Skip to main content Accessibility help

Massively Parallel Sequencing (MPS) of Cell-Free Fetal DNA (cffDNA) for Trisomies 21, 18, and 13 in Twin Pregnancies

  • Erqiu Du (a1) (a2), Chun Feng (a1) (a2), Yuming Cao (a1) (a2), Yanru Yao (a1) (a2), Jing Lu (a1) (a2) and Yuanzhen Zhang (a1) (a2)...


Massively parallel sequencing (MPS) technology has become increasingly available and has been widely used to screen for trisomies 21, 18, and 13 in singleton pregnancies. This study assessed the performance of MPS testing of cell-free fetal DNA (cffDNA) from maternal plasma for trisomies 21, 18, and 13 in twin pregnancies. Ninety-two women with twin pregnancies were recruited. The results were identified through karyotypes of amniocentesis or clinical examination and follow-up of the neonates. Fluorescent in-situ hybridization was used to examine the placentas postnatally in cases of false-positive results. The fetuses with autosomal trisomy 21 (n = 2) and trisomy 15 (n = 1) were successfully detected via MPS testing of cffDNA. There was one false-positive for trisomy 13 (n = 1), and fluorescence in-situ hybridization (FISH) identified confined placental mosaicism in this case. For twin pregnancies undergoing second-trimester screening for trisomy, MPS testing of cffDNA is feasible and can enhance the diagnostic spectrum of non-invasive prenatal testing, which could effectively reduce invasive prenatal diagnostic methods. In addition to screening for trisomy 21, 18, and 13 by cffDNA, MPS can detect fetal additional autosomal trisomy. False-positive results cannot completely exclude confined placental mosaicism.

  • View HTML
    • Send article to Kindle

      To send this article to your Kindle, first ensure is added to your Approved Personal Document E-mail List under your Personal Document Settings on the Manage Your Content and Devices page of your Amazon account. Then enter the ‘name’ part of your Kindle email address below. Find out more about sending to your Kindle. Find out more about sending to your Kindle.

      Note you can select to send to either the or variations. ‘’ emails are free but can only be sent to your device when it is connected to wi-fi. ‘’ emails can be delivered even when you are not connected to wi-fi, but note that service fees apply.

      Find out more about the Kindle Personal Document Service.

      Massively Parallel Sequencing (MPS) of Cell-Free Fetal DNA (cffDNA) for Trisomies 21, 18, and 13 in Twin Pregnancies
      Available formats

      Send article to Dropbox

      To send this article to your Dropbox account, please select one or more formats and confirm that you agree to abide by our usage policies. If this is the first time you use this feature, you will be asked to authorise Cambridge Core to connect with your <service> account. Find out more about sending content to Dropbox.

      Massively Parallel Sequencing (MPS) of Cell-Free Fetal DNA (cffDNA) for Trisomies 21, 18, and 13 in Twin Pregnancies
      Available formats

      Send article to Google Drive

      To send this article to your Google Drive account, please select one or more formats and confirm that you agree to abide by our usage policies. If this is the first time you use this feature, you will be asked to authorise Cambridge Core to connect with your <service> account. Find out more about sending content to Google Drive.

      Massively Parallel Sequencing (MPS) of Cell-Free Fetal DNA (cffDNA) for Trisomies 21, 18, and 13 in Twin Pregnancies
      Available formats


Corresponding author

address for correspondence: Yuanzhen Zhang, No. 169 East Lake Road, Wuchang, Wuhan, China. E-mail:


