Projects

In humans, female infertility is an ongoing problem with an estimated prevalence of around 10%. Worldwide, obesity and overweight reached epidemic proportions and there is strong evidence for their link with infertility. The metabolic profile of obese women is commonly characterized by altered levels of blood parameter values. The latter is well reflected in the reproductive fluids and is believed to negatively affect fertility. Recent studies are shedding light on new forms of embryo–maternal communication via the delivery and/or exchange of extracellular vesicles, newly identified information carriers that exist in the uterine fluid. Animal models are valuable for basic and applied research in the field of reproductive biology especially due to ethical limitations and restricted availability of human biological material. Due to many similarities between bovine and human reproductive physiology, the cow has become a well-established and relevant model for human reproductive research. We hypothesise metabolic stress, as similarly described between obese women and lactating cow, is a key role player negatively affecting the uterine environment during early pregnancy. To proof this theory, we aim to perform novel in vivo and in vitro studies to unravel factors affecting the embryo-maternal cross talk in metabolically stressed dairy cows, focusing on the role of extracellular vesicles

There is currently an urgent need for new screening approaches to identify thyroid hormone disrupting chemicals, both in ecotoxicology and human toxicology. Toxicity testing in the 21st century increasingly relies on high throughput animal-free assays screening chemicals for their interaction with biological targets. While the thyroid system is highly conserved across vertebrate classes, conservation of the chemical susceptibility of the components of the thyroid system (enzymes, receptors) remains to be investigated. In this project, we will compare the deiodinase (the enzyme responsible for thyroid hormone (de)activation) inhibition potential of a set of test chemicals across zebrafish, pig, rat and man. This information is essential to bridge the gap between ecotoxicology and human toxicology and support the exchange of information.

According to WHO reports, over 10% of women in reproductive age (20-44y) are infertile or subfertile. Oocyte quality is one of the major causes of infertility, as evident in several metabolic diseases such as obesity and type II diabetes, as well as aging. During the last 5-7 years a substantial scientific evidence has been built in our research unit focusing on maternal metabolic health in relation to oocyte and embryo quality. We showed evidence that the ovarian follicular microenvironment is significantly altered under the influence of metabolic disorders leading to a direct detrimental lipotoxic impact on the oocyte, manifested by oxidative stress, mitochondrial dysfunction, endoplasmic reticulum (ER) stress and apoptosis. This fundamental knowledge should now form the basis to design a sustainable treatment or prevention strategy. There is a large need for safe drug formulations that can reach the oocyte and particularly influence mitochondrial and ER functions, with high bioavailability and efficiency. Targeting these pathways and organelles usually require the use of hydrophobic compounds. In addition, the oocyte is surrounded by several cumulus cell layers and a glycoprotein layer (zona pellucida) which form biological barriers. At the subcellular level, drug delivery to the mitochondria (such as antioxidants) is another challenge due to the complex inner membrane network structure, unusual phospholipid, Cardiolipin, and a strong negative charge. Information about clathrin- and caveolin-mediated endocytosis in mammalian oocytes, and subsequent formation of endosomes and liposomal degradation are very scarce. Studying these mechanisms and understanding the role gap junctional communication in delivering molecules from cumulus cells (CCs) to the oocyte may facilitate delivery of therapeutics or protective compounds to the ooplasm. This can be combined with strategically designed formulations of e.g. antioxidants to target defective organelles, such as mitochondrial and endoplasmic reticulum, and to reduce oxidative stress at the subcellular level. Our preliminary proteomics data show that proteins related to these endocytosis mechanisms are expressed in oocytes and cumulus cells, and are affected by oocyte quality, cellular metabolic stress and the microenvironment in which an oocyte is matured. The present project proposal aims to take the first steps in understanding the role of these proteins in mediating uptake of nutrients and other compounds in oocytes and CCs. This will be investigated by gene and protein expression analysis at different time points, uptake of trace molecules and using specific inhibitors of each mechanism. The effect of the latter on oocyte developmental competence will be examined. With this approach we aim to provide new fundamental knowledge that may increase treatment efficiency of enhance oocyte quality and fertility.

ERGO presents a new approach that will support a paradigm shift in the regulatory use of standardized test guidelines (TGs) by breaking the existing wall between mammalian and non-mammalian vertebrate testing and assessment of endocrine disrupting chemicals (EDCs). The highly conserved thyroid system will be used as the "proof of concept", but also other conserved endocrine axes/systems such as the Retinoid X Receptor (RXR) and the Hypothalamus Pituitary Gonadal (HPG) axis can be adapted to the cross-vertebrate class approach. ERGO will investigate a battery of draft in vitro assays and evaluate thyroid-responsive biomarkers and endpoints (B/E) suitable for extrapolation of effects from fish and amphibian tests to humans and other mammals (and vice versa) and finally validate successful B/E for inclusion in existing in vivo or new in vitro OECD TGs. A cross-class adverse outcome pathway (AOP) network will provide the scientifically plausible and evidence-based foundation for the selection of B/E in lower vertebrate assays predictive of human health outcomes. In silico modeling and biotransformation data will support cross-vertebrate class effect extrapolation. Major outcomes of ERGO will be: 1) New thyroid-related B/E for inclusion in OECD TGs for improved identification of TDC. 2) An Integrated Approach to Testing and Assessment (IATA) of chemicals for TD based on a multi-class vertebrate AOP network connecting endocrine mechanisms in one vertebrate class to adverse outcomes in another class for safer regulation of EDCs. 3) A tool for TG end users, such as regulators and industry, to extrapolate thyroid effects between vertebrate classes. Implementation of the ERGO IATA strategy in regulations of EDC will make hazard and risk assessment faster, cheaper, simpler and safer and support industry in the development of EDC-free products beneficial for environmental and human safety.

