Official reprint from UpToDate®
www.uptodate.com ©2015 UpToDate®

Noninvasive prenatal testing using cell-free nucleic acids in maternal blood

Adam Wolfberg, MD
Aaron B Caughey, MD, PhD
Section Editor
Louise Wilkins-Haug, MD, PhD
Deputy Editor
Vanessa A Barss, MD, FACOG


Current methods of fetal genetic testing typically involve obtaining samples of amniotic fluid, placenta, fetal blood or, rarely, other fetal tissues or fluids. The invasive techniques required for obtaining fetal samples (eg, amniocentesis, chorionic villus biopsy, fetal umbilical vessel venipuncture, fetoscopy-guided biopsy) place the fetus at risk of injury or death. Therefore, development of accurate, safe, rapid, noninvasive tests for prenatal diagnosis is an area of active investigation.

Fetal genetic material can be found in the maternal circulation, raising the possibility of using maternal blood to diagnose fetal disease. Intact fetal cells can be identified in maternal blood, but are not a reliable source of fetal genetic material because these cells are extremely rare [1,2] and may persist for years after prior pregnancies [3]. By comparison, fetal "cell-free (cf)" nucleic acids not contained within cell membranes (cfDNA and cfRNA) are plentiful in the maternal circulation and unique to the current pregnancy. Thus, they have great potential for use in prenatal diagnosis. It is possible to noninvasively sequence the entire fetal genome [4-6].


Where do cell-free nucleic acids come from?

DNA — Both the mother and the fetal-placental unit produce cfDNA. The primary source of non-maternal cfDNA in the maternal circulation is thought to be apoptosis of placental cells (syncytiotrophoblast), while maternal hematopoietic cells are the source of most maternal cfDNA [7-9]. There is some evidence that apoptosis of fetal erythroblasts also generates fetal cfDNA, which can cross the placenta and enter the maternal circulation [7,10,11]. Because placental nucleic acids are highly representative of fetal nucleic acids, we will use the term fetal cfDNA throughout the rest of the topic.

RNA — Both fetal and placental cells contribute cell-free messenger RNA (cfmRNA) to the maternal circulation [12,13].

When are fetal cell-free nucleic acids found in maternal blood?

DNA — The fetal fraction of cell free DNA increases with gestational age and decreases with maternal weight [14]. These are the most important variables affecting fetal fraction. In addition, the fetal fraction appears to be increased in trisomy 21-positive pregnancies and decreased in trisomy 18-positive and trisomy 13-positive samples, compared with euploid samples.


