21 st Workshop Molecular Biology of

  the German Society for Reproductive Medicine(DGRM)

	Tagungsort:
	Weimar

21. Arbeitskreis Molekularbiologie

der Deutschen Gesellschaft für Reproduktionsmedizin

(DGRM)

09.12.-10.12.2022 in Weimar Programm Friday, 09.10.2022 16:00

						11:45	Hamers	Ilsa	Bonn	Generation of a novel protamine allele allows
		Come together						testing of current hypotheses on protamine function
	17:00	Welcome and opening			12:00	Herbicht	Rebecca	Mariensee	Evaluation of a new sperm purification device
									for preparing bovine frozen-thawed semen for
	Invited Key Note Lecture 17:10 Chavatte-	Pascale	Jouy-en-Josas,	The sinuous paths for a career in reproductive	12:15	Lunch		in vitro fertilization
		Palmer		France	biology
	18:00	Group discussions and snacks		Session Placenta, Preeclampsia I
						13:30	Raja-Xavier Janet	Tübingen	Placental Growth Factor mediated maternal
	Saturday, 10.12.2022							NFAT5-SGK1-VEGF signaling axis in the pathogenesis of preeclampsia
	Session Ovary and Endometrium 09:00 Wolff von Gudenberg Helena 09:15 Altgilbers Stefanie 09:30 Yang Zhiqi 09:45 Heukamp Mariam 10:00 Obermeyer Ulla	Heidelberg Mariensee Tübingen Münster Münster	Extractions from ovarian granulosa cells may have the potential to change the receptivity of the endometrium during the implantation window Genome editing in primordial germ cells of domestic chicken -blue eggshell color Loss of endometrial DJ-1 (PARK7) is associated with recurrent pregnancy loss Establishment of a cell culture based 3D-in vitro-endometrial-model to explore endometrial receptivity in the context of female infertility Impact of cytokines and transgelin on proreceptive gene expression and trophoblast adhesion in a novel 3D model of the window of implantation	13:45 14:00 14:15 14:30 14:45	Mittel-berger Valdes Reisch Riedel Coffee break	Johanna Daniela Beatrix Alina	Augsburg Berlin Essen Essen	The programmed cell death protein 1 (PD1) and its ligand (PD-L1) are significantly downregulated on macrophages and hofbauer cells in the placenta of preeclampsia patients Impaired placental cell communication at the maternal foetal interface in gestational hypertension and environmental stress Therapeutische Ansätze zur Entfernung des anti-angiogenen sFLT-1 bei der Präeklampsie -Daten aus der Klinik und im Mausmodell Erhöhte Level von Anti-Angiogenese während der Schwangerschaft verändern die maternalen Immunantworten und die lymphatische Mimikry in den Spiralarterien in einem transgenen sFLT-1 Mausmodell der Schwangerschaftserkrankung Präeklampsie
	10:15	Coffee break
						Session Placenta, Preeclampsia II
	Session Embryo and Male fertility 10:45 Nikolou Iliana 11:00 Neufeld Gregor 11:15 Kovacevic Andela 11:30 Wiesejahn Christoph	Tübingen Mariensee Bonn Bonn	Embryo-derived trypsin-induced Ca2+ entry is blocked by infertility factor, LEFTY2 Dissecting the influence of IVF on bovine embryo development through single cell sequencing Cylicins are a structural component of the sperm calyx being indispensable for male fertility in mice and human Prm1+/-Prm2+/-male mice are infertile and display various defects in sperm, as well as early arrest during preimplantation development	15:00 15:15 15:30 15:45 16:00	Murrieta-Coxca Ujcic Große Fuentes Zacarías Election of the best presentation José Jena Martin Kaja Jena Silke Jena Paulina Jena Award ceremony End and farewell	Effect of preeclampsia-derived EVs on the blood-brain barrier integrity and microglia activation using static and microfluidic models Placental extracellular vesicles could modify hematopoiesis during pregnancy Multiplexing senescence markers in the placenta: elucidating premature aging in pregnancy pathologies by spatial phenotyping Placental transfer of PETN by ex vivo human placenta perfusion


Davis Laundon

Neil Gostling

Shelley Harris

Pascale Chavatte-Palmer

Rohan Lewis

Multiscale

Multiscale correlative imaging of horse and zebra placental villi

Despite having a single evolutionary origin, the mammalian placenta exhibits wide interspecific morphological and structural variation. Equine (horses and their kin) placentas display branching villi which sit in apposition with maternal tissue and represent the site of fetomaternal nutrient and waste exchange. Three-dimensional imaging techniques have recently identified and quantified novel structures in human placental villi, however similar tools have yet to be broadly applied to other species. Such approaches have the potential to both expand our understanding of comparative placentation and better resolve the structural composition of the studied taxa. Using scanning electron microscopy (SBF-SEM) of horse and zebra placenta, we demonstrated the presence of stromal macrovesicles previously only observed in human placental villi. Here, we also present a workflow for correlative threedimensional imaging of equine placental villi by combining x-ray microtomography (microCT) and SBF-SEM. This allows calculation of the total surface area of the equine placenta including microvilli. Through this workflow, we quantify the villus structure across multiple orders of magnitude in horse and zebra placentas. These morphometric data, including volume, surface area, and branching angle, help us to better resolve equine placental organization and contribute towards a holistic understanding of equine placental function.


Danger Michael

Alexandre Bec

Jérôme Spitz

Perga Marie-Elodie

Questioning the roles of resources nutritional quality in Ecology Editorial Special issue -Oikos

Keywords: literature analysis, ecological stoichiometry, nutritional geometry, macro and micro-nutrients, trophic ecology

Our understanding of ecosystem functioning is strongly linked to the study of predator-prey relationships and food web structures. However, trophic ecology has often focused on identifying taxonomic relationships and quantifying the biomass or energy ingested by consumers, but has often failed to integrate the importance of the nutritional quality of resources in ecological dynamics. Underlying this gap is the multi-dimensional nature of resource quality which has hampered any consensus on the definition of resource nutritional quality. In this special issue, we aimed at gathering a subset of articles exemplifying the diversity of variables by which resources quality is quantified, the diversity of research topics that can be tackled in ecology -from physiological or evolutionary aspects to ecosystem processes -and propose some perspectives on the integration of nutritional quality within broader ecological concepts. Using a semi-automated literature analysis, we map the current landscape of the "resources nutritional quality" research of the last 30 years. We depict how it has been quantified through physical, biological or chemical indicators, the use of these parameters being largely dependent on the type of ecosystem studied and on the investigated ecological process. We then position the articles published in this special issue of Oikos within this landscape, showing they cover a small but relatively well representative subset of the domains of resources quality-related issues. Articles in this special issue browse a range of individual and population-level approaches (embracing evolutionary questions) to community related questions, include methodological issues and ecosystem-wide approaches using trophic quality indicators as tracers of resources origin. Based on these studies and on the literature review, we identify a non-exhaustive list of challenges and perspectives of research that we consider of highest priority in the large topic of trophic ecology.

Introduction

The nutrition of consumers has long been proposed as a major determinant of community structures and ecological processes in ecosystems (Elton 1927[START_REF] Lindeman | The trophic-dynamics aspect of ecology[END_REF]. Resources availability regulates numerous individual or population parameters such as consumers' physiology, life history traits, or some behavioural parameters, while ultimately shaping communities and affecting ecosystem processes. First based on a quantitative approach (e.g. how much biomass/basal resources is/are available in the ecosystem and transferred to upper trophic levels?), it has progressively included more qualitative aspects of resources (e.g. are the resources edible, digestible, bringing essential compounds?; [START_REF] Odum | Fundamentals of Ecology[END_REF][START_REF] Boyd | Nutritive quality of food in ecological systems[END_REF]Scriber & Slansky 1981). Thus, in different ecosystems and trophic levels, it appeared that certain dynamics of populations, communities or ecosystems could depend on the sole quantity of available resources but also on the quality of these resources [START_REF] Österblom | Junk-food in marine ecosystems[END_REF][START_REF] Boyd | Nutritive quality of food in ecological systems[END_REF]. While the need to consider the "resources quality" in ecological questions has been acknowledged, there is however no consensus on how to define this quality as it can refer to a wide range of parameters.

