Skip to main content
SpringerLink
Log in

A prospective study of non-invasive preimplantation genetic testing for aneuploidies (NiPGT-A) using next-generation sequencing (NGS) on spent culture media (SCM)

  • Assisted Reproduction Technologies
  • Published:
Journal of Assisted Reproduction and Genetics Aims and scope Submit manuscript

Abstract

Purpose

This study was to evaluate if spent culture media (SCM) of embryos could be used as a non-invasive tool to achieve aneuploidy screening. Ploidy calls, as well as concordance rates between PGT-A results from trophectoderm (TE) and SCM, were compared. Clinical outcomes of single euploid transfers were also evaluated.

Methods

The study was conducted from March 2017 to June 2018 in a university-based ART center. SCM of day 3 to the day(s) of TE biopsy of all biopsied blastocysts were collected for testing. PGT-A results of SCM were compared with the standard results of TE, with clinical relevance and outcomes examined.

Results

NiPGT-A using SCM gave a sensitivity of 81.6%, specificity of 48.3%, positive predictive value of 82.6%, and negative predictive value of 46.7% in ploidy calling. The concordance rates for autosomes and sex determination were 62.1% and 82.4%, respectively. There were 14 single embryo transfer cycles of euploids as determined by TE biopsy. Clinical outcomes not only confirmed 3 false positive results from SCM but also reflected the true ploidy status of the transferred embryo in one case. If ploidy calls were dichotomized without mosaic embryos, the sensitivity and NPV would increase to 91.0% and 66.7% (p = 0.60 and p = 0.25), respectively.

Conclusions

Cell-free DNA found in SCM could provide ploidy information of an embryo as in PGT-A from its TE. Given its potential to reflect the comprehensive chromosomal profile of the whole embryo, more research based on clinical outcomes is required to determine if SCM could be a reliable selection tool in PGT-A.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2

Similar content being viewed by others

References

  1. Ferraretti AP, Nygren K, Andersen AN, de Mouzon J, Kupka M, Calhaz-Jorge C, et al. Trends over 15 years in ART in Europe: an analysis of 6 million cycles. Hum Reprod. 2017;2017(2):hox012. Available from. https://doi.org/10.1093/hropen/hox012.

    Article  Google Scholar 

  2. Human Fertilisation & Embryology Authority. Fertility treatment 2014–2016 trends and figures. HFEA, March 2018. Available from, https://www.hfea.gov.uk/about-us/publications/research-and-data/

  3. Division of Reproductive Health, Centers for Disease Control and Prevention. 2015 Assisted reproductive technology fertility clinic success rates report. CDC, October 2017. Available from: https://www.cdc.gov/art/reports/2015/fertility-clinic.html.

  4. Baltaci V, Satiroglu H, Kabukçu C, Unsal E, Aydinuraz B, Uner O, et al. Relationship between embryo quality and aneuploidies. Reprod BioMed Online. 2006;12(1):77–82.

    Article  PubMed  CAS  Google Scholar 

  5. Capalbo A, Rienzi L, Cimadomo D, Maggiulli R, Elliott T, Wright G, et al. Correlation between standard blastocyst morphology, euploidy and implantation: an observational study in two centers involving 956 screened blastocysts. Hum Reprod. 2014;29(6):1173–81.

    Article  PubMed  Google Scholar 

  6. Kuliev A, Cieslak J, Ilkevitch Y, Verlinsky Y. Chromosomal abnormalities in a series of 6,733 human oocytes in preimplantation diagnosis for age-related aneuploidies. Reprod BioMed Online. 2003;6(1):54–9.

    Article  PubMed  Google Scholar 

  7. Baart EB, Martini E, Eijkemans MJ, Van Opstal D, Beckers NGM, Verhoeff A, et al. Milder ovarian stimulation for in-vitro fertilization reduces aneuploidy in the human preimplantation embryo: a randomized controlled trial. Hum Reprod. 2007;22(4):980–8.

    Article  PubMed  Google Scholar 

  8. Massie JAM, Shahine LK, Milki AA, Westphal LM, Lathi RB. Ovarian stimulation and the risk of aneuploid conceptions. Fertil Steril. 2011;95(3):970–2.

    Article  PubMed  Google Scholar 

  9. Ata B, Kaplan B, Danzer H, Glassner M, Opsahl M, Tan SL, et al. Array CGH analysis shows that aneuploidy is not related to the number of embryos generated. Reprod BioMed Online. 2012;24(6):614–20.

