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Comparison of DNA methylation profiles of human embryos cultured in either uninterrupted or interrupted incubators

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

Abstract

Purpose

We aimed to compare the DNA methylation profiles of human embryos cultured in uninterrupted or interrupted incubators.

Methods

This study included 9 women, ≤ 30 years old (range: 20–30 years), without a history of genetic diseases or smoking, undergoing ICSI treatment, and each woman donated one oocyte. Embryos were randomly assigned to culture in either time-lapse imaging or standard incubators after ICSI. We compared the DNA methylation profiles of human eight-cell embryos cultured in uninterrupted condition using time-lapse imaging (TLI) incubator (EmbryoScope) to those cultured in interrupted culture model using standard incubators (SI, G185 K-System). Nine single-cell whole-genome bisulfite sequencing (WGBS) datasets were analyzed, including four SI-cultured embryos and five TLI-cultured embryos at the eight-cell stage.

Results

A total of 581,140,020 and 732,348,182 clean reads were generated from the TLI and SI groups, respectively. TLI-cultured embryos had similar genome-wide methylation patterns to SI-cultured embryos. There were no significant differences in the methylation and transcription levels of transposable elements and imprinted control regions. Although a total of 198 differentially methylated genes (DMGs) were identified, only five DMGs had significantly different transcription levels between the two groups.

Conclusions

This is the first study to compare the DNA methylation profiles of embryos cultured in TLI and SI and will provide a foundation for evaluating the safety of TLI application in assisted reproductive technologies. However, further study with a larger cohort of samples was needed for the data validation.

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Data availability

The data that support the findings of this study are available from the corresponding author, upon reasonable request.

References

  1. Chambers GM, Dyer S, Zegers-Hochschild F, de Mouzon J, Ishihara O, Banker M, et al. International Committee for Monitoring Assisted Reproductive Technologies world report: assisted reproductive technology, 2014dagger. Hum Reprod. 2021;36(11):2921–34.

    Article  Google Scholar 

  2. Lazaraviciute G, Kauser M, Bhattacharya S, Haggarty P, Bhattacharya S. A systematic review and meta-analysis of DNA methylation levels and imprinting disorders in children conceived by IVF/ICSI compared with children conceived spontaneously. Hum Reprod Update. 2015;21(4):555–7.

    Article  CAS  Google Scholar 

  3. Odom LN, Segars J. Imprinting disorders and assisted reproductive technology. Curr Opin Endocrinol Diabetes Obes. 2010;17(6):517–22.

    Article  CAS  Google Scholar 

  4. DeBaun MR, Niemitz EL, Feinberg AP. Association of in vitro fertilization with Beckwith-Wiedemann syndrome and epigenetic alterations of LIT1 and H19. Am J Hum Genet. 2003;72(1):156–60.

    Article  CAS  Google Scholar 

  5. Sutcliffe AG, Peters CJ, Bowdin S, Temple K, Reardon W, Wilson L, et al. Assisted reproductive therapies and imprinting disorders–a preliminary British survey. Hum Reprod. 2006;21(4):1009–11.

    Article  CAS  Google Scholar 

  6. Li E, Beard C, Jaenisch R. Role for DNA methylation in genomic imprinting. Nature. 1993;366(6453):362–5.

    Article  CAS  Google Scholar 

  7. Zhu P, Guo H, Ren Y, Hou Y, Dong J, Li R, et al. Single-cell DNA methylome sequencing of human preimplantation embryos. Nat Genet. 2018;50(1):12–9.

    Article  CAS  Google Scholar 

  8. Smith ZD, Chan MM, Humm KC, Karnik R, Mekhoubad S, Regev A, et al. DNA methylation dynamics of the human preimplantation embryo. Nature. 2014;511(7511):611–5.

    Article  CAS  Google Scholar 

  9. Guo H, Zhu P, Yan L, Li R, Hu B, Lian Y, et al. The DNA methylation landscape of human early embryos. Nature. 2014;511(7511):606–10.

    Article  CAS  Google Scholar 

  10. Guo H, Zhu P, Wu X, Li X, Wen L, Tang F. Single-cell methylome landscapes of mouse embryonic stem cells and early embryos analyzed using reduced representation bisulfite sequencing. Genome Res. 2013;23(12):2126–35.

