Open main menu

Wikipedia β

Cell-free fetal DNA (cffDNA) is fetal DNA which circulates freely in the maternal blood. Maternal blood is sampled by venipuncture. Analysis of cffDNA is a method of non-invasive prenatal diagnosis frequently ordered for pregnant women of advanced maternal age.



Cell free fetal DNA sheds into the maternal blood circulation

cffDNA originates from placental trophoblasts.[1][2] Fetal DNA is fragmented when placental microparticles are shed into the maternal blood circulation (figure 1).[3]

cffDNA fragments are approximately 200 base pairs (bp) in length. They are significantly smaller than maternal DNA fragments.[4] The difference in size allows cffDNA to be distinguished from maternal DNA fragments.[5][6]

Approximately 11 to 13.4 percent of the cell-free DNA in maternal blood is of fetal origin. The amount varies widely from one pregnant woman to another.[7] cffDNA is present after five to seven weeks gestation. The amount of cffDNA increases as the pregnancy progresses.[8] The quantity of cffDNA in maternal blood diminishes rapidly after childbirth. Two hours after delivery, cffDNA is no longer detectable in maternal blood.[9]

Analysis of cffDNA may provide earlier diagnosis of foetal conditions than current techniques. As cffDNA is found in maternal blood, sampling carries no associated risk of spontaneous abortion.[10][11][12][13][14] cffDNA analysis has the same ethical and practical issues as other techniques such as amniocentesis and chorionic villus sampling.[15]

Some disadvantages of sampling cffDNA include a low concentration of cffDNA in maternal blood; variation in the quantity of cffDNA between individuals; a high concentration of maternal cell free DNA compared to the cffDNA in maternal blood.[16]

New evidence shows that cffDNA test failure rate is higher, fetal fraction (proportion of fetal versus maternal DNA in the maternal blood sample) is lower and PPV for trisomies 18, 13 and SCA is decreased in IVF pregnancies compared to those conceived spontaneously.[17]

Laboratory methodsEdit

A maternal peripheral blood sample is taken by venesection at about ten weeks gestation.[18]

Separation of cffDNAEdit

Blood plasma is separated from the maternal blood sample using a laboratory centrifuge. The cffDNA is then isolated and purified.[19] In 2007, a standardized protocol for doing this was written by Legler et al through an evaluation of the scientific literature. The highest yield in cffDNA extraction was obtained with the "QIAamp DSP Virus Kit".[20]

Addition of formaldehyde to maternal blood samples increases the yield of cffDNA. Formaldehyde stabilizes intact cells, and therefore inhibits the further release of maternal DNA. With the addition of formaldehyde, the percentage of cffDNA recovered from a maternal blood sample varies between 0.32 percent and 40 percent with a mean of 7.7 percent.[21] Without the addition of formaldehyde, the mean percentage of cffDNA recovered has been measured at 20.2 percent. However, other figures vary between 5 and 96 percent.[22][23]

Recovery of cffDNA may be related to the length of the DNA fragments. Another way to increase the fetal DNA is based on physical length of DNA fragments. Smaller fragments can represent up to seventy percent of the total cell free DNA in the maternal blood sample.

Analysis of cffDNAEdit

In real-time PCR, fluorescent probes are used to monitor the accumulation of amplicons. The reporter fluorescent signal is proportional to the number of amplicons generated. The most appropriate real time PCR protocol is designed according to the particular mutation or genotype to be detected. Point mutations are analysed with qualitative real time PCR with the use of allele specific probes. insertions and deletions are analyzed by dosage measurements using quantitative real time PCR.

cffDNA may be detected by finding paternally inherited DNA sequences via polymerase chain reaction (PCR).[24]>[25]

Quantitative real-time PCREdit

In 2010, Hill et al analyzed the sex-determining region Y gene (SRY) and the Y chromosome short tandem repeat "DYS14" in cffDNA from 511 pregnancies using a quantitative real-time PCR (RT-qPCR). In 401 of 403 pregnancies where maternal blood was drawn at seven weeks gestation or more, both segments of DNA were found.[26]

