EC Gynaecology

Background: Non-invasive prenatal testing (NIPT) for fetal aneuploidies using cell-free DNA (cfDNA) which can be isolated from mothers’ plasma has been widely adopted in clinical practice due to its improved accuracy.

Objective: The core goal of cell-free based prenatal testing is to provide minimally invasive, clinically accurate screening for fetal chromosomal aneuploidies in the early stages of pregnancy.

The purpose of this study was to establish a validated NIPT workflow for cell-free fetal DNA (cffDNA) sequencing from maternal plasma for the detection of trisomy 13, 18, 21 and sex chromosomal aneuploidies (SCA) on a semiconductor sequencing instrument.

Method: A total of ninety-one plasma samples from healthy pregnant provided by Yourgene Health; their cfDNA library was prepared and loaded on the Ion 540 chip for sequencing. The sequencing output data was analysed by using the bioinformatics pipeline of Yourgene Health.

Results: Eighty-one samples were successfully validated out of a total of 91 samples. However, 3% of samples did not meet quality, and 3% of sample libraries had low reads and failed validation. Moreover, building the validated workflow based on Ion Torrent Next-Generation Sequencing (NGS) would allow for fetal aneuploidy detection.

Conclusion: This research aims to introduce and set up probably Nepal's first NGS laboratory for NIPT, utilizing the Ion Torrent technology to provide in-house diagnostic solution for pregnant women.

 Keywords: Aneuploidies; Cell-Free DNA (cfDNA); Cell-Free Fetal DNA (cffDNA); Maternal Blood; Non-Invasive Prenatal Genetic Testing (NIPT); Prenatal Testing

1. Bedei I., et al. “Chances and challenges of new genetic screening technologies (NIPT) in prenatal medicine from a clinical perspective: A narrative review”. Genes (Basel)4 (2021): 501.
2. McLennan A., et al. “Noninvasive prenatal testing in routine clinical practice - An audit of NIPT and combined first-trimester screening in an unselected Australian population”. Australian and New Zealand Journal of Obstetrics and Gynaecology1 (2016): 22-28.
3. Taglauer ES., et al. “Review: Cell-free fetal DNA in the maternal circulation as an indication of placental health and disease”. Placenta 35 (2014): 64-68.
4. Pös O., et al. “Recent trends in prenatal genetic screening and testing”. F1000 Research 8 (2019): 1-10.
5. Carlson LM and Vora NL. “Prenatal diagnosis: Screening and diagnostic tools”. Obstetrics and Gynecology Clinics of North America2 (2017): 245-256.
6. Manegold-Brauer G and Lapaire O. “Clinical implementation of next-generation sequencing in the field of prenatal diagnostics”. Next Generation Sequencing - Advances, Applications and Challenges. InTech (2016).
7. Xu XP., et al. “A method to quantify cell-free fetal DNA fraction in maternal plasma using next generation sequencing: Its application in non-invasive prenatal chromosomal aneuploidy detection”. PLoS One1 (2016): e0146997.
8. Norwitz ER and Levy B. “Noninvasive prenatal testing: the future is now”. Reviews in Obstetrics and Gynecology 2 (2013): 48-62.
9. Taylor-Phillips S., et al. “Accuracy of non-invasive prenatal testing using cell-free DNA for detection of Down, Edwards and Patau syndromes: A systematic review and meta-analysis”. BMJ Open 6 (2016): e010002.
10. Swanson A., et al. “Non-invasive prenatal testing: technologies, clinical assays and implementation strategies for women’s healthcare practitioners”. Current Genetic Medicine Reports 2 (2013): 113-121.
11. Chang BC., et al. “Setting up of first NIPT service laboratory in a Thailand hospital”. Journal of Next Generation Sequencing and Applications 03 (2016): 3.
12. Pappachen DM. “54. Implementation of first NIPT service laboratory in an Indian hospital”. Reproductive BioMedicine Online 1 (2019): e59-e60.
13. Xue Y., et al. “Non-invasive prenatal testing to detect chromosome aneuploidies in 57,204 pregnancies”. Molecular Cytogenetics 12 (2019): 29.
14. Minarik G., et al. “Utilization of benchtop next generation sequencing platforms ion torrent PGM and miseq in noninvasive prenatal testing for chromosome 21 trisomy and testing of impact of in silico and physical size selection on its analytical performance”. PLoS One12 (2015): e0144811.
15. Meena N., et al. “A bioinformatics pipeline for whole exome sequencing: Overview of the processing and steps from raw data to downstream analysis”. Bio-Protocol 8 (2018).
16. Chia-Han Chan. “Detecting chromosomal aneuploidy united states patent application 20160026759”. Taiwan: United States Patent (2016).
17. Hartwig TS., et al. “Non-invasive prenatal testing (NIPT) in pregnancies with trisomy 21, 18 and 13 performed in a public setting - factors of importance for correct interpretation of results”. European Journal of Obstetrics and Gynecology and Reproductive Biology 226 (2018): 35-39.
18. Andari VCM., et al. “Noninvasive prenatal testing: Benefits and limitations of the available tests”. Ceska Gynekologie 1 (2020): 41-48.
19. Hartwig TS., et al. “Discordant non-invasive prenatal testing (NIPT) - a systematic review”. Prenatal Diagnosis 6 (2017): 527-539.
20. Allahbadia GN. “Has noninvasive prenatal testing (NIPT) come of age?” Journal of Obstetrics and Gynecology of India 3 (2015): 141-145.
21. Hui L. “Noninvasive prenatal testing for aneuploidy using cell-free DNA - New implications for maternal health”. Obstetric Medicine 4 (2016): 148-152.
22. Idler J., et al. “Pregnancy after bone marrow transplantation for aplastic anemia [37Q]”. Obstetrics and Gynecology 131 (2018): 194S-194S.
23. Kotsopoulou I., et al. “Non-invasive prenatal testing (NIPT): Limitations on the way to become diagnosis”. Diagnosis 2 (2015): 141-158.