Obesity is becoming a global threat, reducing mother's health and reproductive capacities and affecting the offspring's health. Clear preconception care guidelines for obese future mothers are lacking. Clinical studies are flawed and fundamental studies in basic animal models are scarce. The importance of preconception weight loss on reproduction and baby's health is heavily debated and has never been investigated in detail. This project aims to uncover the role and the importance of clinically relevant preconception care advices to obese women planning for pregnancy. To do so, we propose strategically designed fundamental obese mouse models to assess the impact of preconception weight loss, diet normalization, increased physical activity, omega-3 rich diet or the combination thereof. We will focus on four distinct major research challenges: 1) can we find improvements of mother's metabolic profile before conception in relation to the observed weight loss; 2) does the mother's own fertility success increase; 3) can we improve the postnatal health of the offspring and 4) can we safeguard the offspring's reproductive physiology. Physiological and in depth molecular outcome parameters will be combined to generate a clear and integrated view on the effects of preconception care lifestyle interventions. If successful, these novel insights will be the basis for developing future awareness and education programs aiming at improved human maternal health at the time of conception.

Milk production in Cuba struggles to meet the demand of milk in the daily ration of many Cubans. Several factors resulted in a decrease of milk producing potential in dairy cattle herds. The lack of efficient (re)production strategies decreased the number of dairy cows that attain reasonable milk production levels. For this reason, sustainable measures are urgently required to improve the efficiency of dairy cattle management. The general objective of this project is to increase the milk production potential and as a consequence food security and economic well-being in general and of dairy farmers in Camagüey in particular. To do so, the project aims specifically (i) to implement a computer-based herd health management program for dairy herds in the Camagüey province that will permit the follow-up of (re)production, nutrition, and health parame-ters to finally increase milk production efficiency and (ii) to build up ICT capacity and improve digital literacy at the academic as well as the end-user level to stimulate the development of innovative IT solutions for the livestock sector.

The presence of certain toxicants/chemicals can perturb the endocrine system and induce a variety of responses, some of which can cause severe adverse effects in humans and wildlife. In order to protect human health and the environment, it is therefore important to identify those substances that can cause adverse effects via disruption of the endocrine system. Availability of test methods is an important factor in our ability to identify the substances that cause adverse effects via disruption of the endocrine system and in ensuring a high level of protection of human health and the environment. To ensure that test methods are internationally and mutually accepted a test guideline development programme has been established under the auspices of the OECD. Despite the progress achieved on the development and validation of test guidelines for evaluation of endocrine disruptors over the last 20 years, there are still some gaps and weaknesses in the current test methods for evaluation of endocrine disruptors. The objective of this study is to develop study protocols for testing of endocrine disrupting effects in biotic systems to improve the identification of substances disturbing the endocrine system by enhancing already existing test guidelines.

A disturbed maternal metabolism like in obesity or type II diabetes has clearly been associated with disappointing fertility. We extensively showed that such metabolic disorders have direct effects on the micro-environment of the growing and maturing oocyte, ultimately leading to reduced oocyte and embryo quality. Obesity is a global health threatening problem and recent studies indicated that maternal obesity may result in significant health issues in the offspring. More in depth mechanistic research clearly pointed out the importance of uterine programming in early pregnancy. It is not known however whether the metabolic status of obesity as such and/or potential direct effects of the typical fat rich western type diet are responsible for these observations. Based on the epidemiological relevance of obesity and hypercholesterolemia we hypothesize that obesity or an obesogenic diet of the mother around conception or during the entire pregnancy will alter the micro-environment of the growing embryo and fetus. This will change uterine programming ultimately leading to compromised offspring's health and reproductive physiology. To systematically investigate this hypothesis, we will feed female LDLR knock-out mice (LDLR-/-) an obesogenic diet A) several weeks before conception resulting in maternal obesity at conception or B) solely around conception or C) throughout the entire pregnancy. The offspring will be cross-fostered upon birth and will be used to study the general health of the offspring, the ovarian follicular reserve and the process of folliculo- and oogenesis, the offspring's pre-implantation embryo physiology and gene expression pattern and the receptivity of the offspring's uterus to support full pregnancy resulting in healthy offspring. By using this strategic experimental model we will be able to find the most sensitive window during pregnancy for uterine programming of reproduction, and it allows us to study the effects on every specific step on reproductive functioning. We believe that this project proposal may significantly contribute to the concept of "Developmental Origin of Health and Fertility" by further spreading the knowledge that epigenetic effects of maternal metabolism and diet may jeopardize health but also fertility in the offspring.

