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Less-invasive chromosome screening of embryos and embryo assessment by genetic studies of DNA in embryo culture medium

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

Abstract

Purpose

To perform a preliminary exploration of a new embryo rank in clinical practice by combining the embryo chromosome copy number and mitochondrial copy number analysis of DNA extracted from embryo culture medium and blastocoel fluid.

Method

Eighty-three ICSI embryos from day 2 or day 3 were cultured to day 5 or day 6. Thirty-two blastocysts of 3 cc or above were obtained. Culture medium and blastocoel fluid were collected at 24 h before blastocyst formation. The genomic DNA and mitochondrial DNA (mtDNA) from the culture medium combined with blastocoel fluid and the whole blastocyst were amplified and sequenced by MALBAC-NGS. We compared the chromosomal information generated by the new protocol from the culture medium and the information employed by the whole embryo method. A multivariable linear regression was performed to study the impact of the blastocyst morphological score, chromosomal abnormality, embryo mtDNA copy number, and female age on the culture medium mtDNA copy number.

Results

(1) The DNA from 31 blastocysts was successfully amplified, and the successful amplification rate was 96.9% (31/32). The success rate of the amplification of genomic DNA extracted from the culture medium was 87.5% (28/32). (2) There were 18 blastocysts in which the less invasive method and the whole embryo method revealed the same results. The consistency rate was 66.7% (18/27). (3) The culture medium mitochondrial DNA copy number (MCN) had a significantly positive correlation with the blastocyst mitochondrial DNA copy number (P = 0.001), female age (P = 0.012), and blastocyst score (P = 0.014), but there was no obvious correlation with blastocyst chromosome (P = 0.138).

Conclusions

The preliminary exploration result of the less invasive approach for having an embryo rank was not satisfying, which still awaits further long-term evaluation.

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References

  1. Kushnir VA, Darmon SK, Albertini DF, et al. Effectiveness of in vitro fertilization with preimplantation genetic screening: a reanalysis of United States assisted reproductive technology data 2011-2012 [J]. Fertil Steril. 2016;106(1):75–9.

    Article  Google Scholar 

  2. Schoolcraft WB, Treff NR, Stevens JM, et al. Live birth outcome with trophectoderm biopsy, blastocyst vitrification, and single-nucleotide polymorphism microarray-based comprehensive chromosome screening in infertile patients [J]. Fertil Steril. 2011;96(3):638–40.

    Article  Google Scholar 

  3. Scottrt JR, Upham KM, Forman EJ, 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 [J]. Fertil Steril. 2013;100(3):697–703.

    Article  Google Scholar 

  4. Tobler KJ, Brezina PR, Benner AT, du L, Xu X, Kearns WG. Two different microarray technologies for preimplantation genetic diagnosis and screening, due to reciprocal translocation imbalances, demonstrate equivalent euploidy and clinical pregnancy rates [J]. J Assist Reprod Genet. 2014;31(7):843–50.

    Article  Google Scholar 

  5. Caglar GS, Asimakopoulos B, Nikolettos N, et al. Preimplantation genetic diagnosis for aneuploidy screening in repeated implantation failure[J]. Reprod BioMed Online. 2005;10(3):381–8.

    Article  Google Scholar 

  6. Reisesilva AR, Bruno C, Fleurot R, et al. Alteration of DNA demethylation dynamics by in vitro culture conditions in rabbit pre-implantation embryos [J]. Epigenetics. 2012;7(5):440–6.

    Article  CAS  Google Scholar 

  7. Frenandez-Gonzalez R, Ramirez MA, Pericuesta E, et al. Histone modifications at the blastocyst Axin1(Fu) locus mark the heritability of in vitro culture-induced epigenetic alterations in mice [J]. Biol Reprod. 2010;83(5):720–7.

    Article  Google Scholar 

  8. Market-Velker BA, Fernandes AD, Mann MR. Side-by-side comparison of five commercial media systems in a mouse model: suboptimal in vitro culture interferes with imprint maintenance [J]. Biol Reprod. 2010;83(6):938–50.

    Article  CAS  Google Scholar 

  9. Yu Y, Zhao Y, Li R, Li L, Zhao H, Li M, et al. Assessment of the risk of blastomere biopsy during preimplantation genetic diagnosis in a mouse model: reducing female ovary function with an increase in age by proteomics method [J]. J Proteome Res. 2013;12(12):5475–86.

