PROJECTS

Aneuploidy in early human development

Principal Investigators: Karen Sermon, Claudia Spits

PhD-student: Charlotte Janssens

The interest of Karen Sermon in the genetics of human preimplantation embryos has remained a common project together with Claudia Spits. We were the first to look at the chromosomal content of every single blastomere in embryos of three and four days old using microarrays, and have recently demonstrated by functional testing that the spindle attachment checkpoint, an important checkpoint to assure euploidy, is functional in the early human embryo but is uncoupled from apoptosis. This means that even grossly aneuploid blastomeres will not go into apoptosis.

It has been known since the 1990ies that human preimplantation embryos obtained after in vitro fertilization (IVF) and intracytoplasmic sperm injection display an astonishing frequency -by and large 3⁄4 of the embryos analyzed - of chromosomal abnormalities. These abnormalities can be inherited from the oocyte or the sperm, but more frequently they occur after fertilization, during the first cleavages of the embryo. We want to study the origin of these chromosomal abnormalities through the analysis of the genome and the gene expression patterns in early embryos, between the zygote and the 8-cell stage. We will focus on the systems that the cell uses to check mistakes in segregation of chromosomes, however the analysis of all the genes expressed at such early stages, in combination with chromosomal abnormalities, may yield other markers of genetic health in the early embryo, which may improve IVF efficiency. Moreover, the embryo seems to have ways of dealing with these abnormalities, as at day 5, there seem to be much less abnormalities. This has been coined "self-correction" of the embryo. Thus, we want to determine the role played by programmed cell death (apoptosis) during the elimination of abnormal cells in blastocysts.

This project aims at answering two research questions:
1. Do different levels of transcripts in the cleavage stage embryo lead to different types of abnormalities, and to mosaicism?
2. Are aneuploid cells selectively eliminated from day 4-day 5 embryos by apoptosis due to proteotoxic stress?

Genome integrity in human pluripotent stem cells

Principal Investigators: Claudia Spits (main), Karen Sermon

PhD-students: Nusa Krivec, Edouard Couvreu De Deckersberg, Yingnan Lei

The unique characteristics of human pluripotent stem cells (hPSC) such as human embryonic stem cells (hESC) and more recently, induced pluripotent stem cells (hiPSC), have made them not only attractive as a potential source of cells in regenerative medicine, but also as a research tool for studying early human developmental processes and human disorders.

hESC and hiPSC are kept in culture for long periods of time without this apparently affecting their self-renewal and pluripotent capacities. Nevertheless, we and others have found that these cells accumulate numerous genetic and epigenetic abnormalities, some of which are highly recurrent. Despite that chromosome abnormalities may influence the functional characteristics of hPSC, both in the pluripotent state and upon differentiation, only a handful of studies have investigated this topic. Also, there is very limited information on the possible mechanisms behind the origin of these mutations, and the key driver genes of the selective advantage they appear to confer to the cells.

Taking into account the potential applications of hPSC, their significant genome instability raises concerns for their safety in therapy or as suitable research models. In this context, it is necessary to gain a better insight on the biological meaning of the different types of abnormalities and to evaluate their impact on the differentiation capacity and malignant potential of the cells. Furthermore, understanding the causes and mechanisms of selective advantage of this mutations in culture will allow us to develop more optimal hPSC culture conditions that help preventing genetic drift in the cells.

Conversely, this genome instability it also opens new research venues. For instance, hPSC carrying genetic abnormalities can be used to identify novel gene functions. It is likely that chromosome regions recurrently involved in abnormalities harbor genes of high significance for pluripotent cells. In this case, hPSC provide a good model to study early developmental processes, or even tumorigenesis.

Our research focuses on three points:

• We study the causes and extend of genome instability in hPSC
• We investigate the mechanisms and genes behind the selective advantage of these mutant cells
• We study the impact of these abnormalities on the differentiation capacity of the cells

MitoART

Principal Investigator: Claudia Spits

PhD-student: Joke Mertens

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 tounderstanding 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.

Differences between individual hPSC lines: differentiation potential and malignancy

Principal Investigators: Karen Sermon, Claudia Spits (main)

PhD-students: Nusa Krivec

Human pluripotent stem cells represent a valuable in vitro research tool to study early human development and diseases, cell-based drug and toxicity screening and are also widely considered as a promising cell source for regenerative medicine. All of these applications require the availability of normal cells, which are able to reliably, efficiently and stably differentiate into the desired cell types.

Significant variation in differentiation potential and efficiency between individual hPSC lines has been observed by several groups, often with the occurrence of a marked propensity to differentiate into a specific cell-lineage, e.g. pancreatic, hematopoietic or cardiac cells. This variation among hPSC lines might be linked to different stages of embryonic development at derivation, (epi)genetic variation between individual human embryos, varying techniques for derivation, culturing and passaging, chromosomal or other genetic abnormalities and epigenetic changes acquired during prolonged in vitro culture.

