The relationship between clinical-anamnestic data and cell-free fetal DNA level assessed by semiconductor sequencing within non-invasive prenatal testing

Introduction. Currently, non-invasive prenatal testing (NIPT) is widely used to assess a risk of fetal chromosomal anomalies. NIPТ accuracy depends on the cell-free fetal DNA (cffDNA) percentage relative to total cell-free DNA in the pregnant woman's blood (cfDNA fetal fraction, FF). Despite numerous studies, no consensus regarding FF-affecting factors has been reached yet.
Aim: to investigate a relationship between FF and clinical-anamnestic parameters of pregnant women, pregnancy characteristics, and outcomes using the developed NIPТ technology.
Materials and Methods. A prospective observational study was performed by assessing plasma samples from 5459 women with > 9 week-long singleton pregnancies. NIPТ was performed using semiconductor sequencing followed by bioinformatics data processing, including FF determination, according to a previously developed original algorithm.
Results. Median FF was 11.7 [9.5–14.0] %. It was demonstrated that FF depends on blood collection tube type (p < 0.05). FF was found to decrease with woman age and body mass index, and increase with gestational age, elevated early prenatal screening (EPS) biochemical markers – pregnancy-associated plasma protein-A (РАРР-А) and free beta-subunit of human chorionic gonadotropin (β-hCG) levels (p < 0.05). It has been shown that the FF in pregnant women with trisomy 18 is lower than normal (p < 0.05). An increase in FF was observed in pregnant women with fetal congenital anomalies according to ultrasound results (p < 0.05). No association was found between FF and the conception type, first-trimester ultrasound parameters (nuchal translucency, crown-rump length, ultrasound chromosome anomalies markers), fetal trisomy 13 and 21, fetal sex chromosome anomalies, or pregnancy complications – preeclampsia, gestational diabetes, preterm birth, and fetal growth restriction (p > 0.05).
Conclusion. The identified patterns are important to take into consideration while using and interpreting NIPТ.

1. . Sukhikh G.T., Trofimov D.Yu., Barkov I.Yu. et al. Methological recommendations "Conducting Non-invasive prenatal screening (NIPS) for fetal aneuploidies in maternal blood by high-throughput sequencing (NGS)". [Metodicheskie rekomendacii «Provedenie neinvazivnogoprenatal'nogo DNK-skrininga aneuploidij ploda po krovi materi (NIPS) metodom vysokoproizvoditel'nogo sekvenirovaniya»]. Akusherstvo i ginekologiya. 2024;3(Prilozhenie):4–24. (In Russ.). https://doi.org/10.18565/aig.2024.51.

2. Faas B.H., De Ligt J., Janssen I. et al. 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 Suppl 1:S19–26. https://doi.org/10.1517/14712598.2012.670632.

3. Lo Y.M., Zhang J., Leung T.N. et al. Rapid clearance of fetal DNA from maternal plasma. Am J Hum Genet. 1999;64(1):218–24. https://doi.org/10.1086/302205.

4. Rather R.A., Saha S.C. Reappraisal of evolving methods in non-invasive prenatal screening: Discovery, biology and clinical utility. Heliyon. 2023;9(3):e13923. https://doi.org/10.1016/j.heliyon.2023.e13923 .

5. Deng C., Liu S. Factors Affecting the fetal fraction in noninvasive prenatal screening: a review. Front Pediatr. 2022;10:812781. https://doi.org/10.3389/fped.2022.812781.

6. Kozyulina P.Yu., Vashukova E.S., Glotov A.S. et al. Method for non-invasive prenatal screening of fetal aneuploidy. [Sposob neinvazivnogo prenatal'nogo skrininga aneuploidij ploda]. RF patent RU 2712175 C1. 24.01.2020. Bull. No. 3. 16 p. (In Russ.). Available at: https://patents.s3.yandex.net/RU2712175C1_20200124.pdf. [Accessed: 20.05.2024].

7. Tarasenko O.A., Vashukova E.S., Kozyulina P.Yu. et al. Experience of using high-throughput sequencing (NGS) for noninvasive prenatal screening of fetal aneuploidiy at the D.O. Ott Research Institute of Obstetrics, Gynecology and Reproduction. [Opyt primeneniya vysokoproizvoditel'nogo sekvenirovaniya (NGS) dlya provedeniya neinvazivnogo prenatal'nogo skrininga aneuploidij ploda na baze FGBNU «NII AGiR im. D.O. Otta»]. Akusherstvo i ginekologiya. 2022;(10):37–49. (In Russ.). https://doi.org/10.18565/aig.2022.10.37-49.

8. Kondratskaya V.A., Pokrovskaya M.S., Doludin Yu.V. et al. Influence of preanalytical variables on the quality of cell-free DNA. Biobanking of cell-free DNA material. [Vliyanie preanaliticheskih peremennyh na kachestvo vnekletochnoj DNK. Biobankirovanie materiala dlya vydeleniya vnekletochnoj DNK]. Kardiovaskulyarnaya terapiya i profilaktika. 2021;20(8):3114. (In Russ.). https://doi.org/10.15829/1728-8800-2021-3114.