Nested PCREdit

In 2001, Al-Yatama et al evaluated the use of nested polymerase chain reaction (nested PCR) to determine sex by detecting a Y chromosome specific signal in the cffDNA from maternal plasma. Nested PCR detected 53 of 55 male fetuses. The cffDNA from the plasma of 3 of 25 women with female fetuses contained the Y chromosome-specific signal. The sensitivity of nested PCR in this experiment was 96 percent. The specificity was 88 percent.[27]

Digital PCREdit

Microfluidic devices allow the quantification of cffDNA segments in maternal plasma with accuracy beyond that of real-time PCR. Point mutations, loss of heterozygosity and aneuploidy can be detected in a single PCR step.[28][29][30] Digital PCR can differentiate between maternal blood plasma and fetal DNA in a multiplex fashion.[28]

Shotgun sequencingEdit

High throughput shotgun sequencing using tools such as Solexa or Illumina, yields approximately 5 million sequence tags per sample of maternal serum. In 2008, Fan et al identified aneuploid pregnancies such as trisomy when testing at the fourteenth week of gestation. In 2010, fetal whole of genome mapping by parental haplotype analysis was completed using sequencing of cffDNA from maternal serum.[13] Chiu et al. in 2010 studied 753 pregnant females, using a 2-plex massively parallel maternal plasma DNA sequencing and trisomy was diagnosed with z-score greater than 3.[31] The sequencing gave sensitivity of 100 percent, specificity of 97.9 percent, a positive predictive value of 96.6 percent and a negative predictive value of 100 percent.

Mass spectrometryEdit

Matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS) combined with single-base extension after PCR allows cffDNA detection with single base specificity and single DNA molecule sensitivity.[32] DNA is amplified by PCR. Then, linear amplification with base extension reaction (with a third primer) is designed to anneal to the region upstream from the mutation site. One or two bases are added to the extension primer to produce two extension products from wild-type DNA and mutant DNA. Single base specificity provides advantages over hybridization-based techniques using TaqMan hydrolysis probes. In 2008, when assessing the technique, Ding et al found no false positives or negatives when looking for cffDNA to determine fetal sex in sixteen maternal plasma samples.[32] In 2010, Akolekar et al correctly detected the sex of ninety of ninety-one male foetuses using MALDI-TOF mas spectrometry. The technique had accuracy, sensitivity and specificity of over 99 percent.[33]

Epigenetic modificationsEdit

Differences in gene activation between maternal and fetal DNA can be exploited. epigenetic modifications (heritable modifications that change gene function without changing DNA sequence) can be used to detect cffDNA.[34][35] The hypermethylated RASSF1A promoter is a universal fetal marker used to confirm the presence of cffDNA.[36] In 2012, White et al described a technique where cffDNA was extracted from maternal plasma and then digested with methylation-sensitive and insensitive restriction enzymes. Then, real-time PCR analysis of RASSF1A, SRY, and DYS14 was done.[36] The procedure detected 79 out of 90 (88 percent) maternal blood samples where hypermethylated RASSF1A was present.


mRNA transcripts from genes expressed in the placenta are detectable in maternal plasma.[37] In this procedure, plasma is centrifuged so an aqueous layer appears. This layer is transferred and from it RNA is extracted. RT-PCR is used to detect a selected expression of RNA. For example, Human placental lactogen (hPL) and beta-hCG mRNA are stable in maternal plasma and can be detected. (Ng et al. 2002). This can help to confirm the presence of cffDNA in maternal plasma.[16]


Prenatal sex determinationEdit

X-linked genetic disorder

The analysis of cffDNA from a sample of maternal plasma allows the determination of fetal gender. Whether the sex of the fetus is male or female allows the determination of the risk of a particular X-linked recessive genetic disorder in a particular pregnancy, especially where the mother is a genetic carrier of the disorder.[38] Most X-linked diseases are evident in males because of the lack of the second X-chromosome that can compensate for the disease allele. Common X-linked recessive disorders include Duchenne muscular dystrophy, fragile X syndrome and haemophilia.