Differentiation propensity can affect the intended use of a particular hPSC line in biomedical research or regenerative medicine. As complete functional assays are time-consuming and costly, there is a need to develop easy-to-use assays for fast screening of differentiation propensity of hPSC lines. Also, it is essential to gain deeper understanding in the phenomenon of early lineage specification bias in hESC for the further optimization and refinement of differentiation protocols.

Another obstacle still hampering the realization of the full potential of hPSC is that often during differentiation residual undifferentiated stem cells (rSC) are detected. These cells have lost the ability to differentiate and remain mixed within a differentiated population. Upon transplantation into a patient receiving therapeutic cell treatment, they pose a significant tumorigenic risk. Our understanding of the characteristics and mechanisms of formation of these cells is still very poor, despite the danger they pose. A majority of the work involving rSC has focused on purification techniques, which do not yet show perfect efficiency. A sensitive a-priori screening for the presence of rSC in the cell populations prepared for transplantation could significantly increase the safety of hPSC-based regenerative medicine. Additionally, in-depth characterization of these cells could lead to better prevention methods in culturing techniques and differentiation protocols.

Our aim is to chart the differentiation potential of individual hPSC lines and define markers, either genetic or epigenetic, that can reliably predict the differentiation propensity of a specific stem cell line. Similarly, we are interested in exploring whether and why certain stem cell lines display characteristics of a more malignant nature and what kind of phenotypic properties are inherent to this cell state.

Cell sources for hPSC derivation

Principal Investigators: Karen Sermon (main), Hilde Van de Velde

Human iPSC reprogramming: influencing the epigenetic landscape

Even though hESC and hiPSC display a similar chromatin structure and gene expression pattern, observed differences at the epigenomic level and in the differentiation potential point towards fundamental differences and indicate variability in reestablishing the pluripotent epigenetic patterns throughout the genome. This reprogramming variability includes a somatic 'memory' (though this becomes less apparent after longer culture) as well as iPSC-specific aberrantly methylated loci, both at CG islands as in the non CG-context or regional enrichment of particular covalent histone modifications.

The conversion to pluripotency is an epigenomic event in which the ectopic expressed factors help generating a hyperdynamic chromatin state. The observed aberrant epigenetic marks may impart a regional chromatin conformation that is resistant to complete reprogramming. In this project, we aim at loosening the somatic cell chromatin structure in order to increase epigenetic reprogramming.

This research is part of a collaborative project with research groups from Universiteit Gent (Belgium) and Universiteit Maastricht (The Netherlands). The role of our group within this project is to obtain hiPSC in which the epigenetic landscape resembles as closely as possible that of standard inner cell mass-derived hESC, the golden standard in hPSC research. This research should lead to hiPSC that are better applicable for human germ cell derivation, which is the focus point of the collaborative project.

Exploring the potential of single blastomeres for hESC derivation

Embryonic stem cells are generally derived from the inner cell mass of blastocyst-stage human embryos, but also earlier stages of human preimplantation development show hESC derivation potential. In 2009, we reported the derivation of two hESC lines derived from single blastomeres of four-cell stage embryos. While this method would allow the derivation of hESC lines without the destruction of the embryo, it is not known whether the developmental stage of the embryo at derivation influences its pluripotent characteristics. We want to further improve hESC derivation from single blastomeres of cleavage-stage human embryos, and investigate similarities and differences in the epigenetic landscape as well as in the differentiation potential of these cells.

Totipotency and early differentiation in human embryonic cells

Promoter: Hilde Van de Velde (main), Karen Sermon

Human preimplantation development starts with the fertilization of an oocyte which results in a totipotent zygote. Following fertilization, the zygote undergoes a number of cleavage divisions leading to compaction on day 4 and blastulation on day 5. Compaction is a process during which the cell-cell contacts increase. The cells also acquire apical/basal polarity. A blastocyst is composed of an outer epithelial layer of cells called the trophectoderm (TE) that encloses a fluid-filled cavity with a clump of undifferentiated cells called the inner cell mass (ICM). These two cell types represent the first differentiation during early human embryogenesis. The TE forms extra-embryonic trophoblast structures and is responsible for implantation. The ICM forms the embryo proper and is the source of human embryonic stem cells (hESCs).

Due to the scarcity of human research materials and the ethical objections regarding the use of human embryos, it remains largely unknown how these lineages are established in the human. In mice, lineage segregation is controlled by specific transcription factors and several models have been described to explain how apparently morphologically identical cells differentiate into these distinct cell types. Moreover, it has recently been demonstrated that different signalling pathways play a key role during this differentiation event.