To take into account the importance of resources quality in ecological processes, different frameworks have been developed in the past decades, focusing on distinct physico-chemical or biochemical parameters [START_REF] Wagner | Nutritional indicators and their uses in ecology[END_REF]. Nutritional Geometry [START_REF] Simpson | The Nature of Nutrition: A Unifying Framework From Animal Adaptation to Human Obesity[END_REF] focuses on general descriptors of resources biochemical composition, including energetic macronutrients (proteins, carbohydrates and lipids), micronutrients (vitamins), or allelochemicals and fiber contents. In contrast, the Ecological Stoichiometry framework [START_REF] Sterner | Ecological Stoichiometry: The Biology of Elements from Molecules to the Biosphere[END_REF] focuses on chemical elements. Those elements of focus are mainly carbon (C), nitrogen (N), and phosphorus (P), but they can also be extended to all other essential and non-essential elements [START_REF] Karimi | Beyond macronutrients: element variability and multielement stoichiometry in freshwater invertebrates[END_REF]. More specifically, the Ecological Stoichiometry framework considers the balance between elemental requirements of consumers and elements availability in their resources, explicitly including the consequences of trophic interactions on elemental cycles. Some authors also considered the importance of some specific biochemical compounds (the "biochemical view of trophic interactions; [START_REF] Müller-Navarra | Food Web Paradigms: The Biochemical View on Trophic Interactions[END_REF]. This perspective focuses on important molecules such as Long Chain Poly Unsaturated Fatty Acids -LC PUFAs -sterols, or amino-acids), which, when not present in sufficient amounts in the food resource, are susceptible to limit the consumers' performances or the biological production of ecosystems (e.g. [START_REF] Müller-Navarra | Unsaturated fatty acid content in seston and tropho-dynamic coupling in lakes[END_REF]. Despite the recent attempts to combine these approaches in common conceptual frameworks [START_REF] Sperfeld | Woodstoich III: Integrating tools of nutritional geometry and ecological stoichiometry to advance nutrient budgeting and the prediction of consumer-driven nutrient recycling[END_REF][START_REF] Sperfeld | Bridging ecological stoichiometry and nutritional geometry with homeostasis concepts and integrative models of organism nutrition[END_REF][START_REF] Anderson | Geometric stoichiometry: Unifying concepts of animal nutrition to understand how protein-rich diets can be "too much of a good thing[END_REF][START_REF] Ruiz | Quantifying the energetic cost of food quality constraints on resting metabolism to integrate nutritional and metabolic ecology[END_REF], it still appears that the different visions of nutritional quality are rarely integrated.

One question that arises is whether the difference in parameters selected to describe and quantify resource quality between ecosystems, species, or trophic levels depends on the cultural and historical artefacts of the researchers (e.g. based on the historical development of the different sub-disciplines in different ecosystem types), or reflects true differences in ecological functioning. We also question whether some resources quality-related parameters are more universal than others.

The aim of this Oikos special issue entitled 'The role of the nutritional quality of resources in ecosystem functioning' was to provide an up-to-date, general overview of how ecologists consider resources quality in their studies, and propose some thoughts on the determinants of resource quality and the importance of its consideration in future studies. The contributions published in this special issue are grounded within an exhaustive mapping of the current state of literature, based on a semi-automated literature analysis. We analyse how food quality has been quantified in ecological research and evaluate how much food quality indicators differ between targeted ecosystems, organisms and ecological questions. For full transparency, we detail the approach by which we built this map. The articles published in this special issue are then positioned on the landscape of nutritional quality revealed by the literature map, opening the discussion on the multifactorial nature of resources quality. Finally, based on some articles of the special issue and the results of our literature analysis, a list of future directions of research is proposed.

Mapping the current landscape of resources quality research

Overall methodology

To analyze the diversity of articles dealing with resource quality, we conducted a bibliometric analysis by semi-automatic text mining, using the litsearch R Package [START_REF] Grames | An automated approach to identifying search terms for systematic reviews using keyword co-occurrence networks[END_REF].

The approach included a preliminary Web of Science search (1991-2020) using the terms ("nutrition* quality" OR "quality prey" OR "quality food" OR "quality resource*" OR "diet quality" OR "resource* quality" OR "food quality" OR "prey* quality") and restricting Web of Science categories to fields related to environmental sciences and Ecology. This research, conducted on 20 November 2020, returned 2624 references (to which the 9 articles of this special issue were added). Words or expressions (up to 3 words) that appeared in either the abstract, title or authors' keywords of at least 10 papers were automatically extracted. This initial list of unique terms (3882 in total) was thereafter manually screened and checked to identify and categorize keywords (1060 in total) into four categories. The first category defined a food quality parameter (i.e. as a structural, elemental or biochemical property of the food source) in which keywords were divided in 8 subcategories (Table 1). The second defined endpoints or purposes, i.e. an individual, populational, community or ecosystemic process under focus, and was subcategorized in 10 categories. The third category defined organisms under focus (11 categories) and the fourth an ecosystem type (marine, terrestrial, inland waters or cross-ecosystem) (Table 1). For instance, the terms "arachidonic", "lipid composition" and "polyunsaturated" were categorized as "food quality parameters" and pooled within a "lipids" subcategory of the "food quality parameters" category, (see SI1 for the full list of keywords and keywords allocations to categories and subcategories).

Table 1. List of categories and sub-categories defining food quality parameters, purposes, Organisms and Ecosystems

In a second step, the literature database was automatically screened again for the presenceabsence of the selected keywords in the title, abstract and authors' keywords of each paper.

After this step, 72% of papers could be attributed at least one subcategory of food quality. The list of papers (ca. 600) for which no matching food quality terms had been detected was manually screened again in order to (i) detect additional keywords for food quality parameters, (ii) discard papers which topic was not directly related to food quality aspects, (iii) identify papers actually dealing with food quality but in which food quality was not defined by a structural or biochemical property of the food source itself but rather as the effect of a food source on a consumer life-history traits (subcategory "undefined"). The final dataset contained 2359 papers that were attributed to at least one food quality subcategory.

Binary matrices of keywords presence/absence in the article title, abstracts and authors keywords on the final dataset were automatically computed for each of the four categories.

Presence of at least one keyword of a given subcategory was thereafter used to allocate this subcategory to the paper: for instance, the presence of "arachidonic" would allocate the paper to the subcategory "lipids" in the food quality matrix. A single paper could be allocated to two subcategories within the category matrix. 97% of papers were allocated at least one purpose Co-occurrences within and between subcategories were mapped using non-metric multidimensional scaling (NMDS, vegan package), and clusters were identified using k-means (stats R package). The number of clusters was decided from a broken stick approach and their significance was tested using analysis of similarities (vegan package). Whether two subcategories co-occurred more frequently than what could be expected under the hypothesis of a random distribution was detected using chi-squared tests, using a Bonferroni's correction. When considering all scientific publications in the ecological and environmental science fields, food quality is mostly mentioned as an elemental or stoichiometric property of the food source (39% of all papers, Figure 1a), while lipid or amino-acid/protein content or composition, or an energetically-based reference, are present in about 20% of papers. Isotopes and metals contents are the least frequent references of food quality (<6%). Food quality is not referred as to an explicit parameter of the food source in 23% of papers ("undefined" subcategory). In almost half of the papers (47%), the food quality refers to more than one sub-category. 67%

A map of nutritional quality research

of papers that refer to an elemental-stoichiometric definition also refer to terms from other subcategories (predominantly lipids, amino-acids/proteins and energy). References to carbohydrates, protein compositions and biomechanical properties significantly co-occur as well (Figure SI2a).