Metals play a pivotal role in various biochemical and physiological processes. They are widely found in nature, particularly in mineral deposits and soils, meaning that they are available to be taken up by plants and animals that serve as a food source for humans. Virtually, all metals, including the essential metal micronutrients, are toxic to both fresh and marine organisms as well as humans if exposure levels are sufficiently high. The toxicity of the most important metals such as copper, zinc, nickel, lead, cadmium, etc. have been extensively documented for various species and the results have been used to derive environmental standards. However, the studies on potential toxicity in combined metal scenarios via different routes of exposure and its comparison at different biological levels is scarce. Our study is a first of its kind, in which we will study mixed metal toxicity through different routes of exposure in different organisms. This will not only provide an insight into underlying molecular mechanisms in generic versus compound specific stress responses, but also compare the effects at different biological levels, via different routes of exposure. This will help to understand if the effects are species specific and whether the effects are dependent upon routes of exposure. We expect that with this study we should be able to predict the consequences of metal contamination not only from the toxicological view point but also from ecological point of view.

Obesity is becoming a global threat, reducing mother's health and reproductive capacities and affecting the offspring's health. Clear preconception care guidelines for obese future mothers are completely lacking. Clinical studies are flawed and more fundamental studies in basic animal models are very scarce. The importance of preconception weight loss on reproduction and baby's health is heavily debated and never been investigated in detail. This research project aims to propose strategically designed animal research models to provide relevant scientific evidence for effective and sustainable preconception care advices to obese women planning for pregnancy. To do so, we will assess the impact of preconception weight loss, diet normalization, increased physical activity, omega-3 rich diet or the combination thereof in obese outbred mother mice on four distinct major outcome parameters: 1) improvement of mother's metabolic profile before conception in relation to the observed weight loss; 2) mother's own fertility success; 3) postnatal health of the offspring and 4) offspring's reproductive physiology. Effective guidelines for preconception care lifestyle interventions in obese patients will drastically lower the (public) health care cost associated with assisted reproduction and should maximally safeguard the health of the baby. The data generated will be the basis for awareness and education programs aiming at improved maternal health at the time of conception.

Infertility is a major socio-economic problem affecting millions worldwide and is specifically linked to maternal obesity and other (diet induced) metabolic disorders. Understanding the mechanisms by which altered metabolism affect fertility is crucial for successful interventions. Mitochondria are the power house within the oocyte. Reduced somatic cell mitochondrial function occurs early in the pathogenesis of metabolic diseases. This is mainly due to the lipotoxic effects of elevated free fatty acid concentrations in blood. For the oocyte to be developmentally competent, the number and function of mitochondria should reach a certain threshold. There are several thousands of mitochondria in the mature oocyte derived from about 20 mitochondria in the germ cell. In addition to their bio-energetic roles, mitochondria are also sensors of stress. Oxidative stress and associated cellular damage elicit stress signalling between the mitochondria and the nucleus to start a protective machinery. The effects of metabolic stress on mitochondrial replication and stress responses during oocyte growth and subsequent embryo development are not known. In this project we will use in vitro and in vivo animal models to study mitochondrial functions and stress responses under maternal metabolic stress conditions in growing oocytes. Defect-based protective and rescue interventions will also be tested to investigate opportunities for curative interventions.

Endocrine disruption is a major concern for the health of wildlife populations. Although many studies have shown reproductive impairment as a consequence of endocrine disruption in adult fish, knowledge of the consequences of endocrine disruption for vertebrate embryonic and larval development is scarce. The zebrafish embryo is an ideal model system to investigate the fundamental mechanisms of endocrine disruption. We will first describe the timing of the normal embryonic activation of the hormone synthesis machinery, as well as the hormone profiles, during the early stages of vertebrate development, which has never been done so far. Secondly, using well-described endocrine disrupting compounds, we will specifically disrupt these profiles and propose mechanisms linking the changed profiles to observed developmental effects. We will then validate the proposed mechanisms of toxicity using targeted disruption techniques, including knockout and morpholino knockdown. This project will offer the information that is needed for follow-up projects to develop assays to specifically assess the risk associated with exposure to different classes of EDCs on embryonic and larval development, allowing discrimination among ER and AR agonism and antagonism, as well as aromatase inhibition. Such assays would fit perfectly within both EU and USA regulatory priorities for screening and prioritizing potential EDCs.

Subfertility is a global public health issue. The main causes of subfertility are metabolic diseases such as obesity or type 2 diabetes. Also in farm animals, the link between metabolic stress and subfertility has been acknowledged. Maternal metabolic disorders are associated with upregulated lipolysis leading to a disturbed metabolic profile in the blood (increased non-esterified fatty acids (NEFAs), ketones, altered glucose and insulin). Using the cow as a model, we showed that this altered metabolic profile is reflected in the follicular and oviductal environment in which the oocyte and embryo develop, subsequently altering oviductal functions and jeopardizing oocyte and embryo development and quality. More mechanistic research revealed that increased oxidative stress is one of the key underlying pathways associated with metabolic stress at oocyte and embryo level. Therefore, antioxidants may play an important role in alleviating oxidative stress in metabolically compromised embryos, hereby ameliorating embryo developmental competence and quality and thus fertility. We already showed that circulating antioxidants are reflected in the oocyte's micro-environment and can be altered by the diet. Little is known about the antioxidative environment of the pre-implantation embryo (in the oviduct) and whether these antioxidants can influence the development and quality of metabolically stressed embryos. As such, we hypothesize that dietary antioxidant supplementation in metabolically compromised mothers can change oviductal concentrations of antioxidants hereby changing oviductal cell functions and ameliorating embryo developmental competence and quality. Therefore, the objective of the research proposed here is to culture in vitro derived bovine zygotes in the oviducts of metabolically stressed ewes supplemented with antioxidants and investigate the antioxidative status of the oviductal fluid, the characteristics of the oviduct at the transcriptomic level and the embryo developmental competence and quality. Enhancing the oviductal environment to better support early embryo development is crucial for the embryos to establish successful pregnancy later in development. These results can contribute to drastically reduced medical costs for subfertility treatments. In addition, they may tackle the subfertility problem in high yielding dairy cows hereby improving sustainable and profitable farming.