In this project we aim to analyse transcription factors (NANOG, POU5F1, SOX2 and CDX2) and signalling pathways (WNT, BMP4, Hippo) that contribute to the first lineage differentiation in human embryos. We are also investigating models such as polarisation and the position of the cells during compaction.  This study will contribute to our basic knowledge on human embryogenesis and stem cell biology.

Genetic aspects of male infertility

Principal Investigator: Katrien Stouffs

Infertility is a problem affecting 10-15% of couples with a child wish. For about half of these couples, a male factor is (co-)responsible . Although for a lot of patients the origin of the problems can be detected, still approximately 30% remains undiagnosed. Two main categories of disorders can be distinguished: acquired and congenital. The last category can either be of genetic origin or can be due to a developmental disorder.

Our team is especially interested in men with a genetic cause of male infertility. For infertile men for whom sperm cells can be retrieved either from the ejaculate or by testicular sperm extraction, in vitro fertilization, possibly with intracytoplasmic sperm injection (ICSI) might be a solution for their fertility problems. As a consequence, also potential genetic causes of infertility can be transmitted. Therefore, it is important to identify and study these genetic causes in order to be able to adequately counsel the couples.

The general aims of our studies are:

• To detect changes in the genome that are related to male infertility.
• To get more insight into the underlying mechanism and consequences of known causes of male infertility.
• To study genes expressed in testicular tissues, and therefore potentially involved in spermatogenesis.

Malfomations of cortical development

Principal Investigator: Katrien Stouffs

PhD-student: Hamide Yildirim

The development of the cerebral cortex is extremely complex but can nevertheless be divided into different, partly overlapping stages. Interference with one or more of these processes by genetic or external factors may result in malformations of cortical development (MCD). The most prevalent MCDs include lissencephaly/subcortical band heterotopia, polymicrogyria, periventricular heterotopia and focal cortical dysplasia. MCDs are an important cause of mental and motor impairment, severe epilepsy, intellectual disability, and autism. Patients require a lifelong multidisciplinary follow-up and treatment is restricted to symptom relief. Most MCD have a genetic etiology but there is extensive heterogeneity both with respect to genotypes and phenotypes. For the large majority of patients with MCDs, the exact etiology of their disorder is still unknown, leaving a considerable number of families not having access to counseling or prenatal diagnosis in order to prevent recurrence.

The different projects aim at the further identification of genes involved in the regulation of neuronal migration and the study of the functional consequences of mutations in these genes , starting from a patient-driven approach. This will result in mapping of major pathways involved in cortical development and function.

The general aims of the studies are:

• To search for new genes involved in malformations of cortical development
• To expand the genotype/phenotype knowledge of known genes/diseases involved in malformations of cortical development
• To develop in vitro tools to study functional consequences of genetic alterations
• To study changes in iPSC derived cortical neurons comparing patients and controls

Cardiogenetics

Principal Investigator: Sonia Van Dooren

Co-promotors: Sara Seneca, Maryse Bonduelle

Unravelling the molecular genetic pathways of Brugada syndrome by a next generation sequencing approach

Cardiac arrhythmias are world-wide one of the leading causes of morbidity and mortality. Although environmental factors clearly contribute to the determinants of arrhythmogenesis, familial and population studies both have proven major evidence of genetic susceptibility. Congenital primary cardiac arrhythmias comprise a distinct group of cardiac disorders that result from defects in electrical properties of the heart. The coordinated cardiac activity includes the process of synchronised and successive opening and closing of ion channels in response to the electrical gradient and mediates the action potential in each cardiac compartment. Mutations in genes that either encode or regulate specific cardiac ion channels underlie different forms of inheritable arrhythmogenic disorders that occur in structurally normal hearts. One of common forms is Brugada syndrome (BrS), inherited as an autosomal dominant trait with variable penetrance and expression. The BrS is characterised by coved type ST elevation on the ECG and increased risk of lethal ventricular arrhythmias. One of the proposed mechanisms underlying the typical saddleback and coved-type ECG morphology is the ionic imbalance between inward sodium and calcium currents (INa and ICa) and transient outward potassium currents (Ito) during phase 1 of the cardiac action potential. Although many functional analyses have aided in better understanding the basic arrhythmogenic mechanism of BrS, genetic studies partly fail to further unravel this. Several research groups describe loss-of-function mutations in the gene encoding the pore-forming ion-conduction -subunit of the sodium channel (SCN5A). However, they account for only 20% of Brugada syndrome patients diagnosed following well established rules. Rare mutations have been reported in 2 of the 4 auxiliary function-modifying -subunits of the sodium channel (SCN1B and 3B) and in a panel of recently associated genes (GPD1L, KCNE3 and HCN4).