Articles also preferentially refer to species-level fitness terms (> 54%) and foraging (53%) (Figure 1b), with frequent co-occurrence (foraging is significantly associated to terms related to metabolism and physiology, while fitness terms are significantly associated to contamination and physiology, Figure SI2b). References to community and ecosystemic processes (diversityevolution, production, carbon fluxes, species interactions, development and phenology) are less frequent (18 to 30%). Terms related to contamination occur in 8% of papers.

Research on nutritional quality of food resources overwhelmingly focuses on arthropods (55% of articles), and >35% of articles include at least one primary producer (algae and/or plants and trees; Figure 1c). Arthropods co-occur with algae in 30% of articles, and with plants and trees in 18% of articles, and for 17% of articles with microheterotrophs (Figure SI2c).

Vertebrates and micro-heterotrophs are focus organisms for ca 20% of articles. Other taxonomic groups are present in <10% of articles. There is an almost equal share between terrestrial and aquatic ecosystems, but inland waters represent twice more papers than marine ecosystems (Figure 1d).

Changes in articles distribution within the three clusters over years.

There are clear segregations of the terms used to define resources nutritional quality in between ecosystems and endpoints (Figure 2a) so that articles can be segregated within three clusters (Figure 2b). Elemental, stoichiometric, and lipids descriptors preferentially co-occur in articles focusing on aquatic environments (both marine and continental), where they are related to scientific questions related to carbon fluxes and fitness, foraging and metabolism.

This cluster, figured in purple in Figure 2, is also the one with the highest intra-group similarities and the most dissimilar with the two other clusters. Undefined food quality clusters with purposes such as diversity-evolution, reproduction-recruitment and species interactions, which preferentially cooccur with studies on terrestrial environments (in green in Figure 2).

Current status and perspectives of the research on nutritional quality

Resources quality: what are we talking about?

Our analysis is able to classify most of the articles according to a restricted set of the most common parameters used for describing resources (Table 1). Only 23% of the articles are attributed to the undefined parameters, which either means that the authors used other quality parameters or -for most cases -did not directly referred to any quality parameter, considering some resources of higher quality than others based on a priori knowledge and non-presented or non-measured parameters. This "undefined" category is more represented in terrestrial than in aquatic ecosystems that might be at least partly explained by the long-lasted knowledge of some plant quality gradients for herbivores in agronomy. Among all the parameters used, those referring to the elemental contents of resources (including the stoichiometric approaches) are the most represented, followed by those focusing lipids, amino-acids/proteins and energy. This

Are resources quality indicators used similarly between ecosystems?

Terrestrial and aquatic ecosystems are almost equally represented in our analysis of existing scientific literature, which contrasts with the articles published in the special issue, largely dominated by aquatic studies (only two articles considering terrestrial consumers: Mathieu-

Resuge et al. 2022. Le Gall et al. 2022).

Interestingly, parameters used for describing resources quality significantly differ between ecosystems. Lipids are mainly investigated in aquatic ecosystems -both in freshwaters and marine ecosystems-while macro-nutrients are more frequently investigated in terrestrial ecosystems. This observation directly questions the origin of this difference: are lipids less important in terrestrial ecosystems or is it simply a cultural bias related to the fact that most of the researchers working on lipids come from aquatic ecology? From a biological viewpoint, there is no reason for lipids to play a less relevant role in terrestrial ecosystems, and for example long chain polyunsaturated fatty acids are known as essential for both terrestrial and aquatic consumers (Hixson et al. 2015 and references herein). Since some LC-PUFAs are more abundant in aquatic than in terrestrial ecosystems, terrestrial consumers might benefit from the transfer of these compounds from aquatic ecosystems [START_REF] Hixson | Production, distribution, and abundance of long-chain omega-3 polyunsaturated fatty acids: a fundamental dichotomy between freshwater and terrestrial ecosystems[END_REF]. Elemental approaches are certainly the most shared among ecosystem types, even if these ones are a little bit closer to inland waters. This last observation might be related to the historical development of stoichiometric approaches by limnologists in the late 90's [START_REF] Sterner | Ecological Stoichiometry: The Biology of Elements from Molecules to the Biosphere[END_REF], the transfer to other ecosystems being more recent.

Finally, parameters related to biomechanics and metabolites (including preys chemical defenses) are more typically found in terrestrial ecosystem studies. Aquatic plants may indeed contain less physical defenses -or at least are less studied for that -and plant chemical defenses have long been considered in pioneer works as one of the most important parameter of forage quality for the cattle (e.g. [START_REF] Laycock | Factors influencing forage quality[END_REF].

For which purpose are these quality indicators measured?

As expected, most studies dealing with resources quality consider the consequences of quality to what extent such ecosystem-wide parameters have actually been studied.

How to measure and compare resources quality?

A striking observation in our literature analysis is that a large proportion of articles dealing with resource quality does not investigate any parameter related to the response of consumers to resources consumption/ingestion. A lot of published articles thus focuses on "potential" resource quality parameters and never measures their effective consequences on consumers.

Yet, consumer requirements may vary widely depending between taxa (e.g. [START_REF] Frost | Threshold elemental ratios of carbon and phosphorus in aquatic consumers[END_REF] but also between individuals of a similar taxon [START_REF] Leal | The ecology and evolution of stoichiometric phenotypes[END_REF], Le Gall et al. 2022). Evaluation of consumers' requirements might also greatly depend on the purpose of the study. For example, some fatty acids are especially important for reproduction, less for growth [START_REF] Becker | Differential effects of phosphorus and fatty acids on Daphnia magna growth and reproduction[END_REF][START_REF] Bec | Trophic upgrading of autotrophic picoplankton by the heterotrophic nanoflagellate Paraphysomonas sp[END_REF]. The Nutritional Geometry framework also evidences that the relative amounts of macro-nutrients required for consumers largely vary depending on the life history trait considered or on the sex of the consumer (e.g. [START_REF] Lee | Lifespan and reproduction in Drosophila: new insights from nutritional geometry[END_REF]. Consumers' responses to some quality parameters are also not monotonous and some compounds present in excess in resources can be deleterious for consumers upon a definite threshold [START_REF] Boersma | Too much of a good thing: on stoichiometrically balanced diets and maximal growth[END_REF][START_REF] Anderson | Geometric stoichiometry: Unifying concepts of animal nutrition to understand how protein-rich diets can be "too much of a good thing[END_REF]. Finally, environmental stressors are able to significantly change the nutritional requirements of a consumer. For example, temperature is a major factor impacting the lipidic and stoichiometric requirements of consumers [START_REF] Masclaux | Combined effects of food quality and temperature on somatic growth and reproduction of two freshwater cladocerans[END_REF][START_REF] Ruiz | U-shaped response Unifies views on temperature dependency of stoichiometric requirements[END_REF]. Defining a priori the quality of a resource without evaluating its consequences on consumers' life history traits might thus sometimes lead to large inaccuracies. Another point that deserves attention concerns the way the analyses are conducted, and the discrepancies that arises from methodological differences between studies. In this special issue, Zaguri et al. ( 2022) elegantly demonstrate the difficulty to compare the macro-nutrient contents of different resources originating from different methodologies. This question is crucial for generalizing the results coming from different studies, and this paper shall be cautiously read before starting any meta-analysis on nutritional quality topics (especially those considering macro-nutrients).

Why should we take resources quality into account for studying ecosystem functioning?

First premises of trophic ecology focused on biomass and/or energetic currencies for defining resources quality (Elton 1927[START_REF] Lindeman | The trophic-dynamics aspect of ecology[END_REF]; see [START_REF] Layman | A primer on the history of food web ecology: fundamental contributions of fourteen researchers[END_REF] for an historical review).