In comparison to obstetric ultrasonography which is unreliable for sex determination in the first trimester and amniocentesis which carries a small risk of miscarriage, sampling of maternal plasma for analysis of cffDNA is without risk.[39] The main targets in the cffDNA analysis are the gene responsible for the sex-determining region Y protein (SRY) on the Y chromosome and the DYS14 sequence.[40][41]

Congenital adrenal hyperplasia

In congenital adrenal hyperplasia, the adrenal cortex lacks appropriate corticosteroid synthesis, leading to excess adrenal androgens and affects female fetuses.[42] There is an external masculinization of the genitalia in the female fetuses.[43] Mothers of at risk fetuses are given dexamethasone at 6 weeks gestation to suppress pituitary gland release of androgens.[44]

If analysis of cffDNA obtained from a sample of maternal plasma lacks genetic markers found only on the Y chromosome, it is suggestive of a female fetus. However, it might also indicate a failure of the analysis itself ( a false negative result). Paternal genetic polymorphisms and sex-independent markers may be used to detect cffDNA. An high degree of heterozygosity of these markers must be present for this application.[45]

Paternity testing

Prenatal DNA paternity testing is commercially available. The test can be performed at nine weeks gestation.[citation needed]

Single gene disordersEdit

Autosomal dominant and recessive single gene disorders which have been diagnosed prenatally by analysing paternally inherited DNA include cystic fibrosis, beta thalassemia, sickle cell anemia, spinal muscular atrophy, and myotonic dystrophy.[24][40] Prenatal diagnosis of single gene disorders which are due to an autosomal recessive mutation, a maternally inherited autosomal dominant mutation or large sequence mutations that include duplication, expansion or insertion of DNA sequences is more difficult.[46]

In cffDNA, fragments of 200 – 300 bp length involved in single gene disorders are more difficult to detect.

For example, the autosomal dominant condition, achondroplasia is caused by the FGFR3 gene point mutation.[47] In 2007, a study of two pregnancies with a fetus with achondroplasia found a paternally inherited G1138A mutation from cffDNA from a maternal plasma sample in one and a G1138A de novo mutation from the other.[47]

In studies of the genetics of Huntington's chorea using qRT-PCR of cffDNA from maternal plasma samples, CAG repeats have been detected at normal levels (17, 20 and 24).[48]

cffDNA may also be used to diagnose single gene disorders.[15]Developments in laboratory processes using cffDNA may allow prenatal diagnosis of aneuploidies such as trisomy 21 (Down's syndrome) in the fetus.[49][29]

Hemolytic disease of the fetus and newbornEdit

Incompatibility of fetal and maternal RhD antigens is the main cause of Hemolytic disease of the newborn.[50] Approximately 15 percent of Caucasian women, 3 to 5 percent of black Africa women and less than 3 percent of Asian women are RhD negative.[51]

Accurate prenatal diagnosis is important because the disease can be fatal to the newborn and because treatment including intramuscular immunoglobulin (Anti-D) or intravenous immunoglobulin can be administered to mothers at risk.[52]

In 2010, Cardo et al reported that PCR to detect RHD (gene) gene exons 5 and 7 from cffDNA obtained from maternal plasma between 9 and 13 weeks gestation gave a high degree of specificity, sensitivity and diagnostic accuracy (>90 percent) when compared to RhD determination from newborn cord blood serum.[50] In 2013, Aykute et al found similar results targeting exons 7 and 10.[53] In 2015, Svobodova et al reported that droplet digital PCR in fetal RhD determination was comparable to a routine real-time PCR technique.[54]

Routine determination of fetal RhD status from cffDNA in maternal serum allows early management of at risk pregnancies while decreasing unnecessary use of Anti-D by over 25 percent.[55]


Sex chromosomes

Analysis of maternal serum cffDNA by high-throughput sequencing can detect common fetal sex chromosome aneuploidies such as Turner's syndrome, Klinefelter's syndrome and triple X syndrome but the procedure's positive predictive value is low.[56]

Trisomy 21

Fetal trisomy of chromosome 21 is the cause of Down's syndrome. This trisomy can be detected by analysis of cffDNA from maternal blood by massively parallel shotgun sequencing (MPSS).[57] Another technique is digital analysis of selected regions (DANSR).[57] However, such tests show inconsistent degrees of sensitivity and specificity and therefore may be best used to confirm a positive maternal screening test such as ultrasound markers of the condition.[57].[58]

Trisomy 13 and 18

Analysis of cffDNA from maternal plasma with MPSS looking for trisomy 13 or 18 is possible[59]

