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Epigenetic changes in preimplantation embryos subjected to laser manipulation.

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Abstract

The advantage of using laser for assisted hatching in routine assisted reproductive technology (ART) practice is debatable. Recently, it has been shown that laser-manipulated mouse embryos had compromised genetic integrity. However, the impact of laser-assisted hatching (LAH) on the epigenetic integrity of the preimplantation embryos is not elucidated so far. Since continuous thermal stress on embryos was found to lower mRNA levels of de novo (bovine) methyl transferases in embryos, we hypothesize that thermal energy induced during LAH may alter the epigenetic signature through abnormal de novo methyl transferases (Dnmts) levels. Thus, using mouse model, we made an attempt to look into the expression of Dnmt3a and Dnmt3b in laser-manipulated embryos and their effects on global methylation. This experimental prospective study used mouse embryos from varying developmental stages (2-cell, 6–8-cell, and blastocyst) which were subjected to LAH using a 1480-nm diode laser. Two pulses of 350 μs frequency were applied to breach the zona pellucida, and then, embryos were assessed for the expression of two de novo methyl transferases (Dnmt3a and Dnmt3b) and LINE-1 (long interspersed element-1) methylation when LAH embryos developed to blastocyst stage. Results from this study have shown that blastocysts subjected to LAH at two-cell stage had significantly lower mRNA transcripts of Dnmt3a (P < 0.01) and Dnmt3b (P < 0.05) whereas LAH at six- to eight-cell and blastocyst stages did not affect the mRNA level significantly. On the other hand, LINE-1 methylation did not change significantly between LAH and control group in all the stages studied. These results suggest that two-cell-stage laser manipulation of embryos changes the mRNA level of Dnmts without affecting the global DNA methylation.

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Funding

This work was supported by the Indian Council of Medical Research (ICMR) Grant No. 2010-00020. Funding agency did not have any role in designing the experiment, data analysis and interpretation.

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Authors and Affiliations

  1. Division of Clinical Embryology, Level 2, Central Research Lab, Kasturba Medical College, Manipal University, Manipal, 576104, India

    Sachin D. Honguntikar, Sujith Raj Salian, Fiona D’Souza, Shubhashree Uppangala, Guruprasad Kalthur & Satish Kumar Adiga

Authors
  1. Sachin D. Honguntikar
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  2. Sujith Raj Salian
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  3. Fiona D’Souza
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  4. Shubhashree Uppangala
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  5. Guruprasad Kalthur
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  6. Satish Kumar Adiga
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Contributions

SH has performed the experiments and analyzed the data. SS, FD, and SU have performed the experiment. GK has assisted in designing the study. SKA has designed the study, analyzed the data, and written the manuscript. SH is the guarantor of this work and as such had full access to all the data and takes responsibility for the integrity of the data and the accuracy of the data analysis.

Corresponding author

Correspondence to Satish Kumar Adiga.

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The authors declare that they have no conflict of interest.

Ethical approval

All experiments and animal handling were conducted in accordance with the institutional guidelines for animal experimentation after obtaining prior approval from the Institutional Animal Ethics Committee (IAEC/KMC/110/2012).

Informed consent

Not applicable as no human subjects were involved in this study.

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Honguntikar, S., Salian, S.R., D’Souza, F. et al. Epigenetic changes in preimplantation embryos subjected to laser manipulation.. Lasers Med Sci 32, 2081–2087 (2017). https://doi.org/10.1007/s10103-017-2334-3

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  • DOI: https://doi.org/10.1007/s10103-017-2334-3

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