Hide All
Agarwal, K., & Alfirevic, Z. (2012). Pregnancy loss after chorionic villus sampling and genetic amniocentesis in twin pregnancies: A systematic review. Ultrasound in Obstetrics and Gynecology, 40, 128134.
Alberry, M., Maddocks, D., Jones, M., Abdel Hadi, M., Abdel-Fattah, S., Avent, N., & Soothill, P. W. (2007). Free fetal DNA in maternal plasma in embryonic pregnancies: Confirmation that the origin is the trophoblast. Prenatal Diagnosis, 27, 415418.
Baffero, G. M., Somigliana, E., Crovetto, F., Paffoni, A., Persico, N., Guerneri, S., . . . Fedele, L. (2012). Confined placental mosaicism at chorionic villous sampling: Risk factors and pregnancy outcome., 32, 11021108.
Bevilacqua, E., Gil, M. M., Nicolaides, K. H., Ordoñez, E., Cirigliano, V., Dierickx, H., . . . Jani, J. C. (2015). Performance of screening for aneuploidies by cell-free DNA analysis of maternal blood in twin pregnancies. Ultrasound in Obstetrics and Gynecology, 45, 6166.
Bianchi, D. W. (2004). Circulating fetal DNA: Its origin and diagnostic potential: A review. Placenta, 25, S93S101.
Canick, J. A., Kloza, E. M., Lambert-Messerlian, G. M., Haddow, J. E., Ehrich, M., van den Boom, D., . . . Palomaki, G. E. (2012). DNA sequencing of maternal plasma to identify Down syndrome and other trisomy in multiple gestations. Prenatal Diagnosis, 32, 730734.
Chan Wong, E., Hatakeyama, C., Minor, A., & Ma, S. (2012). Investigation of confined placental mosaicism by CGH in IVF and ICSI pregnancies. Placenta, 33, 202206.
Chen, E. Z., Chiu, R. W., Sun, H., Akolekar, R., Chan, K. C., Leung, T. Y., . . . Lo, Y. M. (2011). Noninvasive prenatal diagnosis of fetal trisomy 18 and trisomy 13 by maternal plasma DNA sequencing. PLoS One, 6, e21791.
Chiu, R. W., Akolekar, R., Zheng, Y. W., Leung, T. Y., Sun, H., Chan, K. C., . . . Lo, Y. M. (2011). Noninvasive prenatal assessment of trisomy 21 by multiplexed maternal plasma DNA sequencing: Large scale validity study. BMJ, 342, c7401.
Choi, S. A., Ko, J. M., Shin, C. H., Yang, S. W., Choi, J. S., & Oh, S. K. (2013). Monozygotic twin discordant for Down syndrome: mos 47, XX, + 21/46, XX and 46, XX. European Journal of Pediatrics, 172, 11171120.
Cleary-Goldman, J., & Berkowitz, R. L. (2005). First trimester screening for Down syndrome in multiple pregnancy. Seminars in Perinatology, 29, 395400.
Dan, S., Wang, W., Ren, J., Li, Y., Hu, H., Xu, Z., . . . Zhang, X. (2012). Clinical application of massively parallel sequencing-based prenatal noninvasive fetal trisomy test for trisomies 21 and 18 in 11,105 pregnancies with mixed risk factors. Prenatal Diagnosis, 32, 12251232.
del Mar Gil, M., Quezada, M. S., Bregant, B., Syngelaki, A., & Nicolaides, K. H. (2014). Cell-free DNA analysis for trisomy risk assessment in first-trimester twin pregnancies. Fetal Diagnosis and Therapy, 35, 204211.
Flori, E., Doray, B., Gautier, E., Kohler, M., Ernault, P., Flori, J., & Costa, J. M. (2004). Circulating cell-free fetal DNA in maternal serum appears to originate from cyto- and syncytio-trophoblastic cells. Case report. Human Reproduction, 19, 723724.
Gahan, P. B., & Swaminathan, R. (2008). Circulating nucleic acids in plasma and serum. Recent developments. Annals of the New York Academy of Sciences, 1137, 16.
Gil, M. M., Quezada, M. S., & Revello, R. (2015). Analysis of cell-free DNA in maternal blood in screening for fetal aneuploidies: Updated meta-analysis. Ultrasound in Obstetrics and Gynecology, 45, 249266.
Gregg, A. R., Gross, S. J., Best, R. G., Monaghan, K. G., Baja, j K., Skotko, B. G., . . . Watson, M. S. (2013). ACMG statement on noninvasive prenatal screening for fetal aneuploidy. Genetics in Medicine, 15, 395398.
Gromminger, S., Yagmur, E., Erkan, S., Nagy, S., Schöck, U., Bonnet, J., . . . Stumm, M. (2014). Fetal aneuploidy detection by cell-free DNA sequencing for multiple pregnancies and quality issues with vanishing twins. Journal of Clinical Medicine, 3, 679692.
Gupta, A., Vaid, A., & Arora, R. (2016). Diachorionic triamniotic triplets — Saline cardiac tamponade for fetal reduction: A novel approach. Journal of Fetal Medicine, 3, 167170.
Hansen, M., Kurinczuk, J. J., Milne, E., de Klerk, N., & Bower, C. (2013). Assisted reproductive technology and birth defects: A systematic review and meta-analysis. Human Reproduction Update, 19, 330353.
Huang, X., Zheng, J., Chen, M., Zhao, Y., Zhang, C., Liu, L., . . . Wang, W. (2014). Noninvasive prenatal testing of trisomies 21 and 18 by massively parallel sequencing of maternal plasma DNA in twin pregnancies. Prenatal Diagnosis, 34, 335340.
Hultén, M. A., Jonasson, J., Iwarsson, E., Uppal, P., Vorsanova, S. G., Yurov, Y. B., & Iourov, I. Y. (2013). Trisomy 21 mosaicism: We may all have a touch of Down syndrome. Cytogenetic and Genome Research, 139, 189192.
