It is by now well known that human embryos carry nummerous chromosome abnormalities during the first days of life, although few is known on the origin of these abnormalities, and their potential impact for future development.
Our research in this domain focuses on two aspects:
- We study the causes and extend of chromosome instability in human embryos
- We investigate the extend and potential impact of mitochondrial genome instability in oocytes and individuals born after assisted reproductive technologies
Origin of chromosomal abnormalities in human embryos
At the start of our studies, human preimplantation embryos were known to carry chromosomal aneuploidies in as much as 40 to 70% of cases, depending on the source of the study. The vast majority of this data came from embryos that underwent blastomere biopsy, followed by FISH for up to 9 chromosomes, during an IVF cycle with preimplantation genetic screening (PGS). PGS has been applied in IVF patients with a bad prognosis to improve IVF results. Although these aneuploidies are probably a key factor influencing the potential of the embryo to implant in the uterus, there were many questions still open.
An interesting observation was the presence of mosaicism in the human embryo, but its frequency remained to be determined. A second point was that the high number of reported aneuploidies predicted an lower rate of implantation after embryo transfer in IVF than seen. This led researchers to suspect that some of these aneuploidies and mosaicism may correct, possibly through selective migration of abnormal cells to the trophectoderm or through apoptosis, during early development of the embryo. However, not much was known at the cellular level on how these abnormalities would arise, how they can possibly correct, and what their ultimate influence on embryonic development is.
Study of the genetic content of preimplantation embryos
Our first aim was to establish the levels of mosaicism in good-quality human embryos using a comprehensive and high-resolution method for cytogenetic analysis. In a first study, we investigated the aneuploidy rates and incidence of mosaicism in top-quality day-3 human preimplantation embryos. We analysed 91 single blastomeres from 14 embryos donated for research by array-based comparative genomic hybridization (aCGH). We found high levels of mosaicism and structural aberrations. This work has been published in Mertzanidou et al. (2013, Human Reproduction), and has become a very well-cited paper.
We found similar results for day-3 embryos obtained from oocytes that have been in-vitro matured (Spits et al., 2014, Human Reproduction). The image above shows part of the embryos included in this last study. All embryos were dissaggregated on day 3 of development and the cells individually analysed by single-cell aCGH. The results are categorized by genetic content as normal cells, cells with a single chromosome gain or loss, cells with two gains or losses and chaotic cells (colour code on the left). These studies on day-3 embryos are two of the very few of its kind, in which the analysis of all cells provides in depth insight into genetic heterogeneity within one human embryo.
In a parallel work, we investigated embryos at day 4 of development. we analysed the aneuploidy and mosaicism in all cells of 13 top-quality Day-4 embryos by aCGH. Our data is the first set of this type, and shows that at this stage, human embryos are still highly abnormal. The data is so complete that it allowed us to virtually reconstruct the developmental history of each embryo (Mertzanidou et al., 2013 Human Reproduction). The image below shows one of these reconstructions, the colour code is as in the box above. Grey cells with a crossing line are hypothetical cells necessary to explain the final outcome. During the second cleavage, one of the cells of embryo 1 underwent DNA replication without cell division (E, endoreduplication). In the next cleavage, the cell underwent a tetrapolar division, resulting in cells A, B, C and D. This is the first time that a solid model is provided for the origin of chaotic cells in human embryos. The cellular mechanisms of these events are currently subject of our research.
Ongoing research: mechanisms behind genome instability in human embryos
We hypothesize that,during the first cleavages, human preimplantation embryos have a defective spindle attachment checkpoint (SAC), the mechanism responsible for safeguarding a proper sister chromatid separation during anaphase. This would explain how the rapidly cleaving blastomeres accumulate aneuploidies, without being arrested or cleared from the embryo by apoptosis. We are currently testing this hypothesis using functional tests.
MitoART - Do mitochondrial DNA mutations play a role in the outcome of assisted reproductive technologies?
MitoARt is the newest of research lines of the team. The project itself was conceived during the writing of the discussion of our publication in Nature Biotechnology in 2013. We found that human embryonic stem cells carried numerous deletions in their mtDNA and that time in culture did not seem to correlate to these mutations. A literature search retrieved a number of works showing that human embryos also carried mtDNA mutations, which supported the idea that the mutations we saw in stem cells actually came from the cells from which we derived the lines rather than being culture-induced. This left us wondering on what their origin could be in the embryos and on their biological relevance, thoughts that were strongly influenced by the setting of our work: a university hospital in which reproductive medicine has a prominent role. The elegant work of Ross and co-workers (Nature, 2013) showing the impressive impact of low-frequency mtDNA mutations on the health of mice further triggered a thorough literature research. This ultimately yielded a coherent model that can finally explain the some of the observations made in children born after assited reproductive technologies (ART), and provides the much-needed information to ensure the correct medical follow-up of these individuals. MitoART has a fascinating balance between technical challenge, fundamental genetics and medical impact.
MitoART in a nutshell
In the past 37 years, assisted reproductive technologies (ART) have helped millions of couples worldwide to become parents. Despite their broad use, there is still uncertainty about their safety. It was soon observed that children born after ART have lower birthweight and more recently there is evidence that they have cardiometabolic abnormalities. Much research has been devoted to understanding these observations, mainly focussing on the search for epigenetic changes, but the question why these children are different still remains open.