Subfertility represents a major problem in domestic animals and humans. In cattle but also in women, up to 40% of total embryonic losses occur between days 7 and 16 of pregnancy, indicating that early embryonic mortality is a major cause of reproductive failure. A finely tuned synchrony between the competent embryo and a receptive endometrium is the key for optimal embryo development and the establishment of a successful pregnancy. Several studies have indicated the existence of complex paracrine and endocrine in vivo communication between early embryo and the maternal tract in mammals. However, so far there is no evidence that the embryo significantly interacts with the endometrium before elongation in the cow (begins 13 days post-mating) or before real implantation in human (day 10-17), while most of the early embryonic losses happen at that time. To understand the pathways of early pregnancy loss we have to elucidate the physiological molecular and biochemical processes underpinning and regulating the earliest maternal-embryonic cross talk upon the moment of embryo arrival in the uterus. In the present project, using the cow as a proven research model to study the very first developmental stages of the pre-implantation embryo, our central aim is to study the embryo/conceptus and endocrine programming of the endometrium to support pregnancy success. In the first part of the PhD research (Milestones 1 and 2, conducted at the University of São Paulo) we hypothesized that exposure to an embryo changes the abundance of specific transcripts and the biochemical composition of the uterine fluid in the cranial region of the pregnant uterine horn. As such an effect is likely to be very local in nature, we developed an in vivo model that allowed sampling the endometrium closer to the embryo (Sponchiado et al., 2017). To limit the use of experimental animals, in the second part of the PhD research (Milestone 3), we propose to use an innovative in vitro co-culture system to investigate the nature of the specific interaction between bovine embryos and endometrial epithelial cells and how this ultimately may affect early embryo quality. This part of the project will be conducted at the University of Antwerp. The proposed combination of in vivo and in vitro systems with state-of-the art analytical tools of transcriptome and metabolomic research should reveal a plethora of candidate genes and bioactive molecules to understand the pathways of the very first embryo-maternal dialogue. The very first communication between the mother and her embryo is undoubtedly one of the most exciting processes in reproductive biology. The knowledge of these mechanisms should help to understand the problem of early embryo mortality and pregnancy loss leading to disappointing fertility results. Only then it will be possible to build on preventive and even therapeutic measures impacting in pregnancy success both in human and agricultural application.

Nano-injection is a promising alternative exposure route for toxicological research using fish embryos. We recently started applying nano-injections and observed differences in toxicity compared to exposure via water. While it is plausible that uptake and biotransformation play a role in these differences, this has never been investigated. The aim of this project is to characterize and understand differences in toxicity following nano-injection compared to aquatic exposure. We will compare accumulation, biotransformation, molecular events and physiological effects after exposure of zebrafish embryos to the same chemical via both exposure routes. This information is essential to evaluate the applicability of this promising alternative exposure route in (eco)toxicology.

Oxidative stress, mitochondrial and endoplasmic reticulum (ER) stress have been recently postulated as main drivers of altered pathways in the oocyte linking maternal metabolic disorders to low fertility outcomes. Upregulated lipolysis, commonly associated with obesity and other metabolic disorders, increases the concentrations of lipotoxic fatty acids in the follicular fluid, which were shown to have a direct detrimental impact on oocyte quality and subsequent embryo development. Oocyte maturation involves complex nuclear, cytoplasmic, and molecular changes that determines its developmental competence. In human IVF clinics, oocytes are usually collected after hormonal stimulation and thus after exposure to metabolic stress during maturation in the follicle in affected mothers. It is not known if the deterioration in developmental competence of these metabolically-compromised oocytes can be rescued by alleviating mitochondrial and endoplasmic stress during or after fertilization, or should the stress be prevented during maturation. This project aims to test sensitive windows for preventative or reparative measures that may improve in vitro embryo production from metabolically-compromised oocytes, and examine the intrinsic quality of the embryos produced after treatment. A well-established bovine in vitro model will be used to generate metabolically-compromised oocytes by exposure to elevated pathophysiological levels of palmitic acid. MitoQ, a highly efficient mitochondria-targeted antioxidant, and Sirolimus, a specific ER-stress inhibitor, will be used to alleviate cellular stress either during fertilization or embryo culture, or during maturation. Embryo development will be recorded and blastocysts quality will be assessed by examining their metabolism, cell proliferation, cell lineage, DNA damage, markers for cellular stress and apoptosis. RNA-seq will be used to detect any persistent alterations in the transcriptomic profile. These studies will help defining windows of sensitivity and efficiency of targeting intracellular stress as a treatment option for infertile patients suffering from metabolic diseases.