    Article  CAS  Google Scholar 

  10. Zhao HC, Zhao Y, Li M, et al. Aberrant epigenetic modification in murine brain tissues of offspring from preimplantation genetic diagnosis blastomere biopsies [J]. Biol Reprod. 2013;89(5):117.

    Article  Google Scholar 

  11. Dawson A, Griesinger G, Diedrich K. Screening oocytes by polar body biopsy[J]. Reprod BioMed Online. 2006;13:104–9.

    Article  CAS  Google Scholar 

  12. Palini S, Galluzzi L, de Stefani S, Bianchi M, Wells D, Magnani M, et al. Genomic DNA in human blastocoele fluid - Reproductive BioMedicine Online. Reprod BioMed Online. 2013;26:603–10.

    Article  CAS  Google Scholar 

  13. Hammond ER, McGillivray BC, Wicker SM, et al. Characterizing nuclear and mitochondrial DNA in spent embryo culture media: genetic contamination identified [J]. Fertil Steril. 2017;107(1):220–228.e5.

    Article  CAS  Google Scholar 

  14. Stigliani S, Anserini P, Venturini PL, Scaruffi P. Mitochondrial DNA content in embryo culture medium is significantly associated with human embryo fragmentation [J]. Hum Reprod. 2013;28(10):2652–60.

    Article  CAS  Google Scholar 

  15. Gianaroli L, et al. Blastocentesis: a source of DNA for preimplantation genetic testing. Results from a pilot study. Fertil Steril. 2014;102:1692–9.

    Article  CAS  Google Scholar 

  16. Gardner DK, Schoolcraft WB. Culture and transfer of human blastocysts. Curr Opin Obstet Gynecol. 1999;11:307.

    Article  CAS  Google Scholar 

  17. Gardner DK, Lane M, Stevens J, Schoolcraft WB. Changing the start temperature and cooling rate in a slow-freezing protocol increases human blastocyst viability. Fertil Steril. 2003;79:407–10.

    Article  Google Scholar 

  18. Zong C, Lu S, Chapman AR, Xie XS. Genome-wide detection of single-nucleotide and copy-number variations of a single human cell. Science. 2012;338:1622–6.

    Article  CAS  Google Scholar 

  19. Belandres D, Shamonki M, Arrach N. Current status of spent embryo media research for preimplantation genetic testing. J Assist Reprod Genet. 2019;36:819–26. https://doi.org/10.1007/s10815-019-01437-6.

    Article  PubMed  Google Scholar 

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

    Article  CAS  Google Scholar 

  21. 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 [J]. Fertil Steril. 2014;102(6):1692–9 e1696.

    Article  CAS  Google Scholar 

  22. 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.

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  24. Feichtinger M, Vaccari E, Carli L, Wallner E, Mädel U, Figl K, et al. Non-invasive preimplantation genetic screening using array comparative genomic hybridization on spent culture media: a proof-of-concept pilot study. Reprod BioMed Online. 2017;34(6):583–9.

    Article  Google Scholar 

  25. 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  CAS  Google Scholar 

  26. Chan KC, Jiang P, Sun K, Cheng YK, Tong YK, Cheng SH, et al. Second generation noninvasive fetal genome analysis reveals de novo mutations, single-base parental inheritance, and preferred DNA ends. Proc Natl Acad Sci U S A. 2016;50:E8159–68.

    Article  Google Scholar 

  27. Munné S, 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;108:62.

    Article  Google Scholar 

  28. Fragouli E, et al. Cytogenetic analysis of human blastocysts with the use of FISH, CGH and aCGH: Scientific data and technical evaluation. Hum Reprod. 2011;26:480.

    Article  CAS  Google Scholar 

  29. Ruttanajit T, et al. Detection and quantitation of chromosomal mosaicism in human blastocysts using copy number variation sequencing. Prenat Diagn. 2015;36:154.

    Article  Google Scholar 

  30. Van EJ, et al. Chromosomal mosaicism in human preimplantation embryos: a systematic review. Hum Reprod Update. 2011;17:620–7.

    Article  Google Scholar 

  31. Vera-Rodriguez M, Rubio C. Assessing the true incidence of mosaicism in preimplantation embryos. Fertil Steril. 2017;107:1107.