The recent technological advances in molecular genomics would allow us to study several proposed and new candidate genes to even the exome in parallel, thereby primarily focusing on proteins involved in INa, ICa and Ito of phase 1 of the cardiac action potential. This next generation sequencing approach has the major advantage of detecting compound and double mutations and/or polymorphisms, which might play a crucial role in the observed reduced penetrance and expressivity. The impact of the discovered mutations and/or polymorphisms will be on the one hand correlated phenotypically and on the other hand further studied in vitro at the transcription and expression level as well on the electrophysiological level.

Clinical practice will greatly benefit from the extrapolation of our mutational analyses and in vitro studies. They will have a substantial impact on patient management, in particular prevention, risk stratification, (presymptomatic, prenatal and pre-implantation) diagnosis and treatment and stratification of subclasses of patients.

Mitochondrial DNA diseases

Principal Investigator: Sara Seneca

Mitochondrial disorders are a very heterogeneous group of devastating diseases, which are cause by defects of the oxidative phosphorylation system. The latter is also known as the respiratory chain and provides the eukaryotic cell of the essential energy supply under the form of cellular ATP molecules. The oxidative phosphorylation system itself is under a unique dual genetic control. Genes located in the mitochondrial and nuclear genomes encode for proteins essential for the structure and functioning of this pathway. At this very moment over 1500 genes are already known to be directly or indirectly involved in mitochondrial function. A mutation in any of these will cause a primary mitochondrial disorder. The defects have now emerged as an important cause of human disease as it is estimated to affect approximately 1 in 5000 of the population. The mitochondrial diseases may cause symptoms in any organ or tissue and present at any age. The understanding of these disease pathogenesis is still limited.

We are interested

• in the identification of the gene defect(s) in patients (mtDNA and nuclear genome)
• the study of these defects and genes in patient material
• the use of model systems to document the consequences of these dysfunctions

Investigations of our team and many others will in the end lead to a more optimal diagnose and potential therapeutic options.

Epigenetics and genomic imprinting, safety of assisted reproductive technologies

Principal Investigator: Martine De Rycke

The general aims of our studies are to gain a more detailed insight into the influence of assisted reproductive technologies (ART) on the epigenome through comparative analysis of epigenetic patterns in human gametes and embryos and in blood and placenta samples from ART or from spontaneous pregnancies.

Background

The different epigenetic modifications such as the DNA methylation at CpG dinucleotides, histone modifications and chromatin remodelling cooperate to regulate gene expression and mediate phenotype characteristics. The two critical periods of genome-wide epigenetic reprogramming during gametogenesis and in the early embryo coincide with the time windows of ART. The small subset of imprinted genes, which have a crucial role in embryonic and placental growth, are reset in the gametes but escape the second reprogramming wave after fertilisation. There is concern that certain aspects of ART may interfere with epigenetic reprogramming and with imprinting in particular.

Most data on imprinting and epigenetic mechanisms have been derived from mice. Data from mice show that ovarian hormonal stimulation can interfere with imprint acquisition in oocytes and imprint maintenance in the embryos, while imprint acquisition for in vitro matured oocytes seems a robust process with no or minor defects. Data from animal models also indicate that embryo culture media may induce epigenetic defects that can influence birth weight. Data from human studies are limited, but they indicate that ovarian hormonal stimulation, embryo culture systems as well as underlying subfertility are contributing risk factors for epigenetic defects.

Previous work from our lab has focused on reprogramming of three imprinted genes and revealed germ-line imprints, time of resetting and imprint maintenance in the human embryo (De Rycke et al. 2002; De Rycke, 2003; Geuns et al., 2003; Geuns et al., 2007a and 2007b).

More recent work focused on expression of the DNA methyltransferase family in control of DNA methylation in gametes and among human preimplantation embryos. Comparison of expression patterns in embryos of good morphological quality with a correct developmental timing and embryos which are delayed or of poor morphological quality indicates that aberrant or failed expression of the epigenetic machinery is connected with preimplantation developmental failure in ART (Petrussa et al., 2014). A similar comparison between fresh and frozen/thawed embryos showed that a slow-freezing DMSO cryopreservation protocol does not seem to interfere with DNA methylation epigenetic reprogramming (De Munck et al., 2015).

Current and future research

To further address the role of epigenetics in ART, it is essential to separate an inherited (infertility) epigenetic modification from a secondary one caused by ART factors. Therefore, families with ART and non-ART siblings will be recruited from the extensive children follow-up database at our centre; these families had an ART treatment for preimplantation genetic diagnosis of early embryos since they were at risk for a genetic disease.  Samples from these families will be used for genome-wide epigenetic testing using next generation sequencing technologies. The comparison of the ART and non-ART sibling pairs will allow to separate inherited genetic factors from ART-related factors and to reveal if and to which extend ART factors contribute to epigenetic changes in ART children.