Factors limiting sensitivity and specificity include the levels of cffDNA in the maternal plasma; maternal chromosomes may have mosaicism.[60]

A number of fetal nucleic acid molecules derived from aneuploid chromosomes can be detected including SERPINEB2 mRNA, clad B, hypomethylated SERPINB5 from chromosome 18, placenta-specific 4 (PLAC4), hypermethylated holocarboxylase synthetase (HLCS) and c21orf105 mRNA from chromosome 12.[61] With complete trisomy, the mRNA alleles in maternal plasma isn't the normal 1:1 ratio, but is in fact 2:1. Allelic ratios determined by epigenetic markers can also be used to detect the complete trisomies. Massive parallel sequencing and digital PCR for fetal aneuploidy detection can be used without restriction to fetal-specific nucleic acid molecules. (MPSS) is estimated to have a sensitivity of between 96 and 100%, and a specificity between 94 and 100% for detecting Down syndromeIt can be performed at 10 weeks of gestational age.[62] One study in the United States estimated a false positive rate of 0.3% and a positive predictive value of 80% when using cffDNA to detect Down syndrome.[63]


Preeclampsia is a complex condition of pregnancy involving hypertension and proteinuria usually after 20 weeks gestation.[64] It is associated with poor cytotrophoblastic invasion of the myometrium. Onset of the condition between 20 and 34 weeks gestation, is considered "early".[65] Maternal plasma samples in pregnancies complicated by preeclampsia have significantly higher levels of cffDNA that those in normal pregnancies.[66][67][68] This holds true for early onset preeclampsia.[65]

Future perspectivesEdit

New generation sequencing may be used to yield a whole genome sequence from cffDNA. This raises ethical questions.[69] However, the utility of the procedure may increase as clear associations between specific genetic variants and disease states are discovered.[70][71]