While the total amount of energy contained in basal resources remains important, a large number of studies underlined the importance of taking into account multiple resources quality parameters to improve our understanding of ecosystems functioning (e.g. [START_REF] Marcarelli | Quantity and quality: unifying food web and ecosystem perspectives on the role of resource subsidies in freshwaters[END_REF]. This is especially true for primary consumers facing the largest imbalances between their requirements and what can be found in their resources, as early underlined by [START_REF] Boyd | Nutritive quality of food in ecological systems[END_REF]. Despite the general acknowledgment that food quality largely drives organism performances, numerous studies continue to neglect resources quality as a fundamental parameter ultimately driving the direction and the intensity of numerous ecological processes. In this special issue,

Challenges and future directions

In comparison to our literature review, the articles published in this special issue of Oikos constitute a small but relatively well representative subset of studies dealing with resources quality-related issues. Based on these studies and on the literature review, we identify a nonexhaustive list of challenges and perspectives of research that we consider of the highest priority in the large topic of trophic ecology.

-Both resource quantity and quality matter, but they might not intervene within the same processes, life-stages or even generations of consumers (Leal et -A second lesson of this analysis is that some aspects of trophic ecology may still be too much restricted to some scientific sub-disciplines or to some ecosystem types. -The resource quality should be understood as a multidimensional property and it is hard to imagine explaining all resources constraints in ecosystems while considering only one kind of quality descriptor (Chouvelon et al, 2022). As underlined in this special issue, different life-history traits of consumers can respond differently to deficiencies in various aspects of resource quality (e.g. specific lipids deficiency might affect consumers' reproduction while not affecting their growth, van Deurs et al. 2022).

Taking into account the multidimensional nature of resource quality through the consideration of consumers' responses to the large diversity of diets available for them constitutes a major challenge [START_REF] Ruiz | Quantifying the energetic cost of food quality constraints on resting metabolism to integrate nutritional and metabolic ecology[END_REF]).

Figure 1 .

 1 Figure 1. Distribution of published articles between food quality sub-categories (a), purposes (b), studied organisms (c), and ecosystem types (d). Sums of percentages can reach >100%

Figure 2 .

 2 Figure 2. Map describing the nutritional quality research on the 1991-2020 period. Colors

  Foodquality aspects related to metabolites, overall nutritional geometry (i.e. biochemical composition in amino-acids-proteins and carbohydrates), as well as those addressing energetical aspects, cluster with physiological purposes, and are less dependent of ecosystem type. Articles of the special issue cover well the spectrum of research about nutritional quality of two of the clusters. Because all of the contributions considered at least one explicit variable to quantify the nutritional quality of the resource, none of them falls within the cluster of "undefined food quality" (in green). Five of the articles of the SI (Chouvelon et al. 2022; Sentis et al. 2022; Mathieu-resurge et al. 2022; Lowman et al. 2022; Van Deurs et al. 2022) fall within the cluster Aquatic Ecosystem/Stoichiometry-lipids/Trophodynamics-fitness, a proportion that mirrors well the actual predominance of those themes within the overall research (Figure 2c). Two more articles of this special issue (Zaguri et al. 2022; LeGall et al. 2022) belong to the cluster linking the nutritional geometry to physiological aspects, which is current ranked second in term of contribution to the overall research. Last, two articles of the SI lie at the interface of the dominant cluster (i.e. Aquatic Ecosystem/Stoichiometrylipids/Trophodynamics-fitness) and the cluster including evolutionary-reproduction purposes (Hudson et al. 2022; Leal et al. 2022).

  dominance may partly arise from the technical simplicity of elemental analyses compared to the analysis of more specific compounds (e.g. macronutrients, requiring more complex methodologies; Zaguri et al. 2022). It could also partly come from the long-lasted use of %N (or the C/N ratio) as an indicator of plant quality for cattle in agronomical studies (e.g. Laycock, & Price, 1970) and the more recent development of the Ecological Stoichiometry framework (Sterner & Elser 2002). Biomechanical parameters (including all physical defenses) used to be largely considered in the early 90's but the relative use of this parameter is clearly declining in the recent years. The articles published in this special issue are well representative of these trends since one third of the published articles question resources quality through the consideration of resources elemental content (Sentis et al. 2022; Lowman et al. 2022; Chouvelon et al. 2022), while a second third focuses on resources macro-nutrient contents, alone (Le Gall et al. 2022; Zaguri et al. 2022) or in association with fatty acids (van Deurs et al. 2022). Finally, three articles specifically focus on fatty acids/lipids (Hudson et al. 2022; Leal et al. 2022; Mathieu-Resuge et al. 2022).

  parameters on the fitness and/or on the foraging behavior of consumers. Most authors thus investigate the consequences of resource quality on the growth, on the survival and on the reproduction of the consumers (Le Gall et al. 2022), ultimately permitting to question secondary production and population/community dynamics in ecosystems (e.g.[START_REF] Rosen | Pollock and the decline of Steller sea lions: testing the junk-food hypothesis[END_REF] -even if such studies remain generally limited in the literature. Other studies (for example van Deurs et al 2022 and Le Gall et al. 2022), question food choices of consumers when exposed to resources of different quality, in more or less close relationship to the socalled Optimal Foraging Theory (e.g.[START_REF] Pyke | Optimal foraging: a selective review of theory and tests[END_REF]. Both fitness-and foraging-related approaches are quite equally distributed between ecosystem types. In contrast, approaches explicitly dealing with the role of resources quality in species interactions (e.g. competition or parasitism) are more closely related to terrestrial ecosystems. It is also interesting to note that less than 20% of all published studies, explicitly refer to evolutionary issues, while many interactions between species and the quality of their resources have been shaped on an evolutionary time scale. In this special issue, Hudson et al. (2022) tackle such question through the analysis of different fish lineages and their metabolic capacities of PUFA synthesis, showing how divergences between lineages depend on the history of freshwater colonization of the studied species. Finally, since some elements/molecules used for defining resources quality can be typical from some ecosystems or resource types, such resources quality parameters can be consequently used as ecosystem tracers[START_REF] Majdi | There's no harm in having too much: A comprehensive toolbox of methods in trophic ecology[END_REF]. For example, Leal et al (2022) used fatty acids of land origin to trace the origin of different food subsidized for a targeted marine molluscan species in seashore ecosystems, while documenting the consequences of different nutritional qualities for the larval settlement and dispersion. In contrast, for characterizing the organic matter transfers from aquatic to terrestrial ecosystems,Mathieu-Resuge et al. (2022)analyze LC-PUFAs combined with stable isotopes for determining the origin of trophic resources for spiders in ecosystems more or less close to lakeshores. Out of this special issue, it can be noted that micro-nutrients or pollutants have also been successfully used for tracking the origin of organic matter in ecosystems or determining habitat use by organisms[START_REF] Ramos | Traceme if you can: the use of intrinsic biogeochemical markers in marine top predators[END_REF]. Finally, references linking resources quality to ecosystem functioning parameters (such as carbon-fluxes) account for ca. 30% of all published studies, even if we cannot know from the present literature review

  Sentis et al. (2022) revisite the predictions of the largely used Rosenzweig-MacArthur consumer-resource model through the introduction of explicit stoichiometric constraints within the model parametrization. The introduction of such resource quality constraints radically changes the predictions of the model in response to nutrient enrichment and temperature increase. Such findings underline the necessity of integrating nutritional quality in theoretical but also experimental approaches to properly understand the response of ecosystems to changes in environmental parameters.

  al, 2022, Le Gall et al, 2022, Van Deurs et al, 2022). There is a crucial need to merge those two nutritional aspects within the study of ecological processes. In that sense, the modelling approach developed from field-based data by Van Deurs et al (2022) is certainly inspiring to fully unfold individual trade-offs. Besides, studies with resources quality are largely restricted to individual or population levels approaches. Trying to upscale the impacts of consumers' nutritional constraints, both in terms of availability and quantity, to their consequences on ecosystem functioning certainly stands as a key challenge of this research area. The use of models taking explicitly into account resources quality along with abundance (as in Sentis et al. 2022) certainly represents a promising way to achieve this goal.

