DNA methylation and gene expression changes derived from assisted reproductive technologies can be decreased by reproductive fluids

Abstract

The number of children born since the origin of Assisted Reproductive Technologies (ART) exceeds 5 million. The majority seem healthy, but a higher frequency of defects has been reported among ART-conceived infants, suggesting an epigenetic cost. We report the first whole-genome DNA methylation datasets from single pig blastocysts showing differences between in vivo and in vitro produced embryos. Blastocysts were produced in vitro either without (C-IVF) or in the presence of natural reproductive fluids (Natur-IVF). Natur-IVF embryos were of higher quality than C-IVF in terms of cell number and hatching ability to. RNA-Seq and DNA methylation analyses showed that Natur-IVF embryos have expression and methylation patterns closer to in vivo blastocysts. Genes involved in reprogramming, imprinting and development were affected by culture, with fewer aberrations in Natur-IVF embryos. Methylation analysis detected methylated changes in C-IVF, but not in Natur-IVF, at genes whose methylation could be critical, such as IGF2R and NNAT.

Data availability

The following data sets were generated

Article and author information

Author details

  1. Sebastian Canovas

    Physiology of Reproduction Group, Departamento de Fisiología, Facultad de Veterinaria, Universidad de Murcia, Murcia, Spain
    Competing interests
    The authors declare that no competing interests exist.
  2. Elena Ivanova

    Epigenetics Programme, The Babraham Institute, Cambridge, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  3. Raquel Romar

    Physiology of Reproduction Group, Departamento de Fisiología, Facultad de Veterinaria, Universidad de Murcia, Murcia, Spain
    Competing interests
    The authors declare that no competing interests exist.
  4. Soledad García-Martínez

    Physiology of Reproduction Group, Departamento de Fisiología, Facultad de Veterinaria, Universidad de Murcia, Murcia, Spain
    Competing interests
    The authors declare that no competing interests exist.
  5. Cristina Soriano-Úbeda

    Physiology of Reproduction Group, Departamento de Fisiología, Facultad de Veterinaria, Universidad de Murcia, Murcia, Spain
    Competing interests
    The authors declare that no competing interests exist.
  6. Francisco Alberto A García-Vázquez

    Physiology of Reproduction Group, Departamento de Fisiología, Facultad de Veterinaria, Universidad de Murcia, Murcia, Spain
    Competing interests
    The authors declare that no competing interests exist.
  7. Heba Saadeh

    Epigenetics Programme, The Babraham Institute, Cambridge, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  8. Simon Andrews

    Bioinformatics Group, The Babraham Institute, Cambridge, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  9. Gavin Kelsey

    Epigenetics Programme, The Babraham Institute, Cambridge, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-9762-5634
  10. Pilar Coy

    Physiology of Reproduction Group, Departamento de Fisiología, Facultad de Veterinaria, Universidad de Murcia, Murcia, Spain
    For correspondence
    pcoy@um.es
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-3943-1890

Funding

Research Councils UK

  • Gavin Kelsey

Ministerio de Economía y Competitividad (AGL2012-40180-C03-01 and AGL2015-66341-R)

  • Pilar Coy

Ministerio de Educación, Cultura y Deporte (PRX14/00348)

  • Pilar Coy

Fundación Séneca. Región de Murcia. Spain (20040/GERM/16)

  • Pilar Coy

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Ethics

Animal experimentation: This study was carried out in strict accordance with the recommendations in the Guiding Principles for the Care and Use of Animals (DHEW Publication, NIH, 80-23). The protocol was approved by the Ethical Committee for Experimentation with Animals of the University of Murcia, Spain (Project Code: 192/2015).