See alsoEdit


  1. ^ Alberry, M., et al. (2007). "Free fetal DNA in maternal plasma in anembryonic pregnancies: confirmation that the origin is the trophoblast". Prenatal Diagnosis 27 (5): 415-418.
  2. ^ Gupta, A. K., et al. (2004). "Detection of fetal DNA and RNA in placenta-derived syncytiotrophoblast microparticles generated in vitro". Clinical Chemistry 50 (11): 2187-2190.
  3. ^ Smets, E. M.; Visser, A.; Go, A. T.; van Vugt, J. M.; Oudejans, C. B. (2006). "Novel biomarkers in preeclampsia". Clinica Chimica Acta: International Journal of Clinical Chemistry 364 (1-2): 22-32.
  4. ^ Chan, K. C., et al. (2004). "Size distributions of maternal and fetal DNA in maternal plasma". Clinical Chemistry 50 (1): 88-92.
  5. ^ Li, Y., et al. (2004). "Size separation of circulatory DNA in maternal plasma permits ready detection of fetal DNA polymorphisms". Clinical Chemistry 50 (6): 1002-1011.
  6. ^ Li, Y., et al. (2005). "Detection of paternally inherited fetal point mutations for beta-thalassemia using size-fractionated cell-free DNA in maternal plasma". The Journal of the American Medical Association 293 (7): 843-849.
  7. ^ Wang, Eric; Batey, Annette; Struble, Craig; Musci, Thomas; Song, Ken; Oliphant, Arnold (July 2013). "Gestational age and maternal weight effects on fetal cell-free DNA in maternal plasma". Prenatal Diagnosis. 33 (7): 662–666. doi:10.1002/pd.4119. PMID 23553731. 
  8. ^ Lo, Y. M., et al. (1998). "Quantitative analysis of fetal DNA in maternal plasma and serum: implications for noninvasive prenatal diagnosis". American Journal of Human Genetics. 62 (4): 768-775.
  9. ^ Lo, Y. M., et al. (1999). "Rapid clearance of fetal DNA from maternal plasma". American Journal of Human Genetics 64 (1): 218-224.
  10. ^ Lo, Y. M., et al. (1998). "Prenatal diagnosis of fetal RhD status by molecular analysis of maternal plasma". The New England Journal of Medicine 339 (24): 1734-1738.
  11. ^ Allyse, M.; Sayres, L. C.; King, J. S.; Norton, M. E.; Cho, M. K. (2012). "Cell-free fetal DNA testing for fetal aneuploidy and beyond: clinical integration challenges in the US context". Human Reproduction 27 (11): 3123-3131.
  12. ^ Mujezinovic, F.; Alfirevic, Z. (2007). "Procedure-related complications of amniocentesis and chorionic villous sampling: a systematic review". Obstetrics and Gynecology 110 (3): 687-694.
  13. ^ a b Lo, Y. M. (2008). "Fetal nucleic acids in maternal plasma". Annals of the New York Academy of Sciences 1137: 140-143.
  14. ^ [1] The NHS RAPID project | Reliable Accurate Prenatal non-Invasive Diagnosis
  15. ^ a b Hahn, S; Chitty, LS (April 2008). "Noninvasive prenatal diagnosis: current practice and future perspectives". Current Opinion in Obstetrics and Gynecology. 20 (2): 146–51. doi:10.1097/GCO.0b013e3282f73349. PMID 18388814. 
  16. ^ a b Wright, C. F.; Burton, H. (2009). "The use of cell-free fetal nucleic acids in maternal blood for non-invasive prenatal diagnosis". Human Reproduction Update 15 (1): 139-151.
  17. ^ Lee, T.J., et al. (2018). "Cell-free fetal DNA testing in singleton IVF conceptions". Human Reproduction. doi:10.1093/humrep/dey033 [Epub ahead of print].
  18. ^ Guibert, J., et al. (2003). "Kinetics of SRY gene appearance in maternal serum: detection by real time PCR in early pregnancy after assisted reproductive technique". Human Reproduction 18 (8): 1733-1736.
  19. ^ Chiu, R. W., et al. (2001). "Effects of blood-processing protocols on fetal and total DNA quantification in maternal plasma". Clinical Chemistry 47 (9): 1607-1613.
  20. ^ Legler, T. J., et al. (2007). "Workshop report on the extraction of foetal DNA from maternal plasma". Prenatal Diagnosis 27 (9): 824-829.
  21. ^ Dhallan, R., et al. (2004). "Methods to increase the percentage of free fetal DNA recovered from the maternal circulation". The Journal of the American Medical Association 291 (9): 1114-1119.
