Symposium title: ‘Towards a genetically perfect society’

The aim of the symposium is to bring together both clinicians and researchers in the fields of obstetrics, fertility, embryology and reproduction to present the latest findings in this area and discuss the controversies.

Friday, April 5, 2019,
8.30- 18.00 uur

Location: Gertrudiskapel (near Utrecht Central Station)
Willemsplantsoen 1c
3511 LA Utrecht

By: Dr. Susana Chuva de Sousa Lopes, recipient of the De Snoo award 2017

 

Background

One of the most exciting and controversial discoveries over the past decade is that conditions experienced early in life – even before birth – can have long-lasting impacts on health and ageing 1,2. Longitudinal studies on humans and experimental studies using animal models also suggest that these effects may span several generations 3. There is an increasing body of evidence that shows that maternal effects on health are caused by epigenetic changes in the embryo that remain stable throughout life and possibly could be passed on to subsequent generations 4,5. For example, a longitudinal study of individuals that were in utero during the Dutch Famine at the end of the Second World War has demonstrated local and genome-wide changes in DNA methylation that can be linked to increased risk of cardiovascular and metabolic disease 6,7. These effects appear to primarily affect embryos exposed during the first trimester, and recently studies in humans point to the importance of pre-implantation and early implantation stages, when drastic changes in DNA methylation occur 8,9.

 

Aims of the project

Recent technical developments regarding single cell transcriptomics and (DNA) methylomics 10,11 as well as the development of a protocol to mimic implantation in mice 12 opens exciting opportunities to study hypothesis driven questions related to the theory of developmental origins of health and disease.

 

We aim to achieve two key objectives:

  • To develop a peri-implantation model using human pre-implantation embryos using a novel synthetic 3D-scaffold to allow the embryo to develop in vitro during the first 14 days. Using a novel synthetic hydrogel as 3D-scaffold will would allow us to develop a novel method to keep the embryos in a stiff-environment that can mimic the endometrium and promote physiological implantation. We plan to seed the hydrogel with human endometrium cells (containing endothelial cells and glandular cells) and characterise in detail the implantation during until day 14 of development.
  • If successful, we will study in great detail how (metabolic) changes in the culture environment will influence the epigenetics of the human embryo. For this we will perform single cell transcriptomics and methylomics as well as other molecular techniques (immunostaining and QPCR) to compare standard culture conditions with those that are known to change DNA methylation (folic acid, vitamin A and vitamin C).

 

Results

Use of a 3D-synthetic scaffold to allow embryo development in vitro during 14-days

The extended culture model has been developed under the supervision of one of the researchers that was part of the team that has developed and published the mouse implantation model 12. The human (frozen-thawed) embryos at day 3 post fertilization (dpf) have been first cultured to the blastocyst stage (day 5), graded them using the Gardner and Schoolcraft grading system and then cultured in a two-step protocol to mimic implantation (from day 5 to day 14) in 2D and in the 3D-scaffold. The results of the 2D and 3D-scaffold have been evaluated by immunofluorescence to assess morphology and lineage segregation (extraembryonic lineages, endoderm ectoderm, mesoderm and germline) using different markers. In the 2D- system, the embryo survives better than in the 3D-system. A manuscript with the results obtained from the 2D-system has been submitted and the results of the 3D system is expected to be submitted by the end of 2018.  Interestingly, we noticed that when using our extended culture method, aneuploid blastocysts excluded the abnormal cells by day 14. These important finding, is being explored and a manuscript is being prepared.

 


2-D System

3-D System

 

The benefits of including endometrium cells (endothelial cells and or glandular cells) in the 3D-scaffold, as not been investigated yet. But those experiments will be performed in the continuation of the project, as one PhD has been recruited to continue with this project.

 

Influence of metabolic changes in the epigenetics of the human blastocycst

In collaboration with the Hubrecht Institute (Prof. van Oudenaarden), we are characterising the transcriptomics and methylomics at single cell level during the preimplantation period. Most importantly, we performed single cell isolation of cells from individual sibling and from those we have requested and obtained the genomic DNA (exome) from the parental bucal swabs. This has allowed us to identify the existing SNPs in the embryos. This work is in progress and will be the base line to then investigate the transcriptomics and methylomics using the extended protocol on sibeling embryos and to test 3 culture conditions known to change DNA methylation (folic acid, vitamin A and vitamin C).

 

References

1 Bateson, P. et al. Nature 430, 419-421 (2004); 2 Gluckman, P. D. et al. Lancet 373, 1654-1657 (2009); 3 Daxinger, L. & Whitelaw, E. Nature reviews. Genetics 13, 153-162 (2012); 4 Gluckman, P. D. et al. Nature reviews. Endocrinology 5, 401-408 (2009); 5 Petronis, A. Nature 465, 721-727 (2010); 6 Heijmans, B. T. et al. PNAS 105, 17046-17049 (2008); 7 Tobi, E. W. et al. PloS one 7, e37933 (2012); 8 Guo, H. et al. Nature 511 (2014); 9 Smith, Z. D. et al. Nature 511, 611-615 (2014); 10 Lefevre, A. & Blachere, T. Methods in molecular biology 1222, 209-226 (2015); 11 Yan, L. et al. Nature structural & molecular biology 20, 1131-1139 (2013); 12 Bedzhov, I. et al. Nature protocols 9, 2732-2739 (2014).