Medical follow-up of ART children

Principal Investigators: Maryse Bonduelle, Florence Belva

Research nurse: Andrea Buysse

Background

When in vitro fertilisation (IVF) was introduced at the Centre for Reproductive Medicine, questions regarding the safety for the offspring born after IVF came across. Over the years, new Assisted Reproductive Technologies (ART), such as Intracytoplasmic Sperm Injection (ICSI), embryo biopsy, vitrification of embryos and oocytes, in vitro maturation of oocytes (IVM), … were introduced in clinical practise. A systematic evaluation of children born after IVF and after newly introduced techniques was therefore needed. The database of all children born after ART is also the start for numerous international projects and a tool for long-term follow-up studies. It also allows us to monitor differences in outcome when modifications or new techniques (e.g. vitrification) are introduced in daily practise.

Aim

To evaluate the safety of ART by assessing genetic, clinical and psycho-motor outcomes in the offspring during early childhood.

Method

Data are collected from our extensive database which contains information about the pregnancy, delivery and early neonatal period. Information from questionnaires filled out by obstetricians, pediatricians and parents is added to the results of a thorough clinical examination. Children are examined in the first and the second year of life at the Centre for Medical Genetics by trained pediatricians. Special attention is given to major and minor malformations and evaluation of the psycho-motor development.

Results

Comparative studies indicate differences between the outcome of IVF and ICSI pregnancies and spontaneous pregnancies: dysmaturity rate, prematurity rate and major malformation rate are higher among children born after ART.

No major differences were found between ICSI and IVF children. In ICSI children a higher frequency of de novo chromosomal abnormalities was observed.

Not only the use of ejaculated sperm, but also the use of immature, non-ejaculated sperm, such as testicular and epididymal sperm, has been introduced into daily routine in our Center. However, due to the increased aneuploidy rate in spermatozoa from patients with testicular failure concerns of increased congenital anomaly rate and chromosomal aberrations in children born after the use of non-ejaculated sperm are pertinent. In our large cohort of 530 children born after ICSI with testicular sperm and of 194 children born after ICSI with epididymal sperm, overall neonatal health in terms of birth parameters, major anomalies and chromosomal aberrations were comparable to results in 2516 children born after the use of ejaculated sperm.

In children born after cryopreservation, prenatal and neonatal outcome results were comparable for children born after transfer of crypreserved ICSI versus cryopreserved IVF embryos. Furthermore, a higher mean birthweight in singletons and a higher major malformation rate in liveborns was found in the total cryopreservation group (IVF + ICSI) when compared to the total fresh group (IVF + ICSI). This higher malformation rate was specifically found in children born after transfer of cryopreserved ICSI embryos compared to children born after transfer of fresh ICSI embryos or cryopreserved IVF embryos.

Vitrification, an ultrarapid freezing procedure without ice crystal formation has become gradually the dominant cryopreservation method. As potential risks of vitrification for the embryo include the toxicity of high concentrations of cryoprotectant, the health of children born after this procedure should be closely monitored. Singletons born after vitrification showed a higher birthweight standard deviation score (SDS) and a lower small-for-gestational age rate in comparison with peers born after fresh embryo transfer. Preterm birth rate and perinatal death rate were comparable between the two groups. Reassuringly, the rate of major congenital malformations in live borns was comparable between the vitrified group and the fresh group, both in singletons and in twins.

Follow-up of children born after new and specific procedures is one of the strengths of our research.

Since 2010, almost all oocytes for donation are vitrified in our Center. Outcomes after the use of closed (as opposed to open) devices for oocyte vitrification are rather sparse. In our study of 95 singleton and 22 twin pregnancies, no major adverse obstetric or neonatal outcomes were observed. Continued follow-up of 2-year old children born after oocyte vitrification is ongoing.

Also, calcium ionophore treatment is being used in assisted reproductive technology (ART) for cases with previous low fertilization rate or total absence of fertilization after insemination by ICSI or when a specific indication such as globozoospermia is present. In our study of 47 children (31 singletons and 16 twin children) which is the largest group described hitherto, birth characteristics and congenital malformations are within the expected range but, although reassuring, should be interpreted with caution due to the small number of children included.

Currently, a follow-up study focusing on medical and psychological outcomes in children born after In Vitro Maturation (IVM) of oocytes is ongoing at the Center for Medical Genetics and the Center for Reproductive Medicine. The psychological follow-up concerns the measurement of the cognitive and psychomotor development at the age of two years. Medical outcome will focus on malformations and growth.

By monitoring all children born after any ART performed at the UZ Brussel, we are able to evaluate very efficiently new developments and detect subtle differences between the different technologies.