Subscribers log in here

To continue reading this article, you must log in with your personal, hospital, or group practice subscription. For more information or to purchase a personal subscription, click below on the option that best describes you:
Literature review current through: Sep 2015. | This topic last updated: Aug 13, 2015.
The content on the UpToDate website is not intended nor recommended as a substitute for medical advice, diagnosis, or treatment. Always seek the advice of your own physician or other qualified health care professional regarding any medical questions or conditions. The use of this website is governed by the UpToDate Terms of Use ©2015 UpToDate, Inc.
  1. Bianchi DW, Williams JM, Sullivan LM, et al. PCR quantitation of fetal cells in maternal blood in normal and aneuploid pregnancies. Am J Hum Genet 1997; 61:822.
  2. Hamada H, Arinami T, Kubo T, et al. Fetal nucleated cells in maternal peripheral blood: frequency and relationship to gestational age. Hum Genet 1993; 91:427.
  3. Bianchi DW, Zickwolf GK, Weil GJ, et al. Male fetal progenitor cells persist in maternal blood for as long as 27 years postpartum. Proc Natl Acad Sci U S A 1996; 93:705.
  4. Fan HC, Gu W, Wang J, et al. Non-invasive prenatal measurement of the fetal genome. Nature 2012; 487:320.
  5. Kitzman JO, Snyder MW, Ventura M, et al. Noninvasive whole-genome sequencing of a human fetus. Sci Transl Med 2012; 4:137ra76.
  6. Lau TK, Cheung SW, Lo PS, et al. Non-invasive prenatal testing for fetal chromosomal abnormalities by low-coverage whole-genome sequencing of maternal plasma DNA: review of 1982 consecutive cases in a single center. Ultrasound Obstet Gynecol 2014; 43:254.
  7. Sekizawa A, Samura O, Zhen DK, et al. Apoptosis in fetal nucleated erythrocytes circulating in maternal blood. Prenat Diagn 2000; 20:886.
  8. Tjoa ML, Cindrova-Davies T, Spasic-Boskovic O, et al. Trophoblastic oxidative stress and the release of cell-free feto-placental DNA. Am J Pathol 2006; 169:400.
  9. Lui YY, Chik KW, Chiu RW, et al. Predominant hematopoietic origin of cell-free DNA in plasma and serum after sex-mismatched bone marrow transplantation. Clin Chem 2002; 48:421.
  10. Lo YM, Lo ES, Watson N, et al. Two-way cell traffic between mother and fetus: biologic and clinical implications. Blood 1996; 88:4390.
  11. Zhong XY, Holzgreve W, Hahn S. Cell-free fetal DNA in the maternal circulation does not stem from the transplacental passage of fetal erythroblasts. Mol Hum Reprod 2002; 8:864.
  12. Wataganara T, LeShane ES, Chen AY, et al. Plasma gamma-globin gene expression suggests that fetal hematopoietic cells contribute to the pool of circulating cell-free fetal nucleic acids during pregnancy. Clin Chem 2004; 50:689.
  13. Wataganara T, Leshane ES, Chen AY, et al. Circulating cell-free fetal nucleic acid analysis may be a novel marker of fetomaternal hemorrhage after elective first-trimester termination of pregnancy. Ann N Y Acad Sci 2004; 1022:129.
  14. Kinnings SL, Geis JA, Almasri E, et al. Factors affecting levels of circulating cell-free fetal DNA in maternal plasma and their implications for noninvasive prenatal testing. Prenat Diagn 2015; 35:816.
  15. Guibert J, Benachi A, Grebille AG, et al. Kinetics of SRY gene appearance in maternal serum: detection by real time PCR in early pregnancy after assisted reproductive technique. Hum Reprod 2003; 18:1733.
  16. Wang E, Batey A, Struble C, et al. Gestational age and maternal weight effects on fetal cell-free DNA in maternal plasma. Prenat Diagn 2013; 33:662.
  17. Chiu RW, Akolekar R, Zheng YW, et al. Non-invasive prenatal assessment of trisomy 21 by multiplexed maternal plasma DNA sequencing: large scale validity study. BMJ 2011; 342:c7401.
  18. Palomaki GE, Kloza EM, Lambert-Messerlian GM, et al. DNA sequencing of maternal plasma to detect Down syndrome: an international clinical validation study. Genet Med 2011; 13:913.
  19. Nygren AO, Dean J, Jensen TJ, et al. Quantification of fetal DNA by use of methylation-based DNA discrimination. Clin Chem 2010; 56:1627.
  20. Lo YM, Tein MS, Lau TK, et al. Quantitative analysis of fetal DNA in maternal plasma and serum: implications for noninvasive prenatal diagnosis. Am J Hum Genet 1998; 62:768.