This project focuses on how strategically applied anti-oxidants in the ratio can improve dairy cow fertility through an improved oocyte and embryo quality. The research will focus on very specific parameters of the oocyte's and embryo's micro-environment and of the quality of the female gamete.

Metabolic disorders have a profound effect on many aspects of reproduction. A growing number of animal in vivo studies indicate that maternal metabolic diseases not only jeopardize embryo implantation, but also program the well-being of the fetus. Awareness grows on the connection between maternal metabolic health, embryo development and blastocyst cell differentiation. This link is conceptualized as the developmental programming hypothesis whereby environmental influences during critical periods of developmental plasticity can elicit effects from mother-toembryo and even from mother-to-child. During the critical first week of development, the embryo has a remarkable metabolic flexibility that allows to grow and develop, even under suboptimal nutrient conditions. However, altered metabolic strategies in the early embryo might come at a cost. Stem cell studies continue to elicit the intriguing link between nutrient abundance and processes that drive cell differentiation. In this context, we propose that the early bovine embryo, developing in a metabolically compromised individual, will contend a severely impaired capacity to support a normal maternal-embryonic crosstalk upon arrival in the uterus. Thus far, no studies enlighten the link between nutrient sensing paths and blastocyst cell lineage differentiation, although understanding of the underlying pathogenesis will be indispensable for designing effective treatments.

A disturbed maternal metabolism like in obesity or type II diabetes has clearly been associated with disappointing fertility. We extensively showed that such metabolic disorders have direct effects on the micro-environment of the growing and maturing oocyte, ultimately leading to reduced oocyte and embryo quality. Obesity is a global health threatening problem and recent studies indicated that maternal obesity may result in significant health issues in the offspring. More in depth mechanistic research clearly pointed out the importance of uterine programming in early pregnancy. It is not known however whether the metabolic status of obesity as such and/or potential direct effects of the typical fat rich western type diet are responsible for these observations. Based on the epidemiological relevance of obesity and hypercholesterolemia we hypothesize that obesity or an obesogenic diet of the mother around conception or during the entire pregnancy will alter the micro-environment of the growing embryo and fetus. This will change uterine programming ultimately leading to compromised offspring's health and reproductive physiology. To systematically investigate this hypothesis, we will feed female LDLR knock-out mice (LDLR-/-) an obesogenic diet A) several weeks before conception resulting in maternal obesity at conception or B) solely around conception or C) throughout the entire pregnancy. The offspring will be cross-fostered upon birth and will be used to study the general health of the offspring, the ovarian follicular reserve and the process of folliculo- and oogenesis, the offspring's pre-implantation embryo physiology and gene expression pattern and the receptivity of the offspring's uterus to support full pregnancy resulting in healthy offspring. By using this strategic experimental model we will be able to find the most sensitive window during pregnancy for uterine programming of reproduction, and it allows us to study the effects on every specific step on reproductive functioning. We believe that this project proposal may significantly contribute to the concept of "Developmental Origin of Health and Fertility" by further spreading the knowledge that epigenetic effects of maternal metabolism and diet may jeopardize health but also fertility in the offspring.

This project aims to develop an alternative testing strategy to reduce the need for fish early life-stage toxicity tests (FELS) for the assessment of chronic toxicity of chemicals to fish, using the adverse outcome pathway (AOP) framework as a guidance for assay development. Currently, the FELS test (OECD TG 210) is one of the primary testing guidelines used to estimate the chronic toxicity of chemicals to fish. Results obtained using this TG are used to support risk assessment around the world. However, important limitations of this animal test are being recognized, including (1) the high numbers of fish used, (2) the relatively low-throughput, and (3) the lack of mechanistic information. In order to comply with the 3R principles, we are developing a non-animal testing strategy which includes both in vitro tests and in vivo whole organism alternative 120 hpf (hours post fertilization) ZFET (zebrafish embryo acute toxicity test) assays. One of the main research questions of this project is to what extent an assay development approach based on the AOP framework is capable of offering a mechanistic basis for selecting assays to develop an alternative testing strategy that allows the prediction of chronic FELS toxicity. This project is a follow-up of Cefic LRI-ECO20, and aims to validate assays that were developed during that project.

This project aims to develop an alternative testing strategy to reduce the need for fish early life-stage toxicity tests (FELS) for the assessment of chronic toxicity of chemicals to fish, using the adverse outcome pathway (AOP) framework as a guidance for assay development. Currently, the FELS test (OECD TG 210) is one of the primary testing guidelines used to estimate the chronic toxicity of chemicals to fish. Results obtained using this TG are used to support risk assessment around the world. However, important limitations of this animal test are being recognized, including (1) the high numbers of fish used, (2) the relatively low-throughput, and (3) the lack of mechanistic information. In order to comply with the 3R principles, we are developing a non-animal testing strategy which includes both in vitro tests and in vivo whole organism alternative 120 hpf (hours post fertilization) ZFET (zebrafish embryo acute toxicity test) assays. One of the main research questions of this project is to what extent an assay development approach based on the AOP framework is capable of offering a mechanistic basis for selecting assays to develop an alternative testing strategy that allows the prediction of chronic FELS toxicity.