    Article  PubMed  CAS  Google Scholar 

  10. Sekhon L, Shaia K, Santistevan A, Cohn KH, Lee JA, Beim PY, et al. The cumulative dose of gonadotropins used for controlled ovarian stimulation does not influence the odds of embryonic aneuploidy in patients with normal ovarian response. J Assist Reprod Genet. 2017;34(6):749–58.

    Article  PubMed  PubMed Central  Google Scholar 

  11. Yang Z, Liu J, Collins GS, Salem SA, Liu X, Lyle SS, et al. Selection of single blastocysts for fresh transfer via standard morphology assessment alone and with array CGH for good prognosis IVF patients: results from a randomized pilot study. Mol Cytogenet. 2012;5(1):24.

    Article  PubMed  PubMed Central  Google Scholar 

  12. Forman EJ, Hong KH, Ferry KM, Tao X, Taylor D, Levy B, et al. In vitro fertilization with single euploid blastocyst transfer: a randomized controlled trial. Fertil Steril. 2013;100(1):100–7.e1.

    Article  PubMed  Google Scholar 

  13. Forman EJ, Hong KH, Franasiak JM, Scott J, Richard T. Obstetrical and neonatal outcomes from the BEST Trial: single embryo transfer with aneuploidy screening improves outcomes after in vitro fertilization without compromising delivery rates. Am J Obstet Gynecol. 2014;210(2):157.e1–6.

    Article  Google Scholar 

  14. Scott RT, Upham KM, Forman EJ, Zhao T, Treff NR. Cleavage-stage biopsy significantly impairs human embryonic implantation potential while blastocyst biopsy does not: a randomized and paired clinical trial. Fertil Steril. 2013;100(3):624–30.

    Article  PubMed  Google Scholar 

  15. Liu J, Wang W, Sun X, Liu L, Jin H, Li M, et al. DNA microarray reveals that high proportions of human blastocysts from women of advanced maternal age are aneuploid and mosaic. Biol Reprod. 2012;87(6):148.

    Article  PubMed  CAS  Google Scholar 

  16. Popovic M, Dheedene A, Christodoulou C, Taelman J, Dhaenens L, Van Nieuwerburgh F, et al. Chromosomal mosaicism in human blastocysts: the ultimate challenge of preimplantation genetic testing? Hum Reprod. 2018 33(7):1342–1354.

  17. Capalbo A, Wright G, Elliott T, Ubaldi FM, Rienzi L, Nagy ZP. FISH reanalysis of inner cell mass and trophectoderm samples of previously array-CGH screened blastocysts shows high accuracy of diagnosis and no major diagnostic impact of mosaicism at the blastocyst stage. Hum Reprod. 2013;28(8):2298–307.

    Article  PubMed  CAS  Google Scholar 

  18. Huang J, Yan L, Lu S, Zhao N, Qiao J. Re-analysis of aneuploidy blastocysts with an inner cell mass and different regional trophectoderm cells. J Assist Reprod Genet. 2017;34(4):487–93.

    Article  PubMed  PubMed Central  Google Scholar 

  19. Palini S, Galluzzi L, De Stefani S, Bianchi M, Wells D, Magnani M, et al. Genomic DNA in human blastocoele fluid. Reprod BioMed Online. 2013;26(6):603–10.

    Article  PubMed  CAS  Google Scholar 

  20. Gianaroli L, Magli MC, Pomante A, Crivello AM, Cafueri G, Valerio M, et al. Blastocentesis: a source of DNA for preimplantation genetic testing. Results from a pilot study. Fertil Steril. 2014;102(6):1692–9 e6.

    Article  PubMed  CAS  Google Scholar 

  21. Tobler KJ, Zhao Y, Ross R, Benner AT, Xu X, Du L, et al. Blastocoel fluid from differentiated blastocysts harbors embryonic genomic material capable of a whole-genome deoxyribonucleic acid amplification and comprehensive chromosome microarray analysis. Fertil Steril. 2015;104(2):418–25.

    Article  PubMed  CAS  Google Scholar 

  22. Zhang Y, Li N, Wang L, Sun H, Ma M, Wang H, et al. Molecular analysis of DNA in blastocoele fluid using next-generation sequencing. J Assist Reprod Genet. 2016;33(5):637–45.