    Article  CAS  Google Scholar 

  11. Fujiwara M, Takahashi K, Izuno M, Duan YR, Kazono M, Kimura F, et al. Effect of micro-environment maintenance on embryo culture after in-vitro fertilization: comparison of top-load mini incubator and conventional front-load incubator. J Assist Reprod Genet. 2007;24(1):5–9.

    Article  Google Scholar 

  12. Kelley RL, Gardner DK. Individual culture and atmospheric oxygen during culture affect mouse preimplantation embryo metabolism and post-implantation development. Reprod Biomed Online. 2019;39(1):3–18.

    Article  CAS  Google Scholar 

  13. Kelley RL, Gardner DK. In vitro culture of individual mouse preimplantation embryos: the role of embryo density, microwells, oxygen, timing and conditioned media. Reprod Biomed Online. 2017;34(5):441–54.

    Article  CAS  Google Scholar 

  14. Kelley RL, Gardner DK. Combined effects of individual culture and atmospheric oxygen on preimplantation mouse embryos in vitro. Reprod Biomed Online. 2016;33(5):537–49.

    Article  Google Scholar 

  15. Skiles WM, Kester A, Pryor JH, Westhusin ME, Golding MC, Long CR. Oxygen-induced alterations in the expression of chromatin modifying enzymes and the transcriptional regulation of imprinted genes. Gene Expr Patterns. 2018;28:1–11.

    Article  CAS  Google Scholar 

  16. Chia N, Wang L, Lu X, Senut MC, Brenner C, Ruden DM. Hypothesis: environmental regulation of 5-hydroxymethylcytosine by oxidative stress. Epigenetics. 2011;6(7):853–6.

    Article  Google Scholar 

  17. Li W, Goossens K, Van Poucke M, Forier K, Braeckmans K, Van Soom A, et al. High oxygen tension increases global methylation in bovine 4-cell embryos and blastocysts but does not affect general retrotransposon expression. Reprod Fertil Dev. 2016;28(7):948–59.

    Article  CAS  Google Scholar 

  18. Gaspar RC, Arnold DR, Correa CA, da Rocha CV Jr, Penteado JC, Del Collado M, et al. Oxygen tension affects histone remodeling of in vitro-produced embryos in a bovine model. Theriogenology. 2015;83(9):1408–15.

    Article  CAS  Google Scholar 

  19. Sciorio R, Thong JK, Pickering SJ. Comparison of the development of human embryos cultured in either an EmbryoScope or benchtop incubator. J Assist Reprod Genet. 2018;35(3):515–22.

    Article  CAS  Google Scholar 

  20. Barberet J, Chammas J, Bruno C, Valot E, Vuillemin C, Jonval L, et al. Randomized controlled trial comparing embryo culture in two incubator systems: G185 K-System versus EmbryoScope. Fertil Steril. 2018;109(2):302-9 e1.

    Article  Google Scholar 

  21. Armstrong S, Bhide P, Jordan V, Pacey A, Farquhar C. Time-lapse systems for embryo incubation and assessment in assisted reproduction. Cochrane Database Syst Rev. 2018;5:CD011320.

    Google Scholar 

  22. Alhelou Y, Mat Adenan NA, Ali J. Embryo culture conditions are significantly improved during uninterrupted incubation: A randomized controlled trial. Reprod Biol. 2018;18(1):40–5.

    Article  Google Scholar 

  23. Park H, Bergh C, Selleskog U, Thurin-Kjellberg A, Lundin K. No benefit of culturing embryos in a closed system compared with a conventional incubator in terms of number of good quality embryos: results from an RCT. Hum Reprod. 2015;30(2):268–75.

    Article  CAS  Google Scholar 

  24. Li J, Huang J, Han W, Shen X, Gao Y, Huang G. Comparing transcriptome profiles of human embryo cultured in closed and standard incubators. PeerJ. 2020;8:e9738.

    Article  Google Scholar 

  25. Glujovsky D, Farquhar C. Cleavage-stage or blastocyst transfer: what are the benefits and harms? Fertil Steril. 2016;106(2):244–50.