  22. ^ Benachi, A., et al. (2005). "Impact of formaldehyde on the in vitro proportion of fetal DNA in maternal plasma and serum". Clinical Chemistry 51 (1): 242-244.
  23. ^ Chinnapapagari, S. K.; Holzgreve, W.; Lapaire, O.; Zimmermann, B.; Hahn, S. (2005). "Treatment of maternal blood samples with formaldehyde does not alter the proportion of circulatory fetal nucleic acids (DNA and mRNA) in maternal plasma". Clinical Chemistry 51 (3): 652-655.
  24. ^ a b Traeger-Synodinos, J. (2006). "Real-time PCR for prenatal and preimplantation genetic diagnosis of monogenic diseases". Molecular Aspects of Medicine 27 (2-3): 176-191.
  25. ^ Boon, E. M., et al. (2007). "Y chromosome detection by Real Time PCR and pyrophosphorolysis-activated polymerisation using free fetal DNA isolated from maternal plasma". Prenatal Diagnosis 27 (10): 932-937.
  26. ^ Hill, M., et al. (2010). "Steroid metabolome in fetal and maternal body fluids in human late pregnancy". The Journal of Steroid Biochemistry and Molecular Biology 122 (4): 114-132.
  27. ^ Al-Yatama, M. K., et al. (2001). "Detection of Y chromosome-specific DNA in the plasma and urine of pregnant women using nested polymerase chain reaction". Prenatal Diagnosis 21 (5): 399-402.
  28. ^ a b Zimmermann, B. G., et al. (2008). "Digital PCR: a powerful new tool for noninvasive prenatal diagnosis?". Prenatal Diagnosis 28 (12): 1087-1093.
  29. ^ a b Lo, Y. M., et al. (2007). "Digital PCR for the molecular detection of fetal chromosomal aneuploidy". Proceedings of the National Academy of Sciences of the United States of America 104 (32): 13116-13121.
  30. ^ Quake, S. (2007). "At the interface of physics and biology". BioTechniques 43 (1): 19.
  31. ^ Chiu, R. W.; Lo, Y. M. (2010). "Pregnancy-associated microRNAs in maternal plasma: a channel for fetal-maternal communication?". Clinical Chemistry 56 (11): 1656-1657.
  32. ^ a b Ding, C. (2008). "Maldi-TOF mass spectrometry for analyzing cell-free fetal DNA in maternal plasma". Methods in Molecular Biology 444: 253-267.
  33. ^ Akolekar, R.; Farkas, D. H.; VanAgtmael, A. L.; Bombard, A. T.; Nicolaides, K. H. (2010). "Fetal sex determination using circulating cell-free fetal DNA (ccffDNA) at 11 to 13 weeks of gestation". Prenatal Diagnosis 30 (10): 918-923.
  34. ^ Tong, Y. K.; Chiu, R. W.; Chan, K. C.; Leung, T. Y.; Lo, Y. M. (2012). "Technical concerns about immunoprecipitation of methylated fetal DNA for noninvasive trisomy 21 diagnosis". Nature Medicine 18 (9): 1327-1328; author reply 1328-1329.
  35. ^ Papageorgiou, E. A., et al. (2011). "Fetal-specific DNA methylation ratio permits noninvasive prenatal diagnosis of trisomy 21". Nature Medicine 17 (4): 510-513.
  36. ^ a b White, H. E., Dent, C. L.; Hall, V. J.; Crolla, J. A.; Chitty, L. S. (2012). "Evaluation of a novel assay for detection of the fetal marker RASSF1A: facilitating improved diagnostic reliability of noninvasive prenatal diagnosis". PLoS ONE 7 (9): e45073.
  37. ^ Ng, E. K., et al. (2002). "Presence of filterable and nonfilterable mRNA in the plasma of cancer patients and healthy individuals". Clinical Chemistry 48 (8): 1212-1217.
  38. ^ Baird, P. A.; Anderson, T. W.; Newcombe, H. B.; Lowry, R. B. (1988). "Genetic disorders in children and young adults: a population study". American Journal of Human Genetics 42 (5): 677-693.
  39. ^ Scheffer, P. G., et al. (2010). "Reliability of fetal sex determination using maternal plasma". Obstetrics and Gynecology 115 (1): 117-126.
  40. ^ a b Bustamante-Aragones, A.; Gonzalez-Gonzalez, C.; de Alba, M. R.; Ainse, E.; Ramos, C. (2010). "Noninvasive prenatal diagnosis using ccffDNA in maternal blood: state of the art". Expert Review of Molecular Diagnostics 10 (2): 197-205.
  41. ^ Zimmermann, B.; El-Sheikhah, A.; Nicolaides, K.; Holzgreve, W.; Hahn, S. (2005). "Optimized real-time quantitative PCR measurement of male fetal DNA in maternal plasma". Clinical Chemistry 51 (9): 1598-1604.