To further expand on this intriguing patho-phsyiological role of elevated NEFA in the problem of subfertility, it need to be addressed whether NEFA can induce epigenetic changes and whether this affects further embryo development and postnatal health. Therefore, this research proposal concentrates on the effect of oocyte maturation under elevated NEFA conditions on DNA methylation patterns in Day 7 embryos, on further pre-implantation in vivo development and on postnatal health and growth. A state of the art in vitro embryo culture and embryo transfer protocols will be used combined with advanced molecular techniques. The integration of data on Day 7 embryo physiology, DNA methylation, further development and postnatal health could provide key metabolic information on the role of elevated NEFA concentrations in reproductive failure.

Metabolic disorders have a profound effect on many aspects of reproduction. High rates of early embryonic loss are a major issue. Upon arrival in the uterus, the proportion of surviving embryos drops off rapidly, which points towards troubled first interactions between embryonic and endometrial tissues. Recent insights suggest that embryo energy metabolism can be involved, as nutrient-sensing pathways regulate differentiation, the latter being crucial to guarantee the first interplay between embryonic and endometrial cells. During the critical first week of development, the embryo has a remarkable metabolic plasticity that allows to grow and develop, even under suboptimal nutrient environments. At the blastocyst stage, this early metabolic adaptation may come at a cost, ultimately leading to pregnancy failure. Herein, we hypothesise that suboptimal metabolic conditions in the early environment of the embryo can impact on the differentiation signature of the resultant trophoblast cells. This might be 'sensed' by the endometrium. We also propose that a differentiated endometrium has the potency to reset and rebalance the embryonic metabolism. Embryo and endometrial 'go' or 'no-go' responses will be studied using bovine in vitro and in vivo set-ups. While embryo implantation varies among species, the initial events between trophoblast and endometrial cells are shared among mammals, which implies that crossspecies lessons can be learned from the cow.

Several fish, amphibian and reptile species exhibit non-genetic sex determination, which is currently poorly understood. Recent advances have identified several genes and pathways involved based on expression patterns, but the functional role of these genes in sex determination and differentiation remains unclear. By endocrine disruption of this process, we will investigate the role of selected genes in sex differentiation in the zebrafish.

Pharmaceutical companies are obligated to perform an environmental risk assessment for each new drug that they launch on the market. The mandatory tests for potential endocrine disrupting compounds require a lot of time and laboratory animals. Therefore, the purpose of this study is to develop a zebrafish embryo test, which is not considered as animal test, that is capable of quickly detecting estrogenic properties of pharmaceuticals.

A disturbed maternal metabolism like in obesity or type II diabetes has clearly been associated with disappointing fertility. We extensively showed that such metabolic disorders have direct effects on the micro-environment of the growing and maturing oocyte, ultimately leading to reduced oocyte and embryo quality. Obesity is a global health threatening problem and recent studies indicated that maternal obesity may result in significant health issues in the offspring. More in depth mechanistic research clearly pointed out the importance of uterine programming in early pregnancy. It is not known however whether the metabolic status of obesity as such and/or potential direct effects of the typical fat rich western type diet are responsible for these observations. Based on the epidemiological relevance of obesity and hypercholesterolemia we hypothesize that obesity or an obesogenic diet of the mother around conception or during the entire pregnancy will alter the micro-environment of the growing embryo and fetus. This will change uterine programming ultimately leading to compromised offspring's health and reproductive physiology. To systematically investigate this hypothesis, we will feed female LDLR knock-out mice (LDLR-/-) an obesogenic diet A) several weeks before conception resulting in maternal obesity at conception or B) solely around conception or C) throughout the entire pregnancy. The offspring will be cross-fostered upon birth and will be used to study the general health of the offspring, the ovarian follicular reserve and the process of folliculo- and oogenesis, the offspring's pre-implantation embryo physiology and gene expression pattern and the receptivity of the offspring's uterus to support full pregnancy resulting in healthy offspring. By using this strategic experimental model we will be able to find the most sensitive window during pregnancy for uterine programming of reproduction, and it allows us to study the effects on every specific step on reproductive functioning. We believe that this project proposal may significantly contribute to the concept of "Developmental Origin of Health and Fertility" by further spreading the knowledge that epigenetic effects of maternal metabolism and diet may jeopardize health but also fertility in the offspring.

Thyroid hormones (THs) play a crucial role in development. At present, experimental studies on the cellular and molecular mechanisms underlying TH-dependent development have mainly concentrated on the later stages of development, while the role of THs during early development is less clear. The objective of this study is to further elucidate the role of THs in vertebrate embryonic development using zebrafish embryos.

This project represents a formal research agreement between UA and on the other hand the Federal Public Service. UA provides the Federal Public Service research results mentioned in the title of the project under the conditions as stipulated in this contract.

This research project concentrates on the effect of oocyte maturation, fertilization and embryo culture under high NEFA conditions on fertilization rate, DNA transcription and methylation patterns in Day 7 embryos.

This project represents a formal research agreement between UA and on the other hand the Flemish Public Service. UA provides the Flemish Public Service research results mentioned in the title of the project under the conditions as stipulated in this contract.

To further expand on this intriguing patho-phsyiological role of elevated NEFA in the problem of subfertility, it need to be addressed whether NEFA can induce epigenetic changes and whether this affects further embryo development and postnatal health. Therefore, this research proposal concentrates on the effect of oocyte maturation under elevated NEFA conditions on DNA methylation patterns in Day 7 embryos, on further pre-implantation in vivo development and on postnatal health and growth. A state of the art in vitro embryo culture and embryo transfer protocols will be used combined with advanced molecular techniques. The integration of data on Day 7 embryo physiology, DNA methylation, further development and postnatal health could provide key metabolic information on the role of elevated NEFA concentrations in reproductive failure.