    Article  Google Scholar 

  26. Alpha Scientists in Reproductive M, Embryology ESIGo. The Istanbul consensus workshop on embryo assessment: proceedings of an expert meeting. Hum Reprod. 2011; 26(6): 1270–83

  27. Krueger F, Andrews SR. Bismark: a flexible aligner and methylation caller for Bisulfite-Seq applications. Bioinformatics. 2011;27(11):1571–2.

    Article  CAS  Google Scholar 

  28. Akalin A, Kormaksson M, Li S, Garrett-Bakelman FE, Figueroa ME, Melnick A, et al. methylKit: a comprehensive R package for the analysis of genome-wide DNA methylation profiles. Genome Biol. 2012;13(10):R87.

    Article  Google Scholar 

  29. Court F, Tayama C, Romanelli V, Martin-Trujillo A, Iglesias-Platas I, Okamura K, et al. Genome-wide parent-of-origin DNA methylation analysis reveals the intricacies of human imprinting and suggests a germline methylation-independent mechanism of establishment. Genome Res. 2014;24(4):554–69.

    Article  CAS  Google Scholar 

  30. Heinz S, Benner C, Spann N, Bertolino E, Lin YC, Laslo P, et al. Simple combinations of lineage-determining transcription factors prime cis-regulatory elements required for macrophage and B cell identities. Mol Cell. 2010;38(4):576–89.

    Article  CAS  Google Scholar 

  31. Zaitseva I, Zaitsev S, Alenina N, Bader M, Krivokharchenko A. Dynamics of DNA-demethylation in early mouse and rat embryos developed in vivo and in vitro. Mol Reprod Dev. 2007;74(10):1255–61.

    Article  CAS  Google Scholar 

  32. Tan K, Zhang Z, Miao K, Yu Y, Sui L, Tian J, et al. Dynamic integrated analysis of DNA methylation and gene expression profiles in in vivo and in vitro fertilized mouse post-implantation extraembryonic and placental tissues. Mol Hum Reprod. 2016;22(7):485–98.

    Article  CAS  Google Scholar 

  33. Deshmukh RS, Ostrup O, Ostrup E, Vejlsted M, Niemann H, Lucas-Hahn A, et al. DNA methylation in porcine preimplantation embryos developed in vivo and produced by in vitro fertilization, parthenogenetic activation and somatic cell nuclear transfer. Epigenetics. 2011;6(2):177–87.

    Article  CAS  Google Scholar 

  34. Wright K, Brown L, Brown G, Casson P, Brown S. Microarray assessment of methylation in individual mouse blastocyst stage embryos shows that in vitro culture may have widespread genomic effects. Hum Reprod. 2011;26(9):2576–85.

    Article  CAS  Google Scholar 

  35. de Waal E, Mak W, Calhoun S, Stein P, Ord T, Krapp C, et al. In vitro culture increases the frequency of stochastic epigenetic errors at imprinted genes in placental tissues from mouse concepti produced through assisted reproductive technologies. Biol Reprod. 2014;90(2):22.

    Google Scholar 

  36. Ghosh J, Coutifaris C, Sapienza C, Mainigi M. Global DNA methylation levels are altered by modifiable clinical manipulations in assisted reproductive technologies. Clin Epigenetics. 2017;9:14.

    Article  Google Scholar 

  37. Zhang JQ, Li XL, Peng Y, Guo X, Heng BC, Tong GQ. Reduction in exposure of human embryos outside the incubator enhances embryo quality and blastulation rate. Reprod Biomed Online. 2010;20(4):510–5.

    Article  Google Scholar 

  38. Velker BA, Denomme MM, Mann MR. Embryo culture and epigenetics. Methods Mol Biol. 2012;912:399–421.

    CAS  Google Scholar 

  39. Mani S, Mainigi M. Embryo culture conditions and the epigenome. Semin Reprod Med. 2018;36(3–04):211–20.

    Article  CAS  Google Scholar 

  40. Rivera RM, Stein P, Weaver JR, Mager J, Schultz RM, Bartolomei MS. Manipulations of mouse embryos prior to implantation result in aberrant expression of imprinted genes on day 9.5 of development. Hum Mol Genet. 2008;17(1):1–14.