  Cross-ecosystem studies, as those developed by Leal et al. (2022) and Mathieuresurge et al. (2022) remain rare. Research in trophic ecology would certainly largely benefit from combining the different frameworks used for characterizing resources quality and their impacts -keeping in mind that methodologies should be harmonized or at least inter-calibrated between studies (Zaguri et al. 2022).

-

  As generally observed in ecosystem ecology, evolutionary questions are still too rarely explicitly tested in trophic ecology investigations. Despite a large array of studies have observed and discussed the evolutionary tenets at play behind the selection of consumers nutritional requirements (see for example Hudson et al. 2022 in this issue), the inclusion of such research questions in the general understanding of ecosystem functioning remains limited. -Finally, Lowman et al. (2022). illustrate that the potential nutritional quality of basal producers can be significantly altered by global warming. In the context or the current global changes and considering the multiple stressors organisms are exposed to, understanding the causes and the consequences of changes in basal resource quality in a multistressor context (concomitant changes in temperature, nutrient and water availability, presence of pollutants, …) is an urgent need, as much in terms of prediction of future ecosystem trajectories as in terms of goods and services provision by nature for humanity.

Acknowledgments -Authors wish to thank all the participants of the GDR GRET 3716 who participated to the "Journées du GRET" in Nantes and Metz.

Funding -Scientific discussions and workshops at the origin of this Special Issue benefited from funds from the GDR GRET (GDR CNRS 3716) leaded by A.B., as well as from the Young Researcher ANR StoichioMic (ANR-18-CE32-0003-01) and the SPACE IUF projects attributed to M.D.

Supplementary information

SI1. keywords classification used for the literature analysis SI 2: associations within categories


W Melissa

R Peter

M-L Vignaud

Da Barbosa

N Silva

A Shekoory

I Mahmoudi

N Morin

R Djelidi

T Fournier

A Lackner

; Kunihs

; Haider

; Meinhardt

; Knöfler

Th Maxian

M Knöfler

S Haider

B Cracknell-Hazra

Oliver C Watkins

Mohammad Omedul Islam

Preben Selvam

Reshma Appukattan Pillai

Hannah Ej Yong

Anne K Bendt

Amaury Cazenave-Gassiot

Keith M Godfrey

Markus R Wenk

Wayne Cutfield

Rohan M Lewis

Shiao-Yng Chan

CHARACTERISATION OF PROTEIN O-GLCNACYLATION REVEALS NOVEL REGULATION OF SECRETORY PATHWAY AND DNA DAMAGE PROTEINS DURING SYNCYTIOTROPHOBLAST DIFFERENTIATION

Early stage investigator (up to three years post-PhD) -

Methods: In this study, an in vitro model that mimics placental OS seen in FGR was used. Placental explants from normal pregnancy (N=6) were cultured for 7 days and exposed to 1mM tBOOH or 1mM hydrogen peroxide (H 2 O 2 ) on days 5 and 6. Ouabain-sensitive (transporter-mediated) uptake of 14 C glutamine and 14 C glutamate was measured for 24h between days 6-7 (shown previously to represent steady-state intracellular accumulation). Culture medium was collected daily to measure indicators of endocrine function (hCG) and cellular stress (IL6 secretion and lactate dehydrogenase (LDH) release). Immuno-histochemistry to assess syncytiotrophoblast integrity (Cytokeratin 7 (CK7)), apoptosis (M30) and lipid peroxidation (4HNE) was assessed in explants at day 7. Data are expressed as % of control and analysed by Wilcoxon signed rank test.

Results: Both tBOOH and H 2 O 2 significantly increased lipid peroxidation, consistent with elevated OS (16x and 6x respectively; p<0.05). tBOOH reduced the uptake of glutamine and glutamate by 90(66-100)% and 95(40-99)% of control (median and range: p<0.05), inhibited hCG secretion by 75(5-81)%, andincreased IL6 secretion to 5774 (1420-16365)% of control (p<0.05). tBOOH increased apoptosis to 275 (100-433)% of control (p<0.05) but did not alter CK7 staining or LDH release. Despite elevating OS, H 2 O 2 did not affect glutamine and glutamate uptake or any of the other variables measured.

Conclusion: 1mM tBOOH induced OS and markedly inhibited glutamine and glutamate uptake. In view of the lack of effect of H 2 O 2 on amino acid uptake, the reduction in uptake following tBOOH exposure might not be directly attributed to oxidative stress. Furthermore, 1mM tBOOH also had detrimental effects on other variables that can influence nutrient transport. Future work will investigate the cellular mechanisms through which tBOOH alters placental function, and the concentration dependency, to aid our understanding of whether oxidative stress has direct effects on glutamine and glutamate uptake. M aternal obesity is characterized by chronic, low-grade inflammation, oxidative stress, and dyslipidemia. This lipo- toxic environment have been associated with poor pregnancy outcomes and increased the risk for the offspring to develop metabolic syndrome later in life. At the fetal-maternal interface, the placenta plays an essential role in the exchanges between the mother and the fetus. Different studies have demonstrated that maternal obesity without gestational diabetes (GD) impacts placental structure but also its major functions including endocrine function, nutrient exchanges. However, birthweight is not affected by the maternal obesogenic environment. So, the placenta has the potential to interpret environmental signals, such as excessive nutrient availability, and adapt itself in order to support fetal growth and development. In this context, to better understand molecular mechanisms implicated in this placental adaptation, we compared lipid, inflammatory, and oxidative status of maternal-placental-fetal triad between obese and non-obese women. Firstly, by using a lipidomic approach, we showed i) a higher polyunsaturated fatty acid (PUFA) concentration specifically in maternal plasma and placenta from obese women compared to non-obese group, ii) a lower sphingolipid (ceramide) concentration specifically in maternal plasma from obese women compared to nonobese group, and iii) a sex-specific disruption of some derivate of ceramides (i.e. lactosylceramide, dihydroceramide, and sphingomyelin) in cord plasma and placenta from obese women. Secondly, our preliminary results demonstrated that maternal obesity seemed to increase oxidative stress as suggested by the apparition of a molecular target generated after lipid peroxidation in female placenta specifically. Thirdly, by using a transcriptomic approach, we found a lower expression of enzymes involved in antioxidant defense (as CAT and GPx8) and lipid metabolism (as FADS1/2 and ELOVL1/4) in female placenta from obese women once again. Finally, we showed that maternal obesity significantly reduced placental inflammatory status (cytokine secretion and signaling pathway) in male placenta from obese women only. In conclusion, our present results clearly showed that maternal obesity without GD is associated with placental lipid metabolism, inflammation, and oxidative stress modifications in a sex-specific manner and highlighted, in part, the molecular mechanisms involved in the placenta's adaptations to a harmful maternal environment.