This project represents a formal research agreement between UA and on the other hand the Province of Antwerp. UA provides the Province of Antwerp research results mentioned in the title of the project under the conditions as stipulated in this contract.

The main objective of this project is to use a mechanistic framework to develop and propose a high-throughput tiered-testing strategy for screening and prioritizing chemicals for FELS testing (OECD 210). The main objective can be divided into four parts: 1) Establish a database of toxicologically relevant FELS-specific AOPs, identify molecular initiating events and subsequent intermediate responses resulting into the apical outcome of interest 2) Propose Tier 1 in vitro screening toolbox to test for AOP-specific events and responses predictive for FELS chronic toxicity. 3) Propose Tier 2 whole-organism ZFET assays to test for AOP-specific events and responses predictive for FELS chronic toxicity and assess the potential of a ZFET molecular screening tool to predict cellular, organ and/or organism responses giving rise to FELS chronic toxicity. 4) Offering a proposal for implementation of a tiered-testing strategy in EU regulation. Assessment of usefulness and applicability of tiered testing strategy for global scientific and regulatory community.

This project represents a research contract awarded by the University of Antwerp. The supervisor provides the Antwerp University research mentioned in the title of the project under the conditions stipulated by the university.

Previous research work revealed that metabolic changes, associated with the growing incidence of obese individuals and diabetics, might have harmful repercussions for the reproductive outcome. The consequences of hyperglycemia, due to obesity and diabetes, have already been investigated thoroughly in rats and mice. This research project rather involves another feature of these metabolic pathologies: the high free fatty acid concentrations in blood. The interest of the human assisted reproduction society in a bovine model to assess the influence of free fatty acids on the oocytes developmental competence, on the viability and function of the granulosa cells and the inquisitiveness to the underlying mechanism of these potential effects, imposes us to further research.

This project represents a formal research agreement between UA and on the other hand a private institution. UA provides the private institution research results mentioned in the title of the project under the conditions as stipulated in this contract.

This project represents a formal research agreement between UA and on the other hand UGent. UA provides UGent research results mentioned in the title of the project under the conditions as stipulated in this contract.

Teratogenic chemicals are prioritized under the EU REACH legislation. Within this framework there is a need for routine testing tools to investigate the mechanistic basis of teratogenicity and to determine whether a substance has teratogenic potential. It has become clear that not only genetic factors (DNA nucleotide sequence) but also epigenetic factors such as DNA methylation, regulating gene behaviour, play a crucial role in teratogenicity. During vertebrate early embryonic development a vital epigenetic reprogramming event takes place (the sequential demethylation and de novo remethylation of the embryonic genome). The extent to which disturbance of methylation mechanisms during embryonic stages is involved in the development of teratogenic effects is still unclear and will be the subject of this study. This project will use the zebrafish embryo, which is already an established test system for the study of developmental effects, as an alternative to using mammals for the study of the role of epigenetic factors in teratogenicity. We aim to achieve two goals: first, we will determine to what extent DNA methylation changes are responsible for transcription changes involved in teratogenicity. Secondly, we will identify and confirm key genes and pathways involved in teratogenicity based on the integration of the molecular data with developmental parameters. Zebrafish embryos will be exposed to different concentrations of chemicals with known teratogenic mode of action during different stages of embryonic development, either during or after the de novo remethylation event. The embryos will be monitored individually until hatching. A large set of developmental parameters will be scored (e.g. heart rate and morphological malformations of body shape, somites and tail). Additionally, after pre and post de novo methylation exposure, RNA and DNA will be extracted which will be used in gene expression and DNA methylation microarray analyses. Based on these microarray data we will establish to what extent differential gene expression that is directly involved in teratogenicity is determined by differences in methylation status. Finally, we will statistically link these molecular data with the observed developmental effects. Key genes will thus be identified which are linked to specific developmental abnormalities. The selected key genes will be confirmed by both bisulfite sequencing (gene specific DNA methylation status) and real-time PCR (gene specific mRNA levels). Although a few previously published studies focused on general methylation patterning in zebrafish, we would be the first to study methylation dynamics in zebrafish embryos in detail on a genome wide scale, linking such data to classical gene expression data. Advancing the zebrafish embryo as an alternative test system for epigenomics research will pave the way for its application in several areas of research including the development of a classifier able to identify teratogenic compounds and the identification of targets for disease prevention or therapy.

This project represents a formal service agreement between UA and on the other hand VIB. UA provides VIB research results mentioned in the title of the project under the conditions as stipulated in this contract.

This project represents a formal research agreement between UA and on the other hand KULeuven. UA provides KULeuven research results mentioned in the title of the project under the conditions as stipulated in this contract.

The project focuses on the long term culture of pre-antral follicles opens a whole new approach and frame work making it possible to study the effects of maternal nutrition and metabolism on the pre-ovulatory follicles and growing and maturing oocytes.

