortium. First-trimester or secondtrimester screening, or both, for Down¿s syndrome. N Engl J Med 2005; 353: 2001-2011. Matias A, Gomes C, Flack N, Montenegro N, Nicolaides KH. Screening for chromosomal abnormalities at 10¿14 weeks: the role of ductus venosus blood flow. Ultrasound Obstet Gynecol 1998; 12: 380-384. Maymon R, Jauniaux E, Holmes A, Wiener YM, Dreazen E, Herman A. Nuchal translucency measurement and pregnancy outcome after assisted conception versus spontaneously conceived twins. Hum Reprod 2001; 16: 1999-2004. Merkatz IR, Nitowsky HM, Macri JN, Johnson WE. An association between low maternal serum alpha-fetoprotein and fetal chromosomal abnormalities. Am J Obstet Gynecol 1984; 148: 886-894. Nawroz H, Koch W, Anker P, Stroun M, Sidransky D. Microsatelite alterations in serum DNA of head and neck cancer patients. Nat Med 1996; 2: 1035-1037. Nicolaides KH, Snijders RJM, Gosden RJM, Berry C, Campbell S. Ultrasonographically detectable markers of fetal chromosomal abnormalities. Lancet 1992; 340: 704-707. Nicolaides KH, Spencer K, Avgidou K, Faiola S, Falcon O.Multicenter study of first-trimester screening for trisomy 21 in 75 821 pregnancies: results and estimation of the potential impact of individual risk-orientated two-stage first-trimester screening. Ultrasound Obstet Gynecol 2005; 25: 221-226. Nicolaides KH, Syngelaki A, Ashoor G, Birdir C, Touzet G. Noninvasive prenatal testing for fetal trisomies in a routinely screened first-trimester population. Am J Obstet Gynecol 2012; 207: 374.e1-6. Nicolaides KH. Nuchal translucency and other first-trimester sonographic markers of chromosomal abnormalities. Am J Obstet Gynecol 2004; 191: 45-67. Nicolaides KH. Screening for fetal aneuplodies at 11-13 weeks. Prenat Diagn 2011; 31: 7-15. Nicolaides KH, Syngelaki A, Poon LC, Gil MM, Wright D. First-trimester contingent screening for trisomies 21,18 and 13 by biomarkers and maternal blood cell-free DNA testing. Fetal Diagn Ther 2014; 35: 185-192. Noble PL, Abraha HD, Snijders RJ, Sherwood R, Nicolaides KH. Screening for fetal trisomy 21 in the first trimester of pregnancy: maternal serum free beta-hCG and fetal nuchal translucency thickness. Ultrasound Obstet Gynecol 1995; 6: 390-395. Office for National Statistics. Gestation-specific Infant Mortality, 2012. http:// www.ons.gov.uk/ons/rel/child- health/gestation- specific-infant-mortality-in- england-and- wales/2012/stb- gestation-specific -infant-mortality--2012.html; Accessed on 02.08.2015. Palomaki GE, Kloza EM, Lambert-Messerlian GM, Haddow JE, Neveux LM, Ehrich M, van den Boom D, Bombard AT, Deciu C, Grody WW, Nelson SF, Canick JA: DNA sequencing of maternal plasma to detect Down syndrome: An international clinical validation study. Genet Med 2011; 13: 913-920. Pandya PP, Hilbert F, Snijders RJ, Nicolaides KH. Nuchal translucency thickness and crown-rump length in twin pregnancies with chromosomally abnormal fetuses. J Ultrasound Med 1995; 14: 565-568. Papageorghiou AT, Avgidou K, Spencer K, Nix B, Nicolaides KH. Sonographic screening for trisomy 13 at 11 to 13(6) weeks of gestation. Am J Obstet Gynecol 2006; 194: 397-401. Pergament E, Cuckle H, Zimmermann B, Banjevic M, Sigurjonsson S, Ryan A, Hall MP, Dodd M, Lacroute P, Stosic M, Chopra N, Hunkapiller N, Prosen DE, McAdoo S, Demko Z, Siddiqui A, Hill M, RabinowitzM. Single-nucleotide polymorphism-based noninvasive prenatal screening in a high-risk and low-risk cohort. Obstet Gynecol 2014; 124: 210-218. Poon