18 th EP G m ee tin g | Am ph ith ea te r of Bi ot ec hn ol og ie s, Bu ild in g 44 0 IN RA E Île -d e-Fr an ce -Jo uy -e n-Jo sa s SESSION 3 I t is a common reception that in high-yielding dairy cows a negative energy balance (NEB) and a decreased reproduc- tive performance co-occur. In calf hepatocytes and endometrial cells (BEND) beta-Hydroxybutyrate (BHBA), as one of the primary characteristics of a NEB, increments the levels of NF-κB regulated cytokines. These include, for instance, tumor necrosis factor-α (TNFα) and Interleukin-6 (IL-6), which are prominent for the inflammatory and immunity response. Moreover, it is described that BHBA increases the oxidant status and leads to higher concentrations of reactive oxygen species in the plasma of dairy cows. A bovine caruncular epithelial cell line (BCEC) was used for the experiments. It is suitable to explore fetal-maternal communication, infections, and metabolic modulations of pregnancy-associated diseases. The cell line was isolated and established from the placentomes of pregnant cows, exhibits the morphological and functional characteristics of epithelial cells and forms an epithelial barrier. To investigate the in vitro impact of BHBA during pregnancy, especially in the period of implantation and placentation, the BCEC was stimulated with 0.6mM, 1.2mM, 1.8mM and 2.4mM BHBA for 24h and 36h. For each experimental group, cells incubated in serum-reduced medium served as controls. Examination of cell metabolism, cell motility and the expression of pro-inflammatory factors have been carried out after the BHBA incubation. The cell metabolism was analysed by an MTT-Assay and the motility by a live-cell imaging program. To investigate the mRNA expression of TNFα, IL-6 and NF-κB a quantitative real-time PCR was utilized. The results indicate a significant negative effect on cell metabolism and the motility at a concentration of 2.4mM BHBA. TNFα shows a significant increase in mRNA expression correlating with higher BHBA concentrations. However, IL-6 and NF-κB were not altered in their mRNA expression compared to the control group. The decreased BCEC metabolism and motility as well as the increased TNFα mRNA expression could lead to a restricted function of maternal caruncles. This might result in a poor interaction between maternal caruncles and fetal cotyledons which could cause early pregnancy losses.

Placenta samples from 6 cows (gestation days 120, 150, 205, 266, 278 and 284) were fixated in Karnovsky and embedded in Durcupan. Seven stacks of 3000 sections (60 nm thickness) each were created by serial block face scanning electron microscopy. Editing, analyses and segmentation were accomplished using the software Microscopy Image Browser. Visualization was done with the software Imod and volume calculations with the software Amira. The methodology was supported by the recording of high-resolution transmission electronic microscopy images. TGC analysis included following criteria: contact to fetal basement membrane (FBM), membrane protrusions to FBM, contact to ME, formation of pseudopodia in direction to ME, presence of membrane-bound granules, double lamellar bodies, mitochondria, rER and number of cell nuclei. For three stacks (150, 205, 284) the cell volumes (CV) and total nuclear volumes (TNV) were calculated. The CV displayed a wide range between 2048 and 14708 μm 3 . TNV varied between 853 and 3395 μm 3 . TGC occurred in majority as binuclear cells. Few TGC with one or three nuclei were also observed. Based on morphological criteria, TGC could be distinguished into young, maturing and mature cells. Young TGC still had close large contact with the FBM, whereas in the maturing and mature TGC the contact was only small or no longer present. Maturing and mature TGC with a TNV of approximately 800 μm 3 began to form contacts with ME. Contacts were mostly established by formation of pseudopodia. Some pseudopodia contained a distinct organelle-free area delimited by a membrane-like structure. The contact of TGC with the ME decreased during gestation. A surprisingly large proportion of TGC still had contact to the FBM. This was usually no longer extensive like in the young TGC but in form of membrane protrusions. Contact with FBM initially increased from ~55% in the second half of gestation, to 100% in late pregnancy and decreased again to ~75% at term. Several TGC had contact to the fetal BM and the ME simultaneously. After fusion with one or more UTC, HC were dispersed in the ME, and had two to 17 cell nuclei. HC with two nuclei could be identified as degenerated hybrid cells. The proportion of HC increased towards end of pregnancy. Therefore, contrary to previous studies, TGC can form contacts with the FBM and the ME simultaneously. Not only young but also immature and mature TGC maintain their contacts to the fetal BM longer than expected. In HC, a distinction could be made between vital and degenerated cells.

18 th EP G m ee tin g | Am ph ith ea te r of Bi ot ec hn ol og ie s, Bu ild in g 44 0 IN RA E Île -d e-Fr an ce -Jo uy -e n-Jo sa s SESSION 5 D uring the early stage of placentation in sheep (15-23 Days), normal conceptus development is affected by trophoblast cells (oTCs) functionality whose dysregulation leads to early pregnancy loss, characterised by inadequate nourishment and oxygen supply. In this crucial developing period, oTCs metabolism is mostly supported by endometrial histotrophic factors, including FGF2, involved in cell differentiation and function through the modulation of specific cellular mechanisms. Among the others, mTOR is known as cellular "nutrient sensor", but its downstream regulation in oTCs is still poorly understood. Therefore, our hypothesis was that oTCs adapt to overcome poor-growth factors in the uterine environment through mTOR signalling pathway modulation. The main aim was to set up an in vitro culture system from early sheep placenta in order to study trophoblast adaptive response in suboptimal environments occurring in impaired pregnancy. Firstly, primary oTCs from 21-days old sheep placenta collected at the slaughterhouse were characterised by cell morphology, immunofluorescence and PCR to detect specific trophoblast markers. Then, oTCs primary cells and oTr cell line were subjected to different treatment (50 ng/ml FGF2, 100 nM rapamycin -mTOR inhibitor -for 24h) to study their effects on cell functionality, gene and protein expression profile. oTCs showed mainly mononuclear cells with epithelial cell-like growth and placental morphological properties, such as bi-and multinucleated syncytium-plaques, expressing peculiar trophoblast markers and progesterone release in culture medium. Cell functionality tests relieved similar results in both cell systems. FGF2 promoted cell proliferation and migration in normal culture conditions, whereas mTOR-inhibition induced a significant decrease. Invasion activity wasn't influenced by FGF2 when mTOR signalling pathway was activated. On the contrary, when it was inhibited, cell invasiveness was affected by FGF2. mTOR-inhibition leaded to a significant low-cell motility both in presence and absence of FGF2, but its supplementation seemed to restored oTCs activity even when mTOR was prevented, as confirmed by mTOR phosphorylation even in presence of the inhibitor. Interestingly, mTOR-inhibition influenced endocrine trophoblast marker regulation, indeed oPL expression wasn't affected by FGF2 supplementation as observed in the control, while IFN-τ was drastically reduced. Present findings support that FGF2 acts to modulate oTCs behaviour by regulating mTOR signalling pathway.

The study provides new insight regarding how mTOR-inhibition influences trophoblast migration and invasion activity, essential for conceptus development. Moreover, mTOR involves in the expression of hormonal trophoblast markers, suggesting that it plays a crucial role in the early placenta growth and foetal-maternal crosstalk.

Methods:

Using CRISPR/Cas9 technology, we deleted two genes, Pigl and Pigf, encoding fundamental proteins for the correct GPI biosynthesis in mouse trophoblast stem cells and studied their role in the regulation of early mouse placentation.

Results:

Phosphatidylinositol Glycan Anchor Biosynthesis Class L (PIGL) and F (PIGF) proteins are essential components of the glycosylphosphatidylinositol (GPI) biosynthetic pathway that culminates in the generation of the GPI glycolipid. The GPI anchor is then post-translationally transferred to proteins in the endoplasmic reticulum (ER) and relocated as GPI-anchored proteins (GPI-APs) to the cell surface where they play essential roles in cell signaling transduction. Our previous work identified the Pigl and Pigf genes as essential for placentation, highlighting the fact that GPI-anchor biosynthesis pathway is critical for early placentation. Here, we analyse the functional contribution of PIGL and PIGF to the GPI biosynthesis pathway in regulating mTSC self-renewal and differentiation. We first characterized the dynamics of Pigl and Pigf expression during trophoblast differentiation by performing a time-course experiment. Whereas Pigf was highly expressed in mTSCs under stem cell conditions and was strongly downregulated during trophoblast differentiation, Pigl expression did not significantly change across this differentiation time course. The deletion of Pigl or Pigf did not affect stemness. However, under differentiation conditions, Pigl -/-and Pigf -/-mTSCs failed to upregulate markers of syncytiotrophoblast, whereas differentiation towards trophoblast giant cells was promoted, indicating that GPI biosynthesis pathway proteins may play a role in the lineage determination towards syncytiotrophoblast. These data point to an essential role for GPI biosynthesis pathway proteins in modulating trophoblast differentiation towards syncytiotrophoblast and identify GPI-APs as novel players in regulating early placentation.