  21. Canick JA, Palomaki GE, Kloza EM, et al. The impact of maternal plasma DNA fetal fraction on next generation sequencing tests for common fetal aneuploidies. Prenat Diagn 2013; 33:667.
  22. Lo YM, Zhang J, Leung TN, et al. Rapid clearance of fetal DNA from maternal plasma. Am J Hum Genet 1999; 64:218.
  23. Yu SC, Lee SW, Jiang P, et al. High-resolution profiling of fetal DNA clearance from maternal plasma by massively parallel sequencing. Clin Chem 2013; 59:1228.
  24. Chiu RW, Lui WB, Cheung MC, et al. Time profile of appearance and disappearance of circulating placenta-derived mRNA in maternal plasma. Clin Chem 2006; 52:313.
  25. Sparks AB, Struble CA, Wang ET, et al. Noninvasive prenatal detection and selective analysis of cell-free DNA obtained from maternal blood: evaluation for trisomy 21 and trisomy 18. Am J Obstet Gynecol 2012; 206:319.e1.
  26. Zimmermann B, Hill M, Gemelos G, et al. Noninvasive prenatal aneuploidy testing of chromosomes 13, 18, 21, X, and Y, using targeted sequencing of polymorphic loci. Prenat Diagn 2012; 32:1233.
  27. Lo YM, Corbetta N, Chamberlain PF, et al. Presence of fetal DNA in maternal plasma and serum. Lancet 1997; 350:485.
  28. Honda H, Miharu N, Ohashi Y, Ohama K. Successful diagnosis of fetal gender using conventional PCR analysis of maternal serum. Clin Chem 2001; 47:41.
  29. Poon LL, Leung TN, Lau TK, et al. Differential DNA methylation between fetus and mother as a strategy for detecting fetal DNA in maternal plasma. Clin Chem 2002; 48:35.
  30. Papageorgiou EA, Karagrigoriou A, Tsaliki E, et al. Fetal-specific DNA methylation ratio permits noninvasive prenatal diagnosis of trisomy 21. Nat Med 2011; 17:510.
  31. Lee da E, Kim SY, Lim JH, et al. Non-invasive prenatal testing of trisomy 18 by an epigenetic marker in first trimester maternal plasma. PLoS One 2013; 8:e78136.
  32. Chan KC, Zhang J, Hui AB, et al. Size distributions of maternal and fetal DNA in maternal plasma. Clin Chem 2004; 50:88.
  33. Lo YM, Lun FM, Chan KC, et al. Digital PCR for the molecular detection of fetal chromosomal aneuploidy. Proc Natl Acad Sci U S A 2007; 104:13116.
  34. Chim SS, Tong YK, Chiu RW, et al. Detection of the placental epigenetic signature of the maspin gene in maternal plasma. Proc Natl Acad Sci U S A 2005; 102:14753.
  35. Chim SS, Jin S, Lee TY, et al. Systematic search for placental DNA-methylation markers on chromosome 21: toward a maternal plasma-based epigenetic test for fetal trisomy 21. Clin Chem 2008; 54:500.
  36. Fairbrother G, Johnson S, Musci TJ, Song K. Clinical experience of noninvasive prenatal testing with cell-free DNA for fetal trisomies 21, 18, and 13, in a general screening population. Prenat Diagn 2013; 33:580.
  37. Gil MM, Quezada MS, Bregant B, et al. Implementation of maternal blood cell-free DNA testing in early screening for aneuploidies. Ultrasound Obstet Gynecol 2013; 42:34.
  38. Bianchi DW, Parker RL, Wentworth J, et al. DNA sequencing versus standard prenatal aneuploidy screening. N Engl J Med 2014; 370:799.
  39. Norton ME, Jacobsson B, Swamy GK, et al. Cell-free DNA analysis for noninvasive examination of trisomy. N Engl J Med 2015; 372:1589.
  40. Lo YM, Lau TK, Zhang J, et al. Increased fetal DNA concentrations in the plasma of pregnant women carrying fetuses with trisomy 21. Clin Chem 1999; 45:1747.
  41. Zhong XY, Bürk MR, Troeger C, et al. Fetal DNA in maternal plasma is elevated in pregnancies with aneuploid fetuses. Prenat Diagn 2000; 20:795.
  42. Wataganara T, LeShane ES, Farina A, et al. Maternal serum cell-free fetal DNA levels are increased in cases of trisomy 13 but not trisomy 18. Hum Genet 2003; 112:204.
  43. Ehrich M, Deciu C, Zwiefelhofer T, et al. Noninvasive detection of fetal trisomy 21 by sequencing of DNA in maternal blood: a study in a clinical setting. Am J Obstet Gynecol 2011; 204:205.e1.
  44. Sehnert AJ, Rhees B, Comstock D, et al. Optimal detection of fetal chromosomal abnormalities by massively parallel DNA sequencing of cell-free fetal DNA from maternal blood. Clin Chem 2011; 57:1042.
  45. Bianchi DW, Platt LD, Goldberg JD, et al. Genome-wide fetal aneuploidy detection by maternal plasma DNA sequencing. Obstet Gynecol 2012; 119:890.
  46. Ashoor G, Syngelaki A, Wagner M, et al. Chromosome-selective sequencing of maternal plasma cell-free DNA for first-trimester detection of trisomy 21 and trisomy 18. Am J Obstet Gynecol 2012; 206:322.e1.