  42. ^ Finning, K. M.; Chitty, L. S. (2008). "Non-invasive fetal sex determination: impact on clinical practice". Seminars in Fetal & Neonatal Medicine 13 (2): 69-75.
  43. ^ Markey, C. M.; Wadia, P. R.; Rubin, B. S.; Sonnenschein, C; Soto, A. M. (2005). "Long-term effects of fetal exposure to low doses of the xenoestrogen bisphenol-A in the female mouse genital tract". Biology of Reproduction 72 (6): 1344-1351.
  44. ^ Sayres, L. C.; Cho, M. K. (2011). "Cell-free fetal nucleic acid testing: a review of the technology and its applications". Obstetrical & Gynecological Survey 66 (7): 431-442.
  45. ^ Hill, M.; Barrett, A. N.; White, H.; Chitty, L. S. (2012). "Uses of cell free fetal DNA in maternal circulation". Best Practice & Research: Clinical Obstetrics & Gynaecology 26 (5): 639-654.
  46. ^ Norbury, G.; Norbury, C. J. (2008). "Non-invasive prenatal diagnosis of single gene disorders: how close are we?". Seminars in Fetal & Neonatal Medicine 13 (2): 76-83.
  47. ^ a b Li, Y.; Page-Christiaens, G. C.; Gille, J. J.; Holzgreve, W.; Hahn, S. (2007). "Non-invasive prenatal detection of achondroplasia in size-fractionated cell-free DNA by MALDI-TOF MS assay". Prenatal Diagnosis 27 (1): 11-17.
  48. ^ [2] De Die-Smulders, C. E. M.; De Wert, G. M. W. R.; Liebaers, I.; Tibben, A.; Evers-Kiebooms, G. (2013). "Reproductive options for prospective parents in families with Huntington's disease: Clinical, psychological and ethical reflections". Human Reproduction Update. 19 (3): 304–315. doi:10.1093/humupd/dms058. PMID 23377865. 
  49. ^ Fan, H. C.; Blumenfeld, Y. J.; Chitkara, U.; Hudgins, L.; Quake, S. R. (2008). "Noninvasive diagnosis of fetal aneuploidy by shotgun sequencing DNA from maternal blood". Proceedings of the National Academy of Sciences of the United States of America 105 (42): 16266-16271.
  50. ^ a b Cardo L. et al "Non-invasive fetal RHD genotyping in the first trimester of pregnancy" Clin Chem Lab Med. 2010 Aug;48(8):1121-6. doi: 10.1515/CCLM.2010.234.
  51. ^ Chinen P. A. et al "Noninvasive determination of fetal rh blood group, D antigen status by cell-free DNA analysis in maternal plasma: experience in a Brazilian population." Am J Perinatol. 2010 Nov;27(10):759-62. doi: 10.1055/s-0030-1253560. Epub 2010 Apr 20.
  52. ^ Okwundu C. and Afolabi B. "Intramuscular versus intravenous anti-D for preventing Rhesus alloimmunization during pregnancy." Cochrane Database Syst Rev. 2013 Jan 31;(1):CD007885. doi: 10.1002/14651858.CD007885.pub2
  53. ^ Ayket A et al "Determination of fetal rhesus d status by maternal plasma DNA analysis" Balkan J Med Genet. 2013 Dec;16(2):33-8. doi: 10.2478/bjmg-2013-0029.
  54. ^ Svobodova I. et al "Performance of Droplet Digital PCR in Non-Invasive Fetal RHD Genotyping - Comparison with a Routine Real-Time PCR Based Approach." PLoS One. 2015 Nov 12;10(11):e0142572. doi: 10.1371/journal.pone.0142572. eCollection 2015.
  55. ^ Papasavva T. et al "Prevalence of RhD status and clinical application of non-invasive prenatal determination of fetal RHD in maternal plasma: a 5 year experience in Cyprus." BMC Res Notes. 2016 Apr 1;9:198. doi: 10.1186/s13104-016-2002-x.
  56. ^ Zhang B. et al "Noninvasive prenatal screening for fetal common sex chromosome aneuploidies from maternal blood." J Int Med Res. 2017 Apr;45(2):621-630. doi: 10.1177/0300060517695008. Epub 2017 Mar 30.
  57. ^ a b c "Down Syndrome: Current Status, Challenges and Future Perspectives." Int J Mol Cell Med. 2016 Summer; 5(3): 125–133. PMCID: PMC5125364
  58. ^ Mersy, E.; Smits, L. J. M.; Van Winden, L. A. A. P.; De Die-Smulders, C. E. M.; Paulussen, A. D. C.; MacVille, M. V. E.; Coumans, A. B. C.; Frints, S. G. M. (2013). "Noninvasive detection of fetal trisomy 21: Systematic review and report of quality and outcomes of diagnostic accuracy studies performed between 1997 and 2012". Human Reproduction Update. 19 (4): 318–29. doi:10.1093/humupd/dmt001. PMID 23396607. 