    Article  CAS  Google Scholar 

  41. Khosla S, Dean W, Brown D, Reik W, Feil R. Culture of preimplantation mouse embryos affects fetal development and the expression of imprinted genes. Biol Reprod. 2001;64(3):918–26.

    Article  CAS  Google Scholar 

  42. Market Velker BA, Denomme MM, Mann MR. Loss of genomic imprinting in mouse embryos with fast rates of preimplantation development in culture. Biol Reprod. 2012;86(5):143–1-16.

    Article  Google Scholar 

  43. Doherty AS, Mann MR, Tremblay KD, Bartolomei MS, Schultz RM. Differential effects of culture on imprinted H19 expression in the preimplantation mouse embryo. Biol Reprod. 2000;62(6):1526–35.

    Article  CAS  Google Scholar 

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Funding

This work was supported by the Natural Science Foundation of Chongqing (cstc2021jcyj-msxmX0785).

Author information

Authors and Affiliations

  1. Chongqing Key Laboratory of Human Embryo Engineering, Center for Reproductive Medicine, Women and Children’s Hospital of Chongqing Medical University, Chongqing, China

    Ling Zhu, Xi Zeng, Weiwei Liu, Wei Han, Guoning Huang & Jingyu Li

  2. Chongqing Clinical Research Center for Reproductive Medicine, Chongqing Health Center for Women and Children, Chongqing, China

    Ling Zhu, Xi Zeng, Weiwei Liu, Wei Han, Guoning Huang & Jingyu Li

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  1. Ling Zhu
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  3. Weiwei Liu
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Contributions

J.L. conceived and designed the study. L.Z. performed the bioinformatics analysis. X.Z, W.L., and W.H. collected and cultured the human embryos. J.L. and G.H. contributed to manuscript drafting with the help from all the authors. All authors read and approved the final manuscript.

Corresponding authors

Correspondence to Guoning Huang or Jingyu Li.

Ethics declarations

Ethics approval

This study was approved by the Institutional Review Board (IRB) of Chongqing Health Center for Women and Children (2016-RGI-01).

Competing interests

The authors declare no competing interests.

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Supplementary information

Below is the link to the electronic supplementary material.

Supplementary file1 Unsupervised hierarchical clustering of DNA methylomes. (PDF 15 KB)

10815_2022_2669_MOESM2_ESM.pdf

Supplementary file2 The distribution of DNA methylation and CpG density in different groups. a, The distribution of DNA methylation and CpG density in the SI group; b, The distribution of DNA methylation and CpG density in the TLI group. (PDF 911 KB)

10815_2022_2669_MOESM3_ESM.pdf

Supplementary file3  Correlation between DNA methylation level of promoter and gene expression. a, Scatter plot of the DNA methylation level of the promoter region and the relative gene expression level of the corresponding gene in the SI group; b, Scatter plot of the DNA methylation level of the promoter region and the relative gene expression level of the corresponding gene in the TLI group. (PDF 1115 KB)

10815_2022_2669_MOESM4_ESM.pdf

Supplementary file4 DNA methylation levels across genomic features of CHG and CHH. a, Plot of CHG methylation levels across genomic features. Different color indicate different groups; b, Plot of CHH methylation levels across genomic features. Different colors indicate different groups. (PDF 888 KB)

Supplementary file5 Number of DMRs vs. different value in real groups and random combinations. (PDF 92 KB)

Supplementary file6 Methylation levels of the imprinted DMRs in the SI and TLI groups. (XLSX 12 KB)

Supplementary file7 Gene expression levels of the imprinted gene in the SI and TLI groups. (XLSX 11 KB)

Supplementary file8 Differentially methylated regions (DMRs) between the SI and TLI groups. (XLSX 46 KB)

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Zhu, L., Zeng, X., Liu, W. et al. Comparison of DNA methylation profiles of human embryos cultured in either uninterrupted or interrupted incubators. J Assist Reprod Genet 40, 113–123 (2023). https://doi.org/10.1007/s10815-022-02669-9

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  • DOI: https://doi.org/10.1007/s10815-022-02669-9

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