    Article  PubMed  PubMed Central  Google Scholar 

  23. Assou S, Aït-Ahmed O, El Messaoudi S, Thierry AR, Hamamah S. Non-invasive pre-implantation genetic diagnosis of X-linked disorders. Med Hypotheses. 2014;83(4):506–8.

    Article  PubMed  CAS  Google Scholar 

  24. Wu H, Ding C, Shen X, Wang J, Li R, Cai B, et al. Medium-based noninvasive preimplantation genetic diagnosis for human α-thalassemias-SEA. Medicine (Baltimore). 2015;94(12):e669.

    Article  CAS  Google Scholar 

  25. Shamonki MI, Jin H, Haimowitz Z, Liu L. Proof of concept: preimplantation genetic screening without embryo biopsy through analysis of cell-free DNA in spent embryo culture media. Fertil Steril. 2016;106(6):1312–8.

    Article  PubMed  CAS  Google Scholar 

  26. Xu J, Fang R, Chen L, Chen D, Xiao JP, Yang W, et al. Noninvasive chromosome screening of human embryos by genome sequencing of embryo culture medium for in vitro fertilization. Proc Natl Acad Sci U S A. 2016;113(42):11907–12.

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  27. Vera-Rodriguez M, Diez-Juan A, Jimenez-Almazan J, Martinez S, Navarro R, Peinado V, et al. Origin and composition of cell-free DNA in spent medium from human embryo culture during preimplantation development. Hum Reprod. 2018;33(4):745–56.

    Article  PubMed  CAS  Google Scholar 

  28. Ho JR, Arrach N, Rhodes-Long K, Ahmady A, Ingles S, Chung K, et al. Pushing the limits of detection: investigation of cell-free DNA for aneuploidy screening in embryos. Fertil Steril. 2018;110(3):467–75 e2.

    Article  PubMed  CAS  Google Scholar 

  29. Saravelos SH, Wong AWY, Chan CPS, Kong GWS, Cheung LP, Chung CHS, et al. Assessment of the embryo flash position and migration with 3D ultrasound within 60 min of embryo transfer. Hum Reprod. 2016;31(3):591–6.

    Article  PubMed  Google Scholar 

  30. Gardner DK, Schoolcraft WB. Culture and transfer of human blastocysts. Curr Opin Obstet Gynecol. 1999;11(3):307–11.

    Article  PubMed  CAS  Google Scholar 

  31. Heitmann RJ, Hill MJ, Richter KS, DeCherney AH, Widra EA. The simplified SART embryo scoring system is highly correlated to implantation and live birth in single blastocyst transfers. J Assist Reprod Genet. 2013;30(4):563–7.

    Article  PubMed  PubMed Central  Google Scholar 

  32. Choy KW, Kwok YK, Cheng YKY, Wong KM, Wong HK, Leung KO, et al. Diagnostic accuracy of the BACs-on-Beads™ assay versus karyotyping for prenatal detection of chromosomal abnormalities: a retrospective consecutive case series. BJOG. 2014;121(10):1245–52.

    Article  PubMed  CAS  Google Scholar 

  33. Scott RTJ, Upham KM, Forman EJ, Hong KH, Scott KL, Taylor D, et al. Blastocyst biopsy with comprehensive chromosome screening and fresh embryo transfer significantly increases in vitro fertilization implantation and delivery rates: a randomized controlled trial. Fertil Steril. 2013;100(3):697–703.

    Article  PubMed  Google Scholar 

  34. Munne S, Kaplan B, Frattarelli JL, Gysler M, Child TJ, Nakhuda G, et al. Global multicenter randomized controlled trial comparing single embryo transfer with embryo selected by preimplantation genetic screening using next-generation sequencing versus morphologic assessment. Fertil Steril. 2017;108(3):e19.

    Article  Google Scholar 

  35. Huang L, Ma F, Chapman A, Lu S, Xie XS. Single-cell whole-genome amplification and sequencing: methodology and applications. Annu Rev Genomics Hum Genet. 2015;16:79–102.

    Article  PubMed  CAS  Google Scholar 

  36. Lane M, Zander-Fox DL, Hamilton H, Jasper MJ, Hodgson BL, Fraser M, et al. Ability to detect aneuploidy from cell free DNA collected from media is dependent on the stage of development of the embryo. Fertil Steril. 2017;108(3):e61.