9. Hidestrand M., Stokowski R., Song K.et al. Influence of temperature during transportation on cell-free DNA analysis. Fetal Diagn Ther. 2012;31(2):122–8. https://doi.org/10.1159/000335020.

10. Medina Diaz I., Nocon A., Mehnert D.H. Performance of streck cfDNA blood collection tubes for liquid biopsy testing. PLoS One. 2016;11(11):e0166354. https://doi.org/10.1371/journal.pone.0166354.

11. Sillence K.A., Roberts L.A., Hollands H.J. et al. Fetal sex and RHD genotyping with digital PCR demonstrates greater sensitivity than realtime PCR. Clin Chem. 2015;61(11):1399–407. https://doi.org/10.1373/clinchem.2015.239137.

12. Revello R., Sarno L., Ispas A. et al. Screening for trisomies by cell-free DNA testing of maternal blood: consequences of a failed result. Ultrasound Obstet Gynecol. 2016;47(6):698–704. https://doi.org/10.1002/uog.15851.

13. Hou Y., Yang J., Qi Y.et al. Factors affecting cell-free DNA fetal fraction: statistical analysis of 13,661 maternal plasmas for non-invasive prenatal screening. Hum Genomics. 2019;13(1):62. https://doi.org/10.1186/s40246-019-0244-0.

14. Kudryavtseva E.V., Kovalev V.V., Dektyarev A.A. Fetal fraction of free-DNA: clinical-diagnostic parallels. [Fetal'naya frakciya free-DNA: klinikodiagnosticheskie paralleli]. Ural'skij medicinskij zhurnal. 2021;20(1):30–5. (In Russ.). https://doi.org/10.52420/2071-5943-2021-20-1-30-35.

15. Deng C., Liu J., Liu S. et al. Maternal and fetal factors influencing fetal fraction: A retrospective analysis of 153,306 pregnant women undergoing noninvasive prenatal screening. Front Pediatr. 2023;11:1066178. https://doi.org/10.3389/fped.2023.1066178.

16. Brar H., Wang E., Struble C. et al. The fetal fraction of cell-free DNA in maternal plasma is not affected by a priori risk of fetal trisomy. J Matern Fetal Neonatal Med. 2013;26(2):143–5. https://doi.org/10.3109/14767058.2012.722731.

17. Hudecova I., Sahota D., Heung M.M. et al. Maternal plasma fetal DNA fractions in pregnancies with low and high risks for fetal chromosomal aneuploidies. PLoS One. 2014;9(2):e88484. https://doi.org/10.1371/journal.pone.0088484.

18. Qiao L., Zhang Q., Liang Y.et al. Sequencing of short cfDNA fragments in NIPT improves fetal fraction with higher maternal BMI and early gestational age. Am J Transl Res. 2019;11(7):4450–9.

19. Guo F.-F., Yang J.-X., Huang Y.-L. et al. Association between fetal fraction at the second trimester and subsequent spontaneous preterm birth. Prenat Diagn. 2019;39(13):1191–7. https://doi.org/10.1002/pd.5566.

20. Talbot A.L., Ambye L., Hartwig T.S. et al. Fetal fraction of cell-free DNA in pregnancies after fresh or frozen embryo transfer following assisted reproductive technologies. Hum Reprod. 2020;35(6):1267–75. https://doi.org/10.1093/humrep/deaa110.

21. Haghiac M., Vora N.L., Basu S. et al. Increased death of adipose cells, a path to release cell-free DNA into systemic circulation of obese women. Obesity (Silver Spring). 2012;20(11):2213–9. https://doi.org/10.1038/oby.2012.138.

22. Lee T.J., Rolnik D.L., Menezes M.A. et al. Cell-free fetal DNA testing in singleton IVF conceptions. Hum Reprod. 2018;33(4):572–8. https://doi.org/10.1093/humrep/dey033.

23. Quezada M.S., 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(1):101–5. https://doi.org/10.1002/uog.14666.

24. Poon L.C., Musci T., Song K. et al. 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(4):215–23. https://doi.org/10.1159/000346806.

25. Lambert-Messerlian G., Kloza E.M., Williams J. et al. Maternal plasma DNA testing for aneuploidy in pregnancies achieved by assisted reproductive technologies. Genet Med. 2014;16(5):419–22. https://doi.org/10.1038/gim.2013.149.

26. Qiao L., Yu B., Liang Y. et al. Sequencing shorter cfDNA fragments improves the fetal DNA fraction in noninvasive prenatal testing. Am J Obstet Gynecol. 2019;221(4):345.e1–345.e11. https://doi.org/10.1016/j.ajog.2019.05.023.

27. Kinnings S.L., Geis J.A., 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(8):816–22. https://doi.org/10.1002/pd.4625.

28. Song Y., Zhou X., Huang S. et al. Quantitation of fetal DNA fraction in maternal plasma using circulating single molecule amplification and re-sequencing technology (cSMART). Clin Chim Acta. 2016;456:151–6. https://doi.org/10.1016/j.cca.2016.03.005.