  47. Norton ME, Brar H, Weiss J, et al. Non-Invasive Chromosomal Evaluation (NICE) Study: results of a multicenter prospective cohort study for detection of fetal trisomy 21 and trisomy 18. Am J Obstet Gynecol 2012; 207:137.e1.
  48. Sparks AB, Wang ET, Struble CA, et al. Selective analysis of cell-free DNA in maternal blood for evaluation of fetal trisomy. Prenat Diagn 2012; 32:3.
  49. Palomaki GE, Deciu C, Kloza EM, et al. DNA sequencing of maternal plasma reliably identifies trisomy 18 and trisomy 13 as well as Down syndrome: an international collaborative study. Genet Med 2012; 14:296.
  50. Nicolaides KH, Syngelaki A, Gil M, et al. Validation of targeted sequencing of single-nucleotide polymorphisms for non-invasive prenatal detection of aneuploidy of chromosomes 13, 18, 21, X, and Y. Prenat Diagn 2013; 33:575.
  51. Porreco RP, Garite TJ, Maurel K, et al. Noninvasive prenatal screening for fetal trisomies 21, 18, 13 and the common sex chromosome aneuploidies from maternal blood using massively parallel genomic sequencing of DNA. Am J Obstet Gynecol 2014; 211:365.e1.
  52. Bianchi DW, Prosen T, Platt LD, et al. Massively parallel sequencing of maternal plasma DNA in 113 cases of fetal nuchal cystic hygroma. Obstet Gynecol 2013; 121:1057.
  53. Ashoor G, Syngelaki A, Wang E, et al. Trisomy 13 detection in the first trimester of pregnancy using a chromosome-selective cell-free DNA analysis method. Ultrasound Obstet Gynecol 2013; 41:21.
  54. Nicolaides KH, Syngelaki A, Ashoor G, et al. Noninvasive prenatal testing for fetal trisomies in a routinely screened first-trimester population. Am J Obstet Gynecol 2012; 207:374.e1.
  55. Lau TK, Jiang FM, Stevenson RJ, et al. Secondary findings from non-invasive prenatal testing for common fetal aneuploidies by whole genome sequencing as a clinical service. Prenat Diagn 2013; 33:602.
  56. Wang Y, Chen Y, Tian F, et al. Maternal mosaicism is a significant contributor to discordant sex chromosomal aneuploidies associated with noninvasive prenatal testing. Clin Chem 2014; 60:251.
  57. Osborne CM, Hardisty E, Devers P, et al. Discordant noninvasive prenatal testing results in a patient subsequently diagnosed with metastatic disease. Prenat Diagn 2013; 33:609.
  58. Grati FR, Malvestiti F, Ferreira JC, et al. Fetoplacental mosaicism: potential implications for false-positive and false-negative noninvasive prenatal screening results. Genet Med 2014; 16:620.
  59. Mao J, Wang T, Wang BJ, et al. Confined placental origin of the circulating cell free fetal DNA revealed by a discordant non-invasive prenatal test result in a trisomy 18 pregnancy. Clin Chim Acta 2014; 433:190.
  60. Snyder MW, Simmons LE, Kitzman JO, et al. Copy-number variation and false positive prenatal aneuploidy screening results. N Engl J Med 2015; 372:1639.
  61. Zhang H, Gao Y, Jiang F, et al. Non-invasive prenatal testing for trisomies 21, 18 and 13: clinical experience from 146,958 pregnancies. Ultrasound Obstet Gynecol 2015; 45:530.
  62. Bianchi DW, Chudova D, Sehnert AJ, et al. Noninvasive Prenatal Testing and Incidental Detection of Occult Maternal Malignancies. JAMA 2015; 314:162.
  63. Lau TK, Jiang F, Chan MK, et al. Non-invasive prenatal screening of fetal Down syndrome by maternal plasma DNA sequencing in twin pregnancies. J Matern Fetal Neonatal Med 2013; 26:434.
  64. Leung TY, Qu JZ, Liao GJ, et al. Noninvasive twin zygosity assessment and aneuploidy detection by maternal plasma DNA sequencing. Prenat Diagn 2013; 33:675.
  65. Canick JA, Kloza EM, Lambert-Messerlian GM, et al. DNA sequencing of maternal plasma to identify Down syndrome and other trisomies in multiple gestations. Prenat Diagn 2012; 32:730.
  66. Huang X, Zheng J, Chen M, et al. Noninvasive prenatal testing of trisomies 21 and 18 by massively parallel sequencing of maternal plasma DNA in twin pregnancies. Prenat Diagn 2014; 34:335.
  67. del Mar Gil M, Quezada MS, Bregant B, et al. Cell-free DNA analysis for trisomy risk assessment in first-trimester twin pregnancies. Fetal Diagn Ther 2014; 35:204.
  68. Struble CA, Syngelaki A, Oliphant A, et al. Fetal fraction estimate in twin pregnancies using directed cell-free DNA analysis. Fetal Diagn Ther 2014; 35:199.
  69. Stanghellini I, Bertorelli R, Capone L, et al. Quantitation of fetal DNA in maternal serum during the first trimester of pregnancy by the use of a DAZ repetitive probe. Mol Hum Reprod 2006; 12:587.
  70. Scheffer PG, van der Schoot CE, Page-Christiaens GC, et al. Reliability of fetal sex determination using maternal plasma. Obstet Gynecol 2010; 115:117.
  71. Akolekar R, Farkas DH, VanAgtmael AL, et al. Fetal sex determination using circulating cell-free fetal DNA (ccffDNA) at 11 to 13 weeks of gestation. Prenat Diagn 2010; 30:918.
  72. Devaney SA, Palomaki GE, Scott JA, Bianchi DW. Noninvasive fetal sex determination using cell-free fetal DNA: a systematic review and meta-analysis. JAMA 2011; 306:627.
  73. Bianchi DW. At-home fetal DNA gender testing: caveat emptor. Obstet Gynecol 2006; 107:216.
  74. Morris JK, Alberman E, Scott C, Jacobs P. Is the prevalence of Klinefelter syndrome increasing? Eur J Hum Genet 2008; 16:163.
  75. Tartaglia NR, Howell S, Sutherland A, et al. A review of trisomy X (47,XXX). Orphanet J Rare Dis 2010; 5:8.
  76. Bianchi DW, Parsa S, Bhatt S, et al. Fetal sex chromosome testing by maternal plasma DNA sequencing: clinical laboratory experience and biology. Obstet Gynecol 2015; 125:375.
  77. Gautier E, Benachi A, Giovangrandi Y, et al. Fetal RhD genotyping by maternal serum analysis: a two-year experience. Am J Obstet Gynecol 2005; 192:666.
  78. Finning K, Martin P, Daniels G. A clinical service in the UK to predict fetal Rh (Rhesus) D blood group using free fetal DNA in maternal plasma. Ann N Y Acad Sci 2004; 1022:119.
  79. Lun FM, Tsui NB, Chan KC, et al. Noninvasive prenatal diagnosis of monogenic diseases by digital size selection and relative mutation dosage on DNA in maternal plasma. Proc Natl Acad Sci U S A 2008; 105:19920.
  80. González-González MC, Trujillo MJ, Rodríguez de Alba M, et al. Huntington disease-unaffected fetus diagnosed from maternal plasma using QF-PCR. Prenat Diagn 2003; 23:232.
  81. Bustamante-Aragones A, Trujillo-Tiebas MJ, Gallego-Merlo J, et al. Prenatal diagnosis of Huntington disease in maternal plasma: direct and indirect study. Eur J Neurol 2008; 15:1338.
  82. Amicucci P, Gennarelli M, Novelli G, Dallapiccola B. Prenatal diagnosis of myotonic dystrophy using fetal DNA obtained from maternal plasma. Clin Chem 2000; 46:301.
  83. Saito H, Sekizawa A, Morimoto T, et al. Prenatal DNA diagnosis of a single-gene disorder from maternal plasma. Lancet 2000; 356:1170.
  84. Lau TK, Lo KW, Chan LY, et al. Cell-free fetal deoxyribonucleic acid in maternal circulation as a marker of fetal-maternal hemorrhage in patients undergoing external cephalic version near term. Am J Obstet Gynecol 2000; 183:712.
  85. Lo YM, Leung TN, Tein MS, et al. Quantitative abnormalities of fetal DNA in maternal serum in preeclampsia. Clin Chem 1999; 45:184.
  86. Zhong XY, Laivuori H, Livingston JC, et al. Elevation of both maternal and fetal extracellular circulating deoxyribonucleic acid concentrations in the plasma of pregnant women with preeclampsia. Am J Obstet Gynecol 2001; 184:414.
  87. Sifakis S, Zaravinos A, Maiz N, et al. First-trimester maternal plasma cell-free fetal DNA and preeclampsia. Am J Obstet Gynecol 2009; 201:472.e1.
  88. Leung TN, Zhang J, Lau TK, et al. Maternal plasma fetal DNA as a marker for preterm labour. Lancet 1998; 352:1904.
  89. Sugito Y, Sekizawa A, Farina A, et al. Relationship between severity of hyperemesis gravidarum and fetal DNA concentration in maternal plasma. Clin Chem 2003; 49:1667.
  90. Zhong XY, Holzgreve W, Li JC, et al. High levels of fetal erythroblasts and fetal extracellular DNA in the peripheral blood of a pregnant woman with idiopathic polyhydramnios: case report. Prenat Diagn 2000; 20:838.
  91. Quezada MS, Francisco C, Dumitrascu-Biris D, et al. Fetal fraction of cell-free DNA in maternal plasma in the prediction of spontaneous preterm delivery. Ultrasound Obstet Gynecol 2015; 45:101.
  92. Rolnik DL, O'Gorman N, Fiolna M, et al. Maternal plasma cell-free DNA in the prediction of pre-eclampsia. Ultrasound Obstet Gynecol 2015; 45:106.
  93. Pergament E, Cuckle H, Zimmermann B, et al. Single-nucleotide polymorphism-based noninvasive prenatal screening in a high-risk and low-risk cohort. Obstet Gynecol 2014; 124:210.