Reviewing Editor

  1. Jessica K Tyler, Weill Cornell Medicine, United States

Publication history

  1. Received: November 26, 2016
  2. Accepted: January 28, 2017
  3. Accepted Manuscript published: January 30, 2017 (version 1)
  4. Accepted Manuscript updated: February 1, 2017 (version 2)
  5. Version of Record published: March 7, 2017 (version 3)
  6. Version of Record updated: April 11, 2017 (version 4)

Copyright

© 2017, Canovas et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

Metrics

  • 3,819
    Page views
  • 881
    Downloads
  • 84
    Citations

Article citation count generated by polling the highest count across the following sources: Scopus, Crossref, PubMed Central.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

  1. Sebastian Canovas
  2. Elena Ivanova
  3. Raquel Romar
  4. Soledad García-Martínez
  5. Cristina Soriano-Úbeda
  6. Francisco Alberto A García-Vázquez
  7. Heba Saadeh
  8. Simon Andrews
  9. Gavin Kelsey
  10. Pilar Coy
(2017)
DNA methylation and gene expression changes derived from assisted reproductive technologies can be decreased by reproductive fluids
eLife 6:e23670.
https://doi.org/10.7554/eLife.23670

Further reading

    1. Developmental Biology
    Qiyan Mao et al.
    Research Article Updated

    Human muscle is a hierarchically organised tissue with its contractile cells called myofibers packed into large myofiber bundles. Each myofiber contains periodic myofibrils built by hundreds of contractile sarcomeres that generate large mechanical forces. To better understand the mechanisms that coordinate human muscle morphogenesis from tissue to molecular scales, we adopted a simple in vitro system using induced pluripotent stem cell-derived human myogenic precursors. When grown on an unrestricted two-dimensional substrate, developing myofibers spontaneously align and self-organise into higher-order myofiber bundles, which grow and consolidate to stable sizes. Following a transcriptional boost of sarcomeric components, myofibrils assemble into chains of periodic sarcomeres that emerge across the entire myofiber. More efficient myofiber bundling accelerates the speed of sarcomerogenesis suggesting that tension generated by bundling promotes sarcomerogenesis. We tested this hypothesis by directly probing tension and found that tension build-up precedes sarcomere assembly and increases within each assembling myofibril. Furthermore, we found that myofiber ends stably attach to other myofibers using integrin-based attachments and thus myofiber bundling coincides with stable myofiber bundle attachment in vitro. A failure in stable myofiber attachment results in a collapse of the myofibrils. Overall, our results strongly suggest that mechanical tension across sarcomeric components as well as between differentiating myofibers is key to coordinate the multi-scale self-organisation of muscle morphogenesis.

    1. Developmental Biology
    2. Stem Cells and Regenerative Medicine
    Marta Perera et al.
    Research Article

    During embryonic development cells acquire identity at the same time as they are proliferating, implying that an intrinsic facet of cell fate choice requires coupling lineage decisions to rates of cell division. How is the cell cycle regulated to promote or suppress heterogeneity and differentiation? We explore this question combining time lapse imaging with single cell RNA-seq in the contexts of self-renewal, priming and differentiation of mouse embryonic stem cells (ESCs) towards the Primitive Endoderm lineage (PrE). Since ESCs are derived from the Inner Cell Mass of the mammalian blastocyst, ESCs in standard culture conditions are transcriptionally heterogeneous containing subfractions that are primed for either of the two ICM lineages, Epiblast and PrE. These subfractions represent dynamic states that can readily interconvert in culture, and the PrE subfraction is functionally primed for endoderm differentiation. Here we find that differential regulation of cell cycle can tip the balance between these primed populations, such that naïve ESC culture conditions promote Epiblast-like expansion and PrE differentiation stimulates the selective survival and proliferation of PrE-primed cells. In endoderm differentiation, we find that this change is accompanied by a counter-intuitive increase in G1 length that also appears replicated in vivo. While FGF/ERK signalling is a known key regulator of ESCs and PrE differentiation, we find it is not just responsible for ESCs heterogeneity, but also cell cycle synchronisation, required for the inheritance of similar cell cycles between sisters and cousins. Taken together, our results point to a tight relationship between transcriptional heterogeneity and cell cycle regulation in the context of lineage priming, with primed cell populations providing a pool of flexible cell types that can be expanded in a lineage-specific fashion while allowing plasticity during early determination.