    Article  Google Scholar 

  37. Hammond ER, Shelling AN, Cree LM. Nuclear and mitochondrial DNA in blastocoele fluid and embryo culture medium: evidence and potential clinical use. Hum Reprod. 2016;31(8):1653–61.

    Article  PubMed  CAS  Google Scholar 

  38. Greco E, Minasi MG, Fiorentino F. Healthy babies after intrauterine transfer of mosaic aneuploid blastocysts. N Engl J Med. 2015;373(21):2089–90.

    Article  PubMed  Google Scholar 

  39. Munné S, Wells D. Detection of mosaicism at blastocyst stage with the use of high-resolution next-generation sequencing. Fertil Steril. 2017;107(5):1085–91.

    Article  PubMed  Google Scholar 

  40. Munné S, Blazek J, Large M, Martinez-Ortiz PA, Nisson H, Liu E, et al. Detailed investigation into the cytogenetic constitution and pregnancy outcome of replacing mosaic blastocysts detected with the use of high-resolution next-generation sequencing. Fertil Steril. 2017;108(1):62–71.e8.

    Article  PubMed  CAS  Google Scholar 

  41. Harper JC, Coonen E, Handyside AH, Winston RM, Hopman AH, Delhanty JD. Mosaicism of autosomes and sex chromosomes in morphologically normal, monospermic preimplantation human embryos. Prenat Diagn. 1995;15(1):41–9.

    Article  PubMed  CAS  Google Scholar 

  42. Delhanty JD, Harper JC, Ao A, Handyside AH, Winston RM. Multicolour FISH detects frequent chromosomal mosaicism and chaotic division in normal preimplantation embryos from fertile patients. Hum Genet. 1997;99(6):755–60.

    Article  PubMed  CAS  Google Scholar 

  43. Magli MC, Jones GM, Gras L, Gianaroli L, Korman I, Trounson AO. Chromosome mosaicism in day 3 aneuploid embryos that develop to morphologically normal blastocysts in vitro. Hum Reprod. 2000;15(8):1781–6.

    Article  PubMed  CAS  Google Scholar 

Download references

Funding

This research was funded by General Research Fund, ref no. 14162417, Research Grant Council (RGC) of Hong Kong, China.

Author information

Author notes

  1. Queenie S. Y. Yeung, Ying Xin Zhang and Jacqueline P. W. Chung contributed equally to this work.

Authors and Affiliations

  1. Assisted Reproductive Technology Unit, Department of Obstetrics and Gynaecology, Faculty of Medicine, The Chinese University of Hong Kong, Hong Kong SAR, China

    Queenie S. Y. Yeung, Jacqueline P. W. Chung, Grace W. S. Kong & Tin Chiu Li

  2. Department of Obstetrics and Gynaecology, The Chinese University of Hong Kong, Hong Kong SAR, China

    Ying Xin Zhang, Wai Ting Lui, Yvonne K. Y. Kwok, Ye Cao & Kwong Wai Choy

  3. Shenzhen Research Institute, The Chinese University of Hong Kong, Shenzhen, 518055, China

    Baoheng Gui & Kwong Wai Choy

  4. Department of Genetics and Metabolism, Maternal and Child Health Hospital of Guangxi Zhuang Autonomous Region, Nanning, 530002, Guangxi, China

    Baoheng Gui

Authors
  1. Queenie S. Y. Yeung
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Ying Xin Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Jacqueline P. W. Chung
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Wai Ting Lui
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Yvonne K. Y. Kwok
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Baoheng Gui
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Grace W. S. Kong
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Ye Cao
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Tin Chiu Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Kwong Wai Choy
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Kwong Wai Choy.

Ethics declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

All procedures performed in this study involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.

Informed consent

Informed consent was obtained from all individual participants included in the study.

Additional information

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Electronic supplementary material

ESM 1

(DOCX 22 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Yeung, Q.S.Y., Zhang, Y.X., Chung, J.P.W. et al. A prospective study of non-invasive preimplantation genetic testing for aneuploidies (NiPGT-A) using next-generation sequencing (NGS) on spent culture media (SCM). J Assist Reprod Genet 36, 1609–1621 (2019). https://doi.org/10.1007/s10815-019-01517-7

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10815-019-01517-7

Keywords

Search

Navigation