    Article  Google Scholar 

  32. Liu J, et al. DNA microarray reveals that high proportions of human blastocysts from women of advanced maternal age are aneuploid and mosaic. Biol Reprod. 2012;87:148.

    Article  Google Scholar 

  33. Fragouli E, Alfarawati S, Spath K, Babariya D, Tarozzi N, Borini A, et al. Analysis of implantation and ongoing pregnancy rates following the transfer of mosaic diploid-aneuploid blastocysts [J]. Hum Genet. 2017;136(7):805–19.

    Article  CAS  Google Scholar 

  34. Greco E, Minasi MG, Fiorentino F. Healthy Babies after Intrauterine Transfer of Mosaic Aneuploid Blastocysts [J]. N Engl J Med. 2015;373(21):2089–90.

    Article  Google Scholar 

  35. Taylor TH, GitlinS A, Patrick JL, et al. The origin, mechanisms, incidence and clinical consequences of chromosomal mosaicism in humans [J]. Hum Reprod Update. 2014;20(4):571–81.

    Article  CAS  Google Scholar 

  36. Werner MD, Hong KH, Franasiak JM, Forman EJ, Reda CV, Molinaro TA, et al. Sequential versus Monophasic Media Impact Trial (SuMMIT): a paired randomized controlled trial comparing a sequential media system to a monophasic medium. Fertil Steril. 2016;105:1215–21. https://doi.org/10.1016/j.fertnstert.2016.01.005.

    Article  CAS  PubMed  Google Scholar 

  37. Kuznyetsov V, Madjunkova S, Antes R, Abramov R, Motamedi G, Ibarrientos Z, et al. Evaluation of a novel non-invasive preimplantation genetic screening approach. PLoS One. 2018;13(5):e0197262.

    Article  Google Scholar 

  38. Mukaida T, Oka C, Goto T, Takahashi K. Artificial shrinkage of blastocoeles using either a micro-needle or a laser pulse prior to the cooling steps of vitrification improves survival rate and pregnancy outcome of vitrified human blastocysts. Hum Reprod. 2006;21(12):3246–52.

    Article  CAS  Google Scholar 

  39. Chan C, Liu V, Lau E, Yeung WS, Ng EH, Ho PC. Mitochondrial DNA content and 4977 bp deletion in unfertilized oocytes. Mol Hum Reprod. 2005;11:843–6.

    Article  CAS  Google Scholar 

  40. Van Blerkom J. Mitochondrial function in the human oocyte and embryo and their role in developmental competence. Mitochondrion. 2011;11:797–813.

    Article  Google Scholar 

  41. Shang W, et al. Comprehensive chromosomal and mitochondrial copy number profiling in human IVF embryos. Reprod BioMed Online. 2017. https://doi.org/10.1016/j.rbmo.2017.10.110.

    Article  CAS  Google Scholar 

  42. Fragouli E, Wells D. Mitochondrial DNA assessment to determine oocyte and embryo viability. Semin Reprod Med. 2015;33(6):401–9.

    Article  CAS  Google Scholar 

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Acknowledgments

We are grateful to our patients for their sincere participation in this study and thank each author for their hard work.

Authors’ roles

X.B. designed and coordinated the study. H.X., C.Y., L.F., and X.S. collected the clinical cases. J.Z. and H.X. performed and analyzed the data. J.Z. wrote the draft of the manuscript. All authors read and approved the final manuscript.

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Authors and Affiliations

  1. Reproductive Medical Center, Department of Obstetrics and Gynecology, General Hospital of Tianjin Medical University, Tianjin, 300052, China

    Jing Zhang, Hong Xia, Haixia Chen, Chenxi Yao, Lizhen Feng, Xueru Song & Xiaohong Bai

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  1. Jing Zhang
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  2. Hong Xia
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Corresponding author

Correspondence to Xiaohong Bai.

Ethics declarations

Ethical approval was obtained from the Scientific and Ethical Committee of Tianjin Medical University for the experimental protocol (IRB2017-155-01). A detailed patient consent form was signed for each embryo sample used in this study.

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The authors declare that they have no conflict of interest.

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Zhang, J., Xia, H., Chen, H. et al. Less-invasive chromosome screening of embryos and embryo assessment by genetic studies of DNA in embryo culture medium. J Assist Reprod Genet 36, 2505–2513 (2019). https://doi.org/10.1007/s10815-019-01603-w

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  • DOI: https://doi.org/10.1007/s10815-019-01603-w

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