  59. ^ Clark-Ganhart C.A. et al "Understanding the Limitations of Circulating Cell Free Fetal DNA: An Example of Two Unique Cases." J Clin Gynecol Obstet. 2014 May;3(2):38-70.
  60. ^ Wataganara, T., et al. (2003). "Maternal serum cell-free fetal DNA levels are increased in cases of trisomy 13 but not trisomy 18". Human Genetics 112 (2): 204-208.
  61. ^ Chiu, R. W.; Lo, Y. M. (2011). "Non-invasive prenatal diagnosis by fetal nucleic acid analysis in maternal plasma: the coming of age". Seminars in Fetal & Neonatal Medicine 16 (2): 88-93.
  62. ^ Noninvasive Prenatal Diagnosis of Fetal Aneuploidy Using Cell-Free Fetal Nucleic Acids in Maternal Blood: Clinical Policy (Effective 05/01/2013) from Oxford Health Plans
  63. ^ Bianchi, D. W.; Parker, R. L.; Wentworth, J.; Madankumar, R.; Saffer, C.; Das, A. F.; Craig, J. A.; Chudova, D. I.; Devers, P. L.; Jones, K. W.; Oliver, K.; Rava, R. P.; Sehnert, A. J. (2014). "DNA Sequencing versus Standard Prenatal Aneuploidy Screening". New England Journal of Medicine. 370 (9): 799–808. doi:10.1056/NEJMoa1311037. PMID 24571752. . A recent study in the New England Journal of Medicine demonstrated the feasibility of using NIPT in a low risk population.
  64. ^ Henderson J. T. et al "Preeclampsia Screening: Evidence Report and Systematic Review for the US Preventive Services Task Force" JAMA. 2017 Apr 25;317(16):1668-1683. doi: 10.1001/jama.2016.18315
  65. ^ a b Seval M. M. et al "Cell free fetal DNA in the plasma of pregnant women with preeclampsia" Clin Exp Obstet Gynecol. 2015;42(6):787-91.
  66. ^ Lo, Y. M., et al. (1999). "Increased fetal DNA concentrations in the plasma of pregnant women carrying fetuses with trisomy 21". Clinical Chemistry 45 (10): 1747-1751.
  67. ^ Leung, T. N.; Zhang, J.; Lau, T. K.; Chan, L. Y.; Lo, Y. M. (2001). "Increased maternal plasma fetal DNA concentrations in women who eventually develop preeclampsia". Clinical Chemistry 47 (1): 137-139.
  68. ^ Zhong, X. Y.; Holzgreve, W.; Hahn, S. (2002). "The levels of circulatory cell free fetal DNA in maternal plasma are elevated prior to the onset of preeclampsia". Hypertension in Pregnancy: Official Journal of the International Society for the Study of Hypertension in Pregnancy 21 (1): 77-83.
  69. ^ Yurkiewicz, I. R.; Korf, B. R.; Lehmann, L. S. (2014). "Prenatal whole-genome sequencing--is the quest to know a fetus's future ethical?". New England Journal of Medicine. 370 (3): 195–7. doi:10.1056/NEJMp1215536. PMID 24428465. 
  70. ^ Wellcome Trust Case Control Consortium; Clayton, David G.; Cardon, Lon R.; Craddock, Nick; Deloukas, Panos; Duncanson, Audrey; Kwiatkowski, Dominic P.; McCarthy, Mark I.; Ouwehand, Willem H.; Samani, Nilesh J.; Todd; Donnelly, Peter; Barrett, Jeffrey C.; Burton, Paul R.; Davison, Dan; Donnelly, Peter; Easton, Doug; Evans, David; Leung, Hin-Tak; Marchini, Jonathan L.; Morris; Spencer, Chris C. A.; Tobin, Martin D.; Cardon, Lon R.; Clayton, David G.; Attwood, Antony P.; Boorman, James P.; Cant, Barbara; Everson, Ursula; Hussey, Judith M. (June 2007). "Genome-wide association study of 14,000 cases of seven common diseases and 3,000 shared controls". Nature. 447 (7145): 661–78. Bibcode:2007Natur.447..661B. doi:10.1038/nature05911. PMC 2719288 . PMID 17554300. 
  71. ^ Mailman MD; Feolo M; Jin Y; Kimura M; Tryka K; Bagoutdinov R; Hao L; Kiang A; Paschall J; Phan L; Popova N; Pretel S; Ziyabari L; Lee M; Shao Y; Wang ZY; Sirotkin K; Ward M; Kholodov M; Zbicz K; Beck J; Kimelman M; Shevelev S; Preuss D; Yaschenko E; Graeff A; Ostell J; Sherry ST (October 2007). "The NCBI dbGaP database of genotypes and phenotypes". Nat. Genet. 39 (10): 1181–6. doi:10.1038/ng1007-1181. PMC 2031016 . PMID 17898773.