LC, Musci T, Song K, Syngelaki A, Nicolaides KH: Maternal plasma cell-free fetal and maternal DNA at 11-13 weeks' gestation: relation to fetal and maternal characteristics and pregnancy outcomes. Fetal Diagn Ther 2013; 33: 215-223. Qu JZ, Leung TY, Jiang P, Liao GJ, Cheng YK, Sun H, Chiu RW, Chan KC, Lo YM. Noninvasive prenatal determination of twin zygosity by maternal plasma DNA analysis. Clin Chem 2013; 59: 427-435. Romero R, Espinoza J, Kusanovic JP, Gotsch F, Hassan S, Erez O, Chaiworapongsa T, Mazor M. The preterm parturation síndrome. BJOG 2006; 113: 17-42. Saigal S, Doyle LW. An overview of mortality and sequelae of preterm birth from infancy to adulthood. Lancet 2008; 371: 261-269. Sebire NJ, Hughes K, D¿Ercole C, Souka A, Nicolaides KH. Increased fetal nuchal translucency at 10¿14 weeks as a predictor of severe twin-to-twin transfusion syndrome. Ultrasound Obstet Gynecol 1997; 10: 86-89. Sebire NJ, Snijders RJM, Hughes K, Sepulveda W, Nicolaides KH. 1996a. Screening for trisomy 21 in twin pregnancies by maternal age and fetal nuchal translucency thickness at 10¿14 weeks of gestation. BJOG 1996a; 103: 999-1003. Sebire NJ, Noble PL, Psarra A, Papapanagiotou G, Nicolaides KH. Fetal karyotyping in twin pregnancies: selection of technique by measurement of fetal nuchal translucency. BJOG1996b; 103: 887-890. Sepulveda W, Sebire NJ, Hughes K, Odibo A, Nicolaides KH. The lambda sign at 10¿14 weeks of gestation as a predictor of chorionicity in twin pregnancies. Ultrasound Obstet Gynecol 1996; 7: 421-423. Snijders RJ, Noble P, Sebire N, Souka A, Nicolaides KH. Fetal Medicine Foundation First Trimester Screening Group. UK multicentre project on assessment of risk of trisomy 21 by maternal age and fetal nuchaltranslucency thickness at 10¿14 weeks of gestation. Lancet 1998; 352: 343-346. Snijders RJM, Holzgreve W, Cuckle H, Nicolaides KH. Maternal age specific risks for trisomies at 9¿14 weeks¿ gestation. Prenat Diagn 1994; 14: 543-552. Snijders RJM, Sebire NJ, Cuckle H, Nicolaides KH. Maternal age and gestational age-specific risks for chromosomal defects. Fetal Diagn Ther 1995; 10: 356-367. Snijders RJM, Sundberg K, Holzgreve W, Henry G, Nicolaides KH. Maternal age and gestation- specific risk for trisomy 21. Ultrasound Obstet Gynecol 1999;13: 167-170. Song Y, Liu C, Qi H, Zhang Y, Bian X, Liu J. Noninvasive prenatal testing of fetal aneuploidies by massively paralle sequencing in a prospective Chinese population. Prenat Diagn 2013; 33: 700-706 Sorenson GD, Pribish DM, Valone FH, Memoli VA, Bzik DJ, Yao SL.Soluble normal and mutated DNA sequences from single-copy genes in human blood. Cancer Epidemiol Biomarkers Prev 1994; 3: 67-71. Spencer K, Kagan KO, Nicolaides KH.Screening for trisomy 21 in twin pregnancies in the first trimester: an update of the impact of chorionicity on maternal serum markers. Prenat Diagn 2008; 28: 49¿52. Spencer K, Souter V, Tul N, Snijders R, Nicolaides KH.A screening program for trisomy 21 at 10¿14 weeks using fetal nuchal translucency, maternal serum free ß-human chorionic gonadotropin and pregnancyassociated plasma protein-A. Ultrasound Obstet Gynecol 1999; 13: 231¿237. Spencer K, Ong C, Skentou H, Liao AW, Nicolaides KH Screening for trisomy 13 by fetal nuchal translucency and maternal serum free beta hCG and PAPP-A at 10¿14 weeks of gestation. Prenat Diagn 2000a; 20: 411-416. Spencer K, Tul N, Nicolaides KH. Maternal serum free beta hCG and PAPP-A in fetal sex chromsome