INTRODUCTION |

The placenta mediates nutrient supply to sustain fetal growth during pregnancy. A large exchange area is created by extensive branching morphogenesis of the trophoblasts. Pivotal to this labyrinth formation is the differentiation and branching of Syncytiumtrophoblast layer II (SynTII). However, little is known regarding the underlying pathways that underpin placentation. Calcium signals are well suited to translate signals from the environment into a cellular response. Previously, we identified a crucial role of the calcium-permeable channel TRPV2 in placental development as Trpv2-/mice display intra uterine growth restriction (IUGR) and lethality. Here, we investigated the role of TRPV2 in placental development and trophoblast branching.

RESULTS |

Global genetic ablation of Trpv2 results in IUGR and embryonic lethality at E18.5. Deletion of Trpv2 in the embryo proper or in the junctional zone of the placenta was compatible with normal development, suggesting that labyrinthine Trpv2 is indispensable for normal fetal development. In the labyrinth, Trpv2 is highly expressed in SynTII trophoblast cells. Moreover, the labyrinth morphology of Trpv2-/-placentas was severely disturbed, with regions where that are void of both SynTII cells and fetal vessels. RNAsequencing of wildtype and Trpv2-/-placentas revealed that branching morphogenesis and angiogenesis pathways were affected. Interestingly, Crispr/Cas9-mediated deletion of Trpv2 in Trophoblast Stem Cells resulted in impaired differentiation of the SynTII labyrinth lineage.

CONCLUSION |

Our observations suggest that loss of TRPV2 in mice disrupts the differentiation of SynTII trophoblasts, which likely contributes to defects in labyrinth morphogenesis, nutrient supply, and thus compromised fetal growth.

Introduction:

Despite advances in cell culture systems, researchers have continued to use culture media based on historic formulations. For example, Dulbecco's modified Eagle's Minimal Essential Medium (DMEM), the most widely used culture media reported in the literature, is based on a formulation in 1959. Standard culture media such as DMEM contain non-physiological levels of nutrients and lack defined concentrations of many metabolites present in vivo. Despite this, nearly all cell culture-based studies on human trophoblasts have been performed using this media. Recently, Tardito et al. (Sci Advances 2019) developed a culture medium (Plasmax), with nutrients and metabolites with concentrations comparable to the human in vivo environment. To understand the impact of the physiological metabolic environment on hTSC growth, differentiation and metabolism, we compared the effects of culturing cells in a physiologically-relevant medium (Plasmax) versus the standard, widelyused medium (DMEM-F12).

Methods: hTSCs (n>4) were cultured in DMEM-F12 or Plasmax and maintained in their undifferentiated state or differentiated into syncytiotrophoblast (STBs) or extravillous trophoblasts (EVTs). Cells were counted daily over 4 days to assess their proliferation rate. Differentiation was determined by qPCR analysis of STB and EVT markers at different stages of differentiation. Energy metabolism was examined by LC-MS analysis of glycolytic and tricarboxylic acid (TCA) cycle intermediates and cellular respiration on an extracellular flux analyser.

Results:

Compared to DMEM-F12, hTSCs grown in Plasmax increased their proliferation by 60% (P=0.003). STB or EVT differentiation, as determined by expression of STB (ERV-W, CYP19A1; P<0.01) and EVT (ERBB2, HLA-G; P<0.001) markers, increased in cells cultured with Plasmax. Lastly, hTSCs in Plasmax demonstrated greater energy metabolism as demonstrated by increased intracellular production of glycolytic and TCA cycle intermediates as well as elevated basal respiration and oxidative phosphorylation.

Conclusion:

Physiological medium (Plasmax) improves the metabolic fidelity of hTSCs resulting in increased proliferation and differentiation compared to standard medium (DMEM-F12). We propose that the physiological concentrations as well as the greater complexity of nutrients and metabolites in Plasmax allow for a better in vitro model system for trophoblast biology. N anoparticles (NPs) are emerging materials enabling a wealth of novel applications, but evidence is increasing that NPs can have adverse effects on pregnancy and health in later life. The mechanisms of developmental toxicity of NPs are poorly understood but likely involve direct toxicity from particles that crossed the placental barrier as well as indirect toxicity from particle accumulation in placental tissue and interference with placental function and signaling. Since the placenta is the most species-specific organ, animal studies may not deliver relevant results for human applications. Consequently, there is a great need for alternative human-based ad-vanced in vitro models for developmental toxicity assessment. We have recently developed a microphysiological model combining a placental barrier model (BeWo tropho-blast cell line) with murine embryoid bodies in a user friendly microfluidic platform1. We currently further im-prove the biological model: 1) for the placenta, we work on establishing co-cultures of primary human cyto-trophoblasts (CTBs) and endothelial cells; 2) as a supportive membrane to cultivate placental cells, we explore highly permeable electrospun membranes with superior permeability to NPs compared to conventional track-etched polymer membranes and 3) we replace murine EBs with human induced pluripotent stem cell (iPSC)-derived EBs. In static insert cultures, we have successfully established placental trophoblast/endothelial cell co-cultures2 and a highly permeable chitosan/PEO electrospun cell culture support3, which will be transferred to the micro-fluidic chip platform. Primary human CTBs were isolated from human term placenta to form a confluent mon-olayer of syncytialized cells. To verify the predictive value of the model for NP transport studies, in particular the impact of flow conditions and a primary syncytiotrophoblast layer, we will study size-dependent transloca-tion of differently sized polystyrene NP in static versus dynamic conditions as well as in BeWo versus primary CTB monolayers and compare the results to those obtained with a human ex vivo placenta perfusion model. Overall, reproducing the extensive interplay between placenta and embryo in a microphysiological model will allow for a systematic assessment of placental transport as well as direct and indirect embryotoxicity to gain novel mechanistic insights, which are important for designing safe NPs.

Background

During the 40 weeks of pregnancy, developing of placenta is associated with aging and an increased expression of senescence associated proteins. Cellular senescence is characterized by a permanent state of cell cycle arrest and shows an upregulation of cyclin-dependent kinase inhibitors like p16 and p21. Cellular senescence can be induced by many stimuli like oxidative stress leading to a higher production of reactive oxygen species (ROS). The ROS production is counteracted by antioxidants like superoxide dismutase (SOD). Although placental aging is a physiological process, premature aging of the placenta can be a pathological state and is a hallmark of several placentaassociated pregnancy complications such as fetal growth restriction (FGR) and preeclampsia (PE). Aims: The study aims to establish a multiplex immunofluorescence (mIF) protocol applicable on placental samples to simultaneously analyze the spatial expression pattern of senescence markers in different tissue compartments. Investigating different placental diseases, the role of dysregulated aging in placental pathologies will be elucidated and putative targets for diagnosis and therapy will be defined. Materials and Methods: Formalin-fixed paraffin-embedded placenta samples (total n=30) including healthy term controls (n=5), healthy term controls delivered by caesarean section (n=5), placentas from late onset (n=5) and early onset FGR-pregnancies (n=5), placentas from pregnancies complicated by PE (n=5), and placentas from pregnancies where PE was accompanied by FGR (n=5) are included in the study. Based on Tyramide Signal Amplification, mIF enables the simultaneous detection of multiple proteins of interest in the same tissue section. Using the Opal™ 6-Plex Detection Kit (AKOYA Bioscience), the expression of the senescence markers p16, p21, SOD and IL6 in different placental cell populations is analyzed. The placental cell populations are differentiated by the following phenotype markers: Vimentin (placental stroma), Cytokeratin (trophoblast), β-hCG (syncytiotrophoblast), E-cadherin (cytotrophoblast), CD31 (endothelium). On each slide, one senescence marker is combined with five phenotype markers. Image acquisition is done with the Vectra Polaris™ fluorescence whole slide scanner and inForm® Tissue Analysis Software (both from AKOYA Bioscience) is used for quantification of senescence marker expression as well as the identification of the different cell types involved in premature aging. Results and discussion: We could successfully establish a 6plex antibody panel for mIF studying placental aging. The quantitative and spatial analysis of six different markers per sample is possible and currently ongoing enabling us to describe different patterns of cells involved in premature placental aging especially in FGR and PE. In PE, the outer layer of the placenta, the syncytiotrophoblast, might be particularly involved, where premature senescence leads to shedding of activating material into the vascular system of the mother, while in FGR fetal parts including fetal endothelium and stroma cells might be predominantly involved as a consequence of reduced placental function.