Medical follow-up of ICSI children

Principal Investigator: Florence Belva

Study nurse: Andrea Buysse

Background

Since the ICSI technique (injection of sperm directly into the oocyt) is indeed a more invasive technique than the previously widely used in vitro fertilization (IVF), concerns about the safety for the offspring with the use of possibly impaired spermatozoa are a matter of debate. In this perspective, a large-scale follow-up study was set up since the introduction of ICSI in 1991. The goal of this follow-up project was to assess the safety of the technique in the worldwide largest cohort.

Aims

• Since Barker's "developmental origins of adult disease" hypothesis which states that responses to environmental stimuli during critical/sensitive periods in early life may have long lasting consequences due to permanent reprogramming, specific critical windows in the prenatal period have been identified both in animals and in humans. It is however not clear if in humans IVF/ICSI can affect early embryonic development with long-term consequences on growth, cardiovascular and metabolic functioning. Therefore, we aimed to evaluate whether children and young adults conceived by ICSI are at increased risk of cardiometabolic disease at older age, by mapping growth patterns and body fat distribution and measuring biomarkers of cardiovascular risk in blood and saliva.

• The importance of the genetic origin of the infertility in males treated by ICSI had not been fully investigated due to the young age of the offspring hitherto. It is however clear that a number of these children will inherit the infertility problem from their father, as ICSI was predominantly performed because of male infertility in the couple. Therefore, we evaluated whether pubertal children and young adults conceived by ICSI have alterations in sexual maturation or reproductive endocrine function.

Results regarding growth and cardiometabolic outcome

Previous studies included childhood development assesment at 5 years through two large multi-center studies where ICSI children were compared with IVF and spontaneously conceived (SC) children. Both studies report reassuring results.

Later, at the age of 8 and 10 years, further psychological and medical outcomes were studied in a long-term follow-up project. ICSI and SC children showed comparable cognitive and motor development until the age of 10. Physical examination including a thorough neurological examination and assessment of the pubertal maturation was comparable between ICSI and SC children, besides a higher blood pressure in ICSI offspring at the age of 8 years.

Further follow-up at the age of 14 years focused on adiposity risk and pubertal development. Blood pressure in resting condition or after a stress test was not found to be increased in 14-year-old ICSI children in comparison with spontaneously conceived controls. It was found that 14-year-old female but not male ICSI teenagers had lower salivary cortisol concentrations in comparison with SC peers. Regarding adiposity and its distribution, ICSI girls were found to have an increased risk of central, peripheral and total adiposity. Only in ICSI boys with more advanced pubertal development, an increased risk for peripheral adiposity was found. Furthermore, at the age of 14 years, pubertal development, characterised by menarche, genital development in males and pubic hair development in males and females was also comparable in the ICSI and the spontaneously conceived group. Breast development was less advanced in ICSI females compared with spontaneously conceived peers.

Continued follow-up at the age of 18 years showed overall reassuring findings in a cohort of 126 longitudinally followed young adults conceived by ICSI in comparsion with 133 controls born after spontaneous conception. Mean concentrations of total cholesterol, triglycerides, insulin and blood pressure were comparable between the ICSI conceived and control participants. While men conceived by ICSI, but not women, had lower mean HDL-cholesterol (“the good cholesterol”) concentrations in comparison to controls, other markers of the metabolic syndrome were not different between the two groups. Furthermore, while men conceived by ICSI, but not women, had a higher peripheral fat deposition, body fat distribution as well as mean levels of adipocytokines were not different when compared to controls.

Results regarding infertility risk

Findings in boys conceived by ICSI

To evaluate the potential risk of testicular seminal dysfunction, inhibin B levels were assessed in pubertal boys. Inhibin B, produced by the Sertoli cells of the testes and by the Sertoli cells and germinal cells during puberty, is known as a marker of spermatogenesis in adults. Normal levels of inhibin B at the age of 8 years in ICSI boys were followed by a significant increase of inhibin B levels at the age of 14 years. In addition, inhibin B levels in ICSI boys were comparable with results from peers in the general population. Secondly, testicular function during puberty was assessed by measurement of salivary testosterone in 14-year-old ICSI boys and were found comparable with results from peers born after spontaneous conception. Reassuringly, serum inhibin B levels and salivary testosterone levels in boys from fathers with severe oligoozospermia (defined as 5 million spermatozoa per ml) were not different from concentrations in boys from fathers without severe oligoozospermia. In order to confirm that a normal progression through subsequent stages of pubertal development occurs and resulting in a normal reproductive capacity, long-term follow-up into adulthood was required.

Therefore, an extensive follow-up project in young adult men and women ranging between 18 and 22 years was set up.