A disturbed maternal metabolism may induce disappointing fertility and may jeopardize the offspring's health. Only recently, the importance of the early developmental stages in life has gained scientific attention in subfertility research. This project focuses on the metabolic and (epi)genetic consequences of long-term elevated non-esterified fatty acid (NEFA) serum concentrations in the dam on ovarian physiology, oocyte and embryo quality. Elevated NEFA cocnetrations are a key factor in several metabolic disorders such as diabetes, negative energy balance and obesitas and is typical for lipolysis. Previous research showed already that these elevated NEFA concentrations are reflected in the follicular fluid of the dominant follicle and that they cause a reduction in the oocyte's developmental competence. To mimic the in vivo situation even better and to substantiate the causative role of elevated NEFA in the pathogenesis of subfertility, a long term exposure model during the process of follicular growth should be developed. Therefore murine secondary follicles will be isolated and will be cultured for 12 days. These growing and maturing follicles will be exposed to elevated NEFA conditions and the effects on folliculogenesis, oocyte developmental competence and embryo quality will be studied in detail.

Proposed by the University, the Francqui Foundation each year awards two Francqui Chairs at the UAntwerp. These are intended to enable the invitation of a professor from another Belgian University or from abroad for a series of ten lessons. The Francqui Foundation pays the fee for these ten lessons directly to the holder of a Francqui Chair.

A disturbed maternal metabolism may induce disappointing fertility and may jeopardize the offspring's health. Only recently, the importance of the early developmental stages in life has gained scientific attention in the study of the pathogenesis of subfertility. This project focuses on the metabolic and (epi)genetic consequences of long-term elevated non-esterified fatty acid serum concentrations in the dam on folliculogenesis, oocyte developmental competence and embryo quality.

This project represents a formal research agreement between UA and on the other hand the Federal Public Service. UA provides the Federal Public Service research results mentioned in the title of the project under the conditions as stipulated in this contract.

Making the fatty acid composition of the human diet more poly-unsaturated is generally proposed as beneficial for health. This combined in vivo and in vitro bovine research model will elucidate the direct consequences of such a fatty acid shift on follicular fluid composition, on oocyte and embryo quality. Earlier work on dairy cows revealed several contradicting results, indicating the need for more in depth research.

While Cuba slowly recovers Gom an economical disaster in the nineties, there are numerous problems left asking for a sustainable solution, one of which is the severe structural shortage in milk production reflected in the absence of milk in the daily ration of many Cubans. A decline in support from former partners and the embargo lead to the loss of milk producing potential in the remaining cattle herds. The lack of operational reproduction techniques to be used as selection tools, severely decreased the absolute number of dairy cows. The current project aims to re-introduce dairy cattle genetics by creating a nucleus herd of cross bred bulls by applying modern cattle breedmg techniques to fertilize oocytes Gom the best local breed founder cows with (imported) dairy cattle sperm. The use of native breeds assures the cross bred products to be adapted to tropical conditions being resistant to relevant parasitic theads. Following this high-tech first phase of the project, the cross bred bulls themselves will propagate dairy cattle genetics in the population by simple, cheap and sustainable Artificial Insemination and natural mating. This way, relevant genetics can preferentially he made available to those farms which have access to the best cattle feeding conditions.

This project represents a formal research agreement between UA and on the other hand the Province of Antwerp. UA provides the Province of Antwerp research results mentioned in the title of the project under the conditions as stipulated in this contract.

Previous research work revealed that metabolic changes, associated with the growing incidence of obese individuals and diabetics, might have harmful repercussions for the reproductive outcome. The consequences of hyperglycemia, due to obesity and diabetes, have already been investigated thoroughly in rats and mice. This research project rather involves another feature of these metabolic pathologies: the high free fatty acid concentrations in blood. The interest of the human assisted reproduction society in a bovine model to assess the influence of free fatty acids on the oocytes developmental competence, on the viability and function of the granulosa cells and the inquisitiveness to the underlying mechanism of these potential effects, imposes us to further research.

Previous research work revealed that metabolic changes, associated with the growing incidence of obese individuals and diabetics, might have harmful repercussions for the reproductive outcome. The consequences of hyperglycemia, due to obesity and diabetes, have already been investigated thoroughly in rats and mice. This research project rather involves another feature of these metabolic pathologies: the high free fatty acid concentrations in blood. The interest of the human assisted reproduction society in a bovine model to assess the influence of free fatty acids on the oocytes developmental competence, on the viability and function of the granulosa cells and the inquisitiveness to the underlying mechanism of these potential effects, imposes us to further research.

Hypercholesterolemia is a human disorder and it has been associated with an unhealthy eat pattern. This combined in vivo and in vitro bovine model will elucidate the effect of nutritionally induced hypercholesterolemia on oocyte and embryo quality. In earlier work we showed that the metabolic changes in dairy cows early post partum and their consequences on oocyte and follicle quality is a valuable model for research in human infertility.

The proposed study will investigate the impact of local treatment with FSH, IGF-I or a combination of both, on follicular dynamics and oocyte quality of bovine, pre-antral follicles in living donors. To do so, a new technique will be developed and validated, to take ovarian biopsies at regular time intervals during treatment, by means of repeated, ultrasound-guided, transvaginal puncture of the ovaries. This procedure, combined with histological examinations and in vitro culture techniques, will help us to investigate on which follicular stage FSH and IGF-I exert their stimulatory effect. The data collected will offer us new insights on the application of hormonal ovarian stimulation and lead to a better understanding of the primordial to primary stage follicle transition.