Background

Fetal growth restriction (FGR) and hypertensive pregnancy diseases including preeclampsia, the most important causes of perinatal mortality and morbidity, are strongly associated with impaired placentation and abnormal utero-placental blood flow. NO-donors like pentaerithrityltetranitrate (PETN) are strong vasodilators, lower the vascular resistance and protect the endothelium from stress. Based on data from a randomized controlled pilot study our aim was to the effects of PETN on reduction of FGR and perinatal death, prematurity and as well as on pregnancy induced hypertension (secondary outcome) in pregnant women with placentation failure. Methods: In a multicenter, randomized, double-blind, placebo controlled trial with two parallel groups pregnant women presenting with a mean uterine artery Pulsatility Index >95th percentile at 19+0 to 22+6 weeks of gestation were randomized to Pentalong® 50 mg or placebo twice daily. Randomization was performed block-wise with a fixed block length stratified by center and high-or low-risk group according to participants' medical history. The primary efficacy endpoint was the composite outcome of perinatal death or development of FGR. Secondary endpoints were neonatal and maternal outcomes. The trial was registered: EudraCT (2016-004396-51), DRKS (DRKS00011374) and clinicaltrials. gov (NCT03669185). Results: Between August 2017 and March 2020 317 women were included in the study and 307 were analysed. The cumulative incidence of the primary outcome was 41.1 % in the PETN group and 45.5 % in the placebo group, (adjusted RR 0.90, 95 %CI 0.69-1.17; p=0.43). Secondary outcomes like preterm birth in 37.7 % vs. 51.9 % (adjusted RR 0.73, 95 %CI 0.56-0.94; p=0.01) and pregnancy induced hypertension in 19.4 % vs. 36.6 % (adjusted RR 0.45, 95 %CI 0.22-0.92; p=0.03) were significantly reduced. Kaplan-Meier analysis revealed that participants in the PETN group might have been less likely to be admitted to the hospital (HR 0.51, 95 % CI 0.30-0.85; p=0.01), had longer time to delivery and were less likely to develop elevated blood pressure. Additionally, angiogenic factors measured during pregnancy as the ratio of soluble Flt-1 and placental growth factor (PlGF) stayed significantly longer within the normal range in the PETN group. Discussion: Although results could not confirm an effect of PETN on the development of FGR and perinatal death, the observed potential effect on maternal blood pressure further suggests PETN as a candidate to improve the outcome in pregnancies complicated by placental dysfunction. Experimental results of our group confirmed the protective effect of PETN on endothelial cells as well as trophoblastic cells exposed to oxidative stress.

Introduction

Adverse events during critical points of pregnancy lead to impaired placental function, which may cause detrimental effects on fetal growth and long-term health issues after birth. The blueprint for placental formation is initiated very early in pregnancy and the periconceptional stage is exquisitely sensitive to the maternal environment. Post-translational modification of proteins by O-GlcNAcylation is involved in the regulation of placental development and is often related to stress-induced epigenetic changes. Here, we investigated the effects of elevated protein O-GlcNAcylation during peri-implantation on placental and fetal development.

Methods

Pregnant mice were injected intraperitoneally with thiamet G (TMG) to manipulate O-GlcNAc levels during the implantation window, i.e. E3.5-E7.5. Genes linked to trophoblast development in early pregnancy were assessed by qPCR. At term (E18.5), fetal and placental weights were taken and placental cell types and nutrient (System A amino acid) transport were assessed by immunohistochemistry and transfer of 14C-methyl-AIB respectively.

Results

As expected, protein O-GlcNAcylation in E7.5 implantation sites was increased by TMG treatment. Expression of two trophoblast lineage-specific transcription factors, eomes and stra13, was reduced at E9.5. At term, fetal weight was significantly decreased by 7% in TMG-treated mice, accompanied by reduced fetal:placental weight ratio. However, placental weight, gross morphology, total area of fetal vessels (immunostained with CD31) and area of trophoblast cells (immunostained with pan-cytokeratin) in labyrinth zone were unaffected at term, nor was System A amino acid transport altered. Results: We observed that intracellular total and free cholesterol levels were elevated in hypoxia and H/R. To investigate the underlying factors responsible for the accumulation of cholesterol, we evaluated several genes controlling cholesterol homeostasis. We found that the mRNA expression of the transcription factors SREBP-2 and SRE-BP-1a, was increased in hypoxia together with HMGCR, the rate limiting gene involved in cholesterol biosynthesis.

Conclusions

The mRNA abundance of ACAT1, an enzyme involved in the esterification of cholesterol, was lower in both hypoxia and H/R, explaining the increase in total cholesterol and the accumulation of free cholesterol in the cells.

To evaluate if the increase of intracellular cholesterol affects cholesterol efflux transporters, we determined the gene and protein expression of ABCA1, ABCG1 and SR-BI. The mRNA expression of ABCA1 and ABCG1 was increased in hypoxia; SR-BI mRNA levels were elevated in both hypoxia and H/R compared with normoxia. The protein abundance of ABCA1 was reduced in both hypoxia and H/R, while the protein expression of SR-BI was increased in hypoxia compared to normoxia. These results suggest that cholesterol transport is altered after the hypoxic insult. This was confirmed in functional experiments where cholesterol efflux to maternal serum was higher in hypoxia and H/R and efflux to fetal serum was increased in H/R compared to normoxic conditions. These findings suggest that in PE there is not only an increased cholesterol concentration in the placenta, but that both the mother and the fetus receive more cholesterol during pregnancy, which may result in an increased cardiovascular risk later in life.

To determine the underlying reasons and potential consequences of the altered cholesterol efflux, we measured the protein secretion of ApoA-I and ApoE (cholesterol acceptors) and progesterone (product of cholesterol metabolism). All these factors were unchanged after hypoxia and H/R.

Conclusion:

Several pathways of cholesterol homeostasis as well as the function of ABCA1, ABCG1 and SRBI are altered in a primary trophoblast model mimicking the phenotype of PE. The increased cholesterol efflux and the observed dysregulation of cholesterol biosynthesis may have not only critical consequences for both the mother and the fetus during pregnancy but also later in life.

18 th 1 Commissariat à l'Energie Atomique et aux Energies Alternatives (CEA), Biosciences and Biotechnology Institute of Grenoble, Grenoble, France; 2 Institut National de la Santé et de la Recherche Médicale U1292, Biosanté, Grenoble, France University Grenoble-Alpes, 38000, Grenoble, France; 3 Centre Hospitalo-Universitaire Grenoble Alpes, Service Obstétrique, CS 10217, 38043 Grenoble Cedex 9, France; Université Grenoble Alpes, GRENOBLE, France.

Abstract

* Presenting author; *early stage investigator -Yes P reeclampsia (