Kalousek, D. K. (2000). Pathogenesis of chromosomal mosaicism and its effect on early human development. American Journal of Medical Genetics, 91, 3945.
Kalousek, D. K., & Vekemans, M. (2000). Confined placental mosaicism and genomic imprinting. Journal of Medical Genetics, 14, 723730.
Klinger, K., Landes, G., Shook, D., Harvey, R., Lopez, L., Locke, P., . . . Dackowski, W. (1992). Rapid detection of chromosome aneuploidies in uncultured amniocytes by using fluorescence in situ hybridization (FISH). American Journal of Human Genetics, 51, 5565.
Lanza, F., Castoldi, G., Castagnari, B., Todd, R. F. 3rd., Moretti, S., Spisani, S., . . . Traniello, S. (1998). Expression and functional role of urokinase-type plasminogen activator receptor in normal and acute leukaemic cells. British Journal of Haematology, 103, 110123.
Lau, T. K., Jiang, F., Chan, M. K., Zhang, H., Lo, P. S., & Wang, W. (2013). Non-invasive prenatal screening of fetal Down syndrome by maternal plasma DNA sequencing in twin pregnancies. Journal of Maternal-Fetal & Neonatal Medicine, 26, 434437.
Leung, T. Y., Qu, J. Z., Liao, G. J., Jiang, P., Cheng, Y. K., Chan, K. C., . . . Lo, Y. M. (2013). Noninvasive twin zygosity assessment and aneuploidy detection by maternal plasma DNA sequencing. Prenatal Diagnosis, 33, 675681.
Machin, G., (2009). Non-identical monozygotic twins, intermediate twin types, zygosity testing, and the non-random nature of monozygotic twinning: A review. American Journal of Medical Genetics Part C: Seminars in Medical Genetics, 151, 110127.
Mao, J., Wang, T., Wang, B. J., Liu, Y. H., Li, H, Zhang, J., . . . Chen, Y. (2014). Confined placental origin of the circulating cell free fetal DNA revealed by a discordant non-invasive prenatal test result in a trisomy 18 pregnancy. Clinica Chimica Acta, 10, 190193.
Nobili, E., Paramasivam, G., & Kumar, S. (2013). Outcome following selective fetal reduction in monochorionic and dichorionic twin pregnancies discordant for structural chromosomaland genetic disorders. Australian and New Zealand Journal of Obstetrics and Gynaecology, 53, 114118.
Odibo, A. O., Lawrence-Cleary, K., & Macones, G. A. (2003). Screening for aneuploidy in twins and higher-order multiples: Is first-trimester nuchal translucency the solution? Obstetrical & Gynecological Survey, 58, 609614.
Papavassiliou, P., York, T. P., Gursoy, N., Hill, G., Nicely, L. V., Sundaram, U., & Jackson-Cook, C. (2009). The phenotype of persons having mosaicism for trisomy 21/Down syndrome reflects the percentage of trisomic cells present in different tissues. American Journal of Medical Genetics Part A, 149, 573583.
Qu, J. Z., Leung, T. Y., Jiang, P., Liao, G. J., Cheng, Y. K., Sun, H., . . . Lo, Y. M. (2013). Noninvasive prenatal determination of twin zygosity by maternal plasma DNA analysis. Clinical Chemistry, 59, 427435.
Ramsey, K. W., Slavin, T. P., Graham, G., Hirata, G. I., Balaraman, V., & Seaver, L. H. (2012). Monozygotic twins discordant for trisomy 13. Journal of Perinatology, 32, 306308.
Reuss, A., Gerlach, H., Bedow, W., Landt, S., Kuhn, U., Stein, A., . . . Eiben, B. (2011). Monozygotic twins discordant for trisomy 18. Ultrasound in Obstetrics & Gynecology, 38, 727728.
Rosner, M., & Hengstschläger, M. (2013). Amniotic fluid stem cells and fetal cell microchimerism. Trends in Molecular Medicine, 19, 271272.
Sarno, L., Revello, R., Hanson, E., Akolekar, R., & Nicolaides, K. H. (2016). Prospective first-trimester screening for trisomies by cell-free DNA testing of maternal blood in twin pregnancies. Ultrasound in Obstetrics & Gynecology, 47, 705711.
Simonazzi, G., Curti, A., Farina, A., Pilu, G., Bovicelli, L., & Rizzo, N. (2010). Amniocentesis and chorionic villus sampling in twin gestations: Which is the best sampling technique? American Journal of Obstetrics and Gynecology, 202, 365.e1–5.
Spencer, K. (2000). Screening for trisomy 21 in twin pregnancies in the first trimester using free beta-hCG and PAPP-A, combined with fetal nuchal translucency thickness. Prenatal Diagnosis, 20, 9195.
Vink, J., Wapner, R., & D'Alton, M. E. (2012). Prenatal diagnosis in twin gestations. Seminars in Perinatology, 36, 169174.
Wright, D., Kagan, K. O., Molina, F. S., Gazzoni, A., & Nicolaides, K. H. (2008). A mixture model of nuchal translucency thickness in screening for chromosomal defects. Ultrasound in Obstetrics & Gynecology, 31, 376383.



Full text views

Total number of HTML views: 0
Total number of PDF views: 0 *
Loading metrics...

Abstract views

Total abstract views: 0 *
Loading metrics...

* Views captured on Cambridge Core between <date>. This data will be updated every 24 hours.

Usage data cannot currently be displayed