Findings in men conceived by ICSI

Results of a single semen sample in 54 young adult ICSI men and 57 spontaneously conceived males were compared. Young ICSI adults had a lower median sperm concentration, lower median total sperm count and lower median total motile sperm count in comparison to spontaneously conceived peers. Furthermore, compared to males born after spontaneous conception, ICSI men were nearly three times more likely to have sperm concentrations below the World Health Organisation (WHO) reference value of 15 million/ml and four times more likely to have total sperm counts below 39 million. Furthermore, ICSI men had comparable mean levels reproductive hormones (FSH, LH, testosterone and inhibin B) in comparison to spontaneously conceived counterparts.

Findings in women conceived by ICSI

In the cohort of 71 young adult women, conceived by ICSI because of male infertility in their parents, antral follicle count and circulating reproductive hormone levels (including AMH, FSH, LH and DHEAS) were found to be comparable with results from peers born after spontaneous conception.

Ongoing studies

Currently, a follow-up study focusing on men conceived by ICSI with use of testicular (hence non-ejaculated) sperm born to fathers with azoospermia (absence of sperm in the ejaculate) is ongoing at the Center for Medical Genetics and the Center for Reproductive Medicine.

Medical and psychological follow-up of PGT children

Principal Investigators: Julie Nekkebroeck (psychological) and Florence Belva (Medical)

Study nurse: Andrea Buysse

Background

Preimplantation Genetic Testing (PGT) allows to determine specific monogenic defects, chromosomal imbalances and the gender of the embryo. PGT requires an embryo biopsy, which removes one or two blastomeres and is performed at the 6- to 8-cell cleavage stage. PGT is a more invasive technique compared to ICSI and conventional IVF and could induce potential risks for the children. PGT combined with HLA (Human Leukocyte Antigen) matching allows the selection of unaffected HLA-compatible embryos in order to cure a severely affected sibling with a transplantation using haematopoietic stem cells from the umbilical cord blood of the newborn. This new application of PGT raises numerous medical, psychological and ethical questions. It is important to evaluate the health of the children born after PGT as well as the psychological well-being of these children and their family.

The Center for Medical Genetics of the UZ Brussel has a longstanding experience and expertise in the follow-up of children born after application of Assisted Reproductive Technology (ART). As one of the few centers in Flanders and worldwide and within the framework of an ongoing commitment to the long-term follow-up of children born after PGT, the research team from the Center for Medical Genetics assesses physical health of these children in terms of growth and neuro-motor development.

Aims

• To assess whether PGT is associated with significant health, growth, mental and psychomotor outcomes in early childhood
• To evaluate parental well-being and the parent-child relationship after PGT
• To evaluate medical outcomes of PGT-HLA children
• To evaluate psychological well-being of children born after PGT-HLA and their siblings and to describe the impact of this treatment on the family relations

Results

Psychological outcome after PGT at 2 years and beyond

Seventy 2 year-old PGT singletons were examined and compared to the same number of ICSI and naturally conceived (NC) children. PGT singletons displayed normal mental and motor development when compared to ICSI and NC children. No differences for language development, temperament, and internalising problem behaviour were found.

PGT and ICSI mothers reported less total behaviour problems and externalising behaviour problems than the mothers in the NC group. ICSI fathers reported less total behaviour problems and externalising behaviour problems than the PGT and NC fathers. There were no differences between the parents on psychological well-being.

Results of the PGT twins were also very comparable with those of the ICSI and NC twins and this for all different domains of development.

Continued follow-up was performed in a group of 47 PGT singletons, 49 ICSI singletons and 48 singletons born after spontaneous conception (SC). The children were matched for age, gender, birth order and educational level of the mother. The cognitive abilities and motor skills of 5 to 6-year-old singletons born after PGT did not differ from results in ICSI or SC singletons.

Analysis of outcomes in PGT-HLA families is still ongoing.

Medical outcome after PGT at 2 years and beyond

Preimplantation Genetic Testing is an invasive procedure due to the embryo biopsy but offers a recourse for parents to prevent the transmission of a heritable disease to their offspring.  Although couples opting for PGT-M or PGT-SR are not necessarily infertile, they still have to run through all steps associated with Assisted Reproductive Technologies (ART), including ovarian stimulation and ICSI. Therefore, the medical outcome of children born after ICSI in combination with an embryo biopsy, should be closely monitored.

In the neonatal period, the outcome in terms of birth weight, low birth weight rate, prematurity rate, and major malformation rate was found comparable between PGT and ICSI singletons.

At the age of 2 years, although overall anthropometrics were comparable between 102 children born after PGT and 102 children born after ICSI, body mass index standard deviation scores tended to be lower in children born after PGT in comparison to children born after ICSI. The prevalence of major as well as minor congenital anomalies, hospital admissions and surgical interventions was similar for PGT and ICSI children.

At the age of 6 years, no differences in anthropometric measurements and blood pressure readings were found in more than 80 PGT singleton children in comparison with a matched cohort of peers born after ICSI without embryo biopsy.

PGT and its influence on children and their parents

Principal Investigator: Julie Nekkebroeck

Researcher: Christiane Winter

Follow-up of families and their children born after Preimplantation Genetic Testing (PGT)

Research questions to be answered

• Is the socio-emotional development of 5 to 6-year old PGT children comparable to ICSI children and spontaneously conceived controls (SC)?
• Do parents in the three groups differ regarding the way they experience stress from parenting and/or do they have different vulnerabilities?

Results

• Regarding their social-emotional development and family functioning, PGT children/families were very much comparable to ICSI and SC children/families.
• PGT and ICSI mothers experienced less stress, felt more competent and were more goal-directed than mothers of SC children.

Pregnancy and the early postpartum period after Preimplantation Genetic Testing (PGT): stress, attachment and coping

Getting pregnant is not that evident for couples with a history of a genetic disease in the family. Therefore, a second research line focused on the transition to parenthood of couples using PGT or ICSI compared to spontaneously conceived controls. This study is situated  against the background of the developmental origings of early brain and behaviour development and mental health (van den Bergh, 2011), stressing the relationship between prenatal and postnatal maternal mental health and diverse levels of their offsprings' health. In this study 59 PGT couples, 58 ICSI couples and 68 SC couples participated in the longitudinal project. Both, women and men, filled out questionnaires at each trimester of the pregnancy and at three months postpartum.

Research questions to be answered

• Do PGT couples differ in their experience of stress (depression, pregnancy specific anxiety) during pregnancy?
• Do conception groups differ in their attachment to the fetus and the baby?
• If there were group differences, do factors such as personality, attachment in partner relationship, attachment to ones’ own parents, idealization of parenthood, coping and life satisfaction function as moderators?

Results

• PGT couples are not more at risk of mental health problems (depression, pregnancy specific anxiety) than ICSI & SC couples
• We found that gender differences exist but they are not conception-group specific
• Pre- and postnatal attachment do not differ between conception groups and analysis of moderators did not reveal additional information

!!!!!This line of research has been discontinued!!!!

Human pluripotent stem cells as models for myotonic dystrophy type I

Principal Investigator: Karen Sermon

From 2002 onwards, REGE developed a new avenue of research: since numerous good-quality preimplantation embryos that carried a monogenic disease were donated for research after PGD, André Van Steirteghem came up with the idea to derive human embryonic stem cell lines from these embryos and to use them as model for monogenic diseases. The main focus of research for Karen Sermon in this field has remained human pluripotent stem cells as a model myotonic dystrophy type I. Both hESC and iPSC (http://emgerege.vub.ac.be/Research_ES_CS.php) have been proposed as tools for in vitro disease modelling, see for instance here. They could be used for the identification of disease biomarkers and to test the efficacy of new drugs or other types of therapies. This is particularly relevant for those diseases where animal models have been disappointing, or even not available. Moreover, if we would be able to replace animal models by in vitro hESC and iPSC models, this would mean a significant reduction in the number of lab animals used in medical research. One type of diseases for which disease modelling in hPSC has often been proposed are a group of neurodegenerative disorders that are caused by so-called dynamic mutations. These diseases such as Huntington’s disease, myotonic dystrophy type 1 (DM1) and fragile X syndrome are caused by the instability displayed by long trinucleotide repeats. For these diseases, there is currently no effective treatment to prevent or delay the progression of the disease. Given the complexity of the findings, it is not surprising that the mechanisms of trinucleotide repeat instability are still a subject of intense investigation. We have a longstanding project to elucidate the mechanisms of instability in DM1 and closely collaborate with Prof Christopher Pearson, one of the most influential researchers in this area (http://www.cepearsonlab.com/pearson.php).
 

The aims of this project are:

To characterise the behaviour of the trinucleotide repeat in the hESC lines derived from preimplantation embryos shown to be affected after preimplantation genetic diagnosis for DM1, and iPSC lines derived from patients who donated a skin biopsy and to dissect the different aspects of trinucleotide instability in DM1 making use of hESC and iPSC. Such aspects are for instance DNA methylation of the neighbouring DNA and the influence of DNA repair proteins that are known to erroneously repair the trinucleotide track. These aspects are studied both in undifferentiated hESC and iPSC and differentiated derivatives. Therefore, we differentiate our hESC and iPSC into disease-relevant cells such as skeletal muscle. The effect of for instance DNA repair proteins is studied through knock-out of these genes using CRISPR/Cas9 technology. Our work is sponsored by the major Flemish granting agencies FWO. We have also received continuous support from the Association Belge contre les Maladies neuro-Musculaires (http://www.abmm.be). We are also deeply indebted to Mrs Mireille Aerens, holder of the Mireille Aerens Chair at the Vrije Universiteit Brussel who generously sponsored the PhD of Lise Barbé