In MitoART, we hypothesize that the low-birth weight and the cardiometabolic differences seen in ART children is due to an increased mitochondrial DNA (mtDNA) mutation load at conception, during gestation, and later in life, which in turn results in mitochondrial dysfunction. I propose that there are two non-mutually exclusive sources for these mutations. The first hypothesis is that some forms of female infertility are linked to an increased mtDNA mutation load, which can be transmitted to the offspring. Secondly, we propose that controlled ovarian stimulation, a procedure used in the majority of ART treatments, leads to mtDNA mutation in the oocytes.
In this project we will test if there are differences in the mtDNA mutation load between individuals conceived after ART or by spontaneous conception at different stages of their development. We will first develop a novel massive parallel sequencing-based approach to simultaneously screen the mtDNA for very low frequency point mutations and rearrangements both in DNA samples and single cells. We will screen material of different origins, all of them unique and precious, and carefully selected to answer the questions of the study. These include DNA samples of ART patients and controls, oocytes obtained in different conditions, placental samples, newborns, 14-year old children and 18-year olds.
MitoART: work in progress
We recently published part of our preliminary work on the technical evaluation of different massively parallel sequencing platforms and methods for the analysis of the mitochondrial genome. (Van Campenhout et al., 2014). We are currently finalizing the setup of a massively parallel sequencing-based approach to study very low frequency point mutations and deletions in DNA samples and single cells, and initializing the pilot studies on human oocytes and on children born after ART.
Chromosome constitution of human embryos generated after in vitro maturation including 3-isobutyl-1-methylxanthine in the oocyte collection medium.Spits C, Guzman L, Mertzanidou A, Jacobs K, Ortega-Hrepich C, Gilchrist RB, Thompson JG, De Vos M, Smitz J, Sermon K. Hum Reprod. 2015, 30:653-63.
A bumpy ride on the diagnostic bench of massive parallel sequencing, the case of the mitochondrial genome.Vancampenhout K, Caljon B, Spits C, Stouffs K, Jonckheere A, De Meirleir L, Lissens W, Vanlander A, Smet J, De Paepe B, Van Coster R, Seneca S. PLoS One. 2014, 9:e112950.
Current issues in medically assisted reproduction and genetics in Europe: research, clinical practice, ethics, legal issues and policy.Harper J, Geraedts J, Borry P, Cornel MC, Dondorp WJ, Gianaroli L, Harton G, Milachich T, Kaariainen H, Liebaers I, Morris M, Sequeiros J, Sermon K, Shenfield F, Skirton H, Soini S, Spits C, Veiga A, Vermeesch JR, Viville S, de Wert G, Macek M Jr; ESHG, ESHRE and EuroGentest2. Hum Reprod. 2014, 29:1603-9.
Current issues in medically assisted reproduction and genetics in Europe: research, clinical practice, ethics, legal issues and policy. European Society of Human Genetics and European Society of Human Reproduction and Embryology. Harper JC, Geraedts J, Borry P, Cornel MC, Dondorp W, Gianaroli L, Harton G, Milachich T, Kaariainen H, Liebaers I, Morris M, Sequeiros J, Sermon K, Shenfield F, Skirton H, Soini S, Spits C, Veiga A, Vermeesch JR, Viville S, de Wert G, Macek M Jr. European Journal of Human Genetics 2013, Suppl 2:S1-S21.
Evolution of aneuploidy up to day 4 of human preimplantation development. Mertzanidou A*, Spits C*, Nguyen H.T., Van de Velde H, Sermon K. Human Reproduction. 2013, 28:1716-24. * Joint first authorship.
Microarray analysis reveals abnormal chromosomal complements in over 70% of 14 normally developing human embryos. Human Reproduction.Mertzanidou A*, Wilton L*, Cheng J*, Spits C, Vanneste E, Moreau Y, Vermeesch JR, Sermon K. 2013, 28:256-64. * Joint first authorship.
PGD for monogenic disorders: aspects of molecular biology. Spits C and Sermon K. Prenatal Diagnosis. 2009, 29:50-6.
Preimplantation genetic diagnosis for cancer predisposition syndromes. Spits C, De Rycke M, Van Ranst N, Verpoest W, Lissens W, Van Steirteghem A, Liebaers I, Sermon K. Prenatal Diagnosis. 2007, 27:447-456.
Single-cell aneuploidy detection by array CGH. Le Caignec C, Spits C, Sermon K, De Rycke M, Thienpont B, Moreau Y, Fryns JP, Van Steirteghem A, Liebaers I, Vermeesch RJ. Nucleic Acids Research. 2006, 34:e68.
Whole genome multiple displacement amplification from single-cells. Spits C*, Le Caignec C*, De Rycke M, Van Haute L, Van Steirteghem A, Liebaers I, Sermon K. Nature Protocols. 2006, 1:1965-70. * Joint first authorship.
Development and evaluation of single-cell whole genome multiple displacement amplification. Spits C*, Le Caignec C*, De Rycke M, Van Haute L, Van Steirteghem A, Liebaers I, Sermon K. Human Mutation. 2006, 27:496-503. * Joint first authorship.
Preimplantation genetic diagnosis for Marfan Syndrome. Spits C, De Rycke M, Verpoest W, Lissens W, Van Steirteghem A, Liebaers I, Sermon K. Fertility and Sterility. 2006, 86:310-20.
Preimplantation genetic diagnosis for Neurofibromatosis type 1. Spits C, De Rycke M, Van Ranst N, Joris H, Verpoest W, Lissens W, Devroey P, Van Steirteghem A, Liebaers I, Sermon K. Molecular Human Reproduction. 2005,11:381-7.
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