By: Ewart Kuijk, recipient of the De Snoo award 2017

Center for Molecular Medicine and Oncode Institute, University Medical Center Utrecht, Utrecht University, Utrecht, the Netherlands

 

First of all, I am highly grateful for having received the De Snoo award in 2017. In addition to having been able to perform the study explained below, the De Snoo award has propelled my research further resulting in additional funding to extend my studies on instability of the embryonic genome, thereby helping me to establish my own independent research line.

Abstract

Sperm DNA damage affects post zygotic genome function and interferes with proper embryo development, thereby impacting on miscarriage rates, fertility and postnatal pathologies. Nevertheless, the direct consequences of sperm DNA damage on the embryonic genome are largely unknown, because this requires single cell and low input sequencing techniques that have only recently been developed. The aim of this study was to elucidate how sperm DNA damage affects embryonic genomes. By absence of homologous templates we hypothesized that damaged sperm will be repaired through the inaccurate process of non-homologous end-joining which will result in the formation of Structural Variants (SVs) such as copy number variants (CNVs), translocations and inversions. Bovine embryos were produced with sperm subjected to different doses of γ-radiation (experimental group) and with untreated sperm (control group). Increasing doses of γ-radiation resulted in decreasing blastocyst rates. Moreover, embryos produced with γ-radiated sperm contained more micronuclei and other nuclear aberrations than control embryos. In follow-up experiments, the resulting zygotes were cultured until the 2-cell stage after which both individual blastomeres were collected and subjected to single-cell sequencing (control group n = 36 embryos, 2.5 Gray group n =42 embryos, 10 Gray group n = 18 embryos). This revealed that embryos produced with damaged sperm contain significantly more chromosomal segmental gains and deletions than control embryos (fig. 1). Additionally, experimental embryos contained more aneuploidies than control embryos, indicating that sperm DNA damage can induce mis-segregations (fig. 1). Strikingly, for all the observed gain, a reciprocal loss was observed in the sister cell (fig. 1, 2). If the 2-cell stage embryo would have been sequenced “in bulk” instead of both blastomeres separately, the genome would have appeared copy neutral and all variants would have been missed.

Figure 1: (Left) Number of copy number changes per cell for control embryos and embryos produced with 2 different doses of γ -radiated sperm, (Right) Number of aneuploidies per cell for control embryos and embryos produced with 2 different doses of γ -radiated sperm.

 

 

 

 

 

 

 

 

 

 

 

 

Figure 2; Single cell sequencing data for individual blastomeres. (Left) Circos plot showing the copy number state for all of the chromosomes for both cells of a 2-cell stage embryo from the control group. (Right) Circos plot showing the copy number state for all of the chromosomes for both cells of a 2-cell stage embryo from the 10 Gray group. Dark green = copy state 2 on the Crick strand, dark purple = copy state 1 on the Crick strand, light green = copy state 2 on the Watson strand, light purple = copy state 1 on the Watson strand. The total copy number state of Watson and Crick strands is indicated by the color in the inner ring for each cell: yellow = copy number state 2 (normal), blue = copy number state 3 (triploid), red = copy number state 1 (monozygous). From this picture, it is clear that the same chromosomes and chromosomal fragments that have a copy number state of 1 in one cell have a copy number state 3 in the sister cell, resulting in an average copy number state of 2 for both cells. Note: the data is derived through Strand-seq, a single cell sequencing technique that sequences the DNA strand that was used as a template in the previous cell division. When the Watson strand has been used as template for both the maternal and the paternal chromosome, the sequencing reads will map to the Watson side of the chromosome, when the Crick strand has been used as template for both the maternal and the paternal chromosome, the sequencing reads will map to the Crick side of the chromosome, if both the Watson and the Crick strands have been used as templates, the sequencing reads will map to both sides of the chromosome.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Single-cell sequencing of all blastomeres of 8-cell stage embryos produced with damaged sperm indicates that the reciprocal gains and losses observed at the 2-cell stage are preserved within their separate cellular lineages. I am currently sequencing entire blastocysts derived from damaged sperm to gain more insight in the effects of sperm DNA damage on the embryonic genome at later stages of development. Together, these results demonstrate that sperm DNA damage leads to reduced developmental competence, probably caused by the observed genomic aberrations. Embryos that succeed to develop further, are at risk to carry de novo structural variation. These findings may be of relevance for fertility treatments of males with high levels of sperm DNA damage. This study delivers the required proof of principle to be extended to study the effects of sperm DNA damage on human embryonic genomes, as was originally proposed. The manuscript for this study is currently in preparation.

 

This study was performed in collaboration with: S.H.A. Middelkamp1, D.C.J. Spierings2, H.T. A. van Tol3, V. Guryev2, P.M. Lansdorp2,4, B.A.J. Roelen3, E.P.J. Cuppen1

1 Center for Molecular Medicine and Oncode Institute, University Medical Center Utrecht, Utrecht University, Utrecht, the Netherlands

2 European Research Institute for the Biology of Ageing (ERIBA), University of Groningen, UMC Groningen, Groningen, The Netherlands.

3 Department of Farm Animal Health, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands

4 Terry Fox Laboratory, BC Cancer Agency, Vancouver, BC, Canada; Department of Medical Genetics, University of British Columbia, Vancouver, BC, Canada