29. Scott F.P., Menezes M., Palma-Dias R. et al. Factors affecting cell-free DNA fetal fraction and the consequences for test accuracy. J Matern Fetal Neonatal Med. 2018;31(14):1865–72. https://doi.org/10.1080/14767058.2017.1330881.

30. Miltoft C.B., Rode L., Ekelund C.K. et al. Contingent first-trimester screening for aneuploidies with cell-free DNA in a Danish clinical setting. Ultrasound Obstet Gynecol. 2018;51(4):470–9. https://doi.org/10.1002/uog.17562.

31. Manokhina I., Singh T.K., Robinson W.P. Cell-free placental DNA in maternal plasma in relation to placental health and function. Fetal Diagn Ther. 2017;41(4):258–64. https://doi.org/10.1159/000448707.

32. Metzenbauer M., Hafner E., Hoefinger D. et al. Three-dimensional ultrasound measurement of the placental volume in early pregnancy: method and correlation with biochemical placenta parameters. Placenta. 2001;22(6):602–5. https://doi.org/10.1053/plac.2001.0684.

33. Palomaki G.E., Kloza E.M., Lambert-Messerlian G.M. et al. Circulating cell free DNA testing: are some test failures informative? Prenat Diagn. 2015;35(3):289–93. https://doi.org/10.1097/01.ogx.0000470818.76451.

34. Suzumori N., Ebara T., Yamada T. et al. Fetal cell-free DNA fraction in maternal plasma is affected by fetal trisomy. J Hum Genet. 2016;61(7):647–52. https://doi.org/10.1038/jhg.2016.25.

35. Rava R.P., Srinivasan A., Sehnert A.J. et al. Circulating fetal cell-free DNA fractions differ in autosomal aneuploidies and monosomy X. Clin Chem. 2014;60:243–50. https://doi.org/10.1373/clinchem.2013.207951.

36. Krishna I., Badell M., Loucks T.L. et al. Adverse perinatal outcomes are more frequent in pregnancies with a low fetal fraction result on noninvasive prenatal testing. Prenat Diagn. 2016;36(3):210–5. https://doi.org/10.1002/pd.4779.

37. Kudryavtseva E.V., Kovalev V.V., Kanivets I.V. et al. Cell-free-fetal DNA: an experience of population screening fjr chromosome pathology in Russia. [Free-DNA ploda: opyt populyacionnogo skrininga hromosomnoj patologii v Rossii]. Voprosy ginekologii, akusherstva i perinatologii. 2019;18(3):46–51. (In Russ.). https://doi.org/10.20953/1726-1678-2019-3-46-51.

38. Arizawa M., Nakayama M. Pathological analysis of the placenta in trisomies 21, 18 and 13. Nihon Sanka Fujinka Gakkai Zasshi. 1992;44(1):9–13. [In Japanese].

39. Chen C.P. Placental abnormalities and preeclampsia in trisomy 13 pregnancies. Taiwan J Obstet Gynecol. 2009;48(1):3–8. https://doi.org/10.1016/S1028-4559(09)60028-0.

40. Becking E.C., Wirjosoekarto S.A.M., Scheffer P.G. et al. Low fetal fraction in cell-free DNA testing is associated with adverse pregnancy outcome: Analysis of a subcohort of the TRIDENT-2 study. Prenat Diagn. 2021;41(10):1296–304. https://doi.org/10.1002/pd.6034.

41. Becking E.C., Scheffer P.G., Henrichs J. et al. Fetal fraction of cell-free DNA in noninvasive prenatal testing and adverse pregnancy outcomes: a nationwide retrospective cohort study of 56,110 pregnant women. Am J Obstet Gynecol. 2023;S0002-9378(23)02128-2. https://doi.org/10.1016/j.ajog.2023.12.008.

42. Bardi F., Bet B.B., Pajkrt E. et al. Additional value of advanced ultrasonography in pregnancies with two inconclusive cell-free DNA draws. Prenat Diagn. 2022;42(11):1358–67. https://doi.org/10.1002/pd.6238.

43. Parsadanyan N.G., Shubina E.S., Tetruashvili N.K. et al. Free embryonic DNA levels in a patient with recurrent miscarriage and severe placental insufficiency. [Uroven' svobodnoj embrional'noj DNK u pacientki s privychnym vykidyshem i tyazheloj placentarnoj nedostatochnost'yu]. Akusherstvo i ginekologiya. 2015;(4):90–4. (In Russ.).

44. Reus A.D., Stephenson M.D., van Dunné F.M. et al. Chorionic villous vascularization related to phenotype and genotype in first trimester miscarriages in a recurrent pregnancy loss cohort. Hum Reprod. 2013;28(4):916–23. https://doi.org/10.1093/humrep/det025.

45. Gracheva M.I., Kan N.E., Krasniy A.M. Role of cell-free fetal DNA in early diagnosis of pregnancy complications. [Rol' vnekletochnoj fetal'noj DNK v rannej diagnostike oslozhnenij beremennosti]. Akusherstvo i ginekologiya. 2016;(10):5–10. (In Russ.). https://doi.org/10.18565/aig.2016.10.5-10.