defects in the first trimester. Prenat Diagn 2000b; 20: 390-394. Spencer K, Liao A, Skentou H, Cicero S, Nicolaides KH. Screening for Triploidy by fetal nuchal translucency and maternal serum free ß-hCG and PAPP-A at 10-14 weeks of gestation. Prenat Diagn 2000c; 20: 495¿499. Spencer K, Spencer CE, Power M, Moakes A, Nicolaides KH One stop clinic for assessment of risk for fetal anomalies; a report of the first year of prospective screening for chromosomal anomalies in the first trimester. BJOG 2000d; 107: 1271-1275. Spencer K, Crossley JA, Aitken DA, Nix AB, Dunstan FD, Williams K. The effect of temporal variation in biochemical markers of trisomy 21 across the first and second trimesters of pregnancy on the estimation of individual patient specific risks and detection rates for Down¿s syndrome. Ann Clin Biochem 2003a; 40: 219¿231. Spencer K, Bindra R, Nix ABJ, Heath V, Nicolaides KH. Delta- NT or NT MoM: which is the most appropriate method for calculating accurate patient-specific risks for trisomy 21 in the first trimester?. Ultrasound Obstet Gynecol 2003b; 22: 142-148. Spencer K, Spencer CE, Power M, Dawson C, Nicolaides KH.Screening for chromosomal abnormalities in the first trimester using ultrasound and maternal serum biochemistry in a one stop clinic: a review of three years prospective experience. Br J Obstet Gynaecol 2003c; 110: 281-286. Stein W, Müller S, Gutensohn K, Emons G, Legler T. Cell-free fetal DNA and adverse outcome in low risk pregnancies. Eur J Obstet Gynecol Reprod Biol 2013: 166 : 10-13. Stroun M, Maurice P, Vasioukhin V, Lyautey J, Lederrey C, Lefort F, Rossier A, Chen XQ, Anker P. The origin and mechanism of circulating DNA. Ann NY Acad Sci 2000; 906: 161-168. Struble CA, Syngelaki A, Oliphant A, Song K, Nicolaides KH. Fetal fraction estimate in twin pregnancies using directed cell-free DNA analysis. Fetal Diagn Ther 2014; 35: 199-203. Valenti C, Schutta EJ, Kehaty T. Prenatal diagnosis of Down¿s syndrome. Lancet 1968; ii: 220. Van Lith JM, Pratt JJ, Beekhuis JR, Mantingh A.Second trimester maternal serum immuno-reactive inhibin as a marker for fetal Down¿s syndrome. Prenat Diagn 1993; 12: 801-806. Vandecruys H, Faiola S, Auer M, Sebire N, Nicolaides KH. Screening for trisomy 21 in monochorionic twins by measurement of fetal nuchal translucency thickness. Ultrasound Obstet Gynecol 2005; 25: 551-553. Vasioukhin V, Anker P, Maurice P, Lyautey J, Lederrey C, Stroun M. Point mutations of the N-ras gene in the blood plasma DNA of patients with myelodysplastic syndrome or acute myelogenous leukaemia. Br J Haematol 1994; 86: 774-779. Wald NJ, Rodeck C, Hackshaw AK, Walters J, Chitty L, Mackinson AM. SURUSS Research Group. First and second trimester antenatal screening for Down¿s syndrome: the results of the Serum, Urine and Ultrasound Screening Study (SURUSS). Health Technol Assess 2003a. 7: 1-88. Wapner R, Thom E, Simpson JL, et al. First trimester maternal serum biochemistry and fetal nuchal translucency screening (BUN) study group. First trimester screening for trisomies 21 and 18. N Engl J Med 2003; 349: 1405-1413. Wøjdemann KR, Larsen SO, Shalmi AC, et al. Nuchal translucency measurements are highly correlated in both mono- and dichorionic twin pairs. Prenat Diagn 2006;26: 218-220. Wright D, Spencer K, Kagan KK, Torring N, Petersen OB, Christou A, Kallikas J, Nicolaides KH.First-trimester combined screening for trisomy 21 at 7-14 weeks gestation. Ultrasound Obstet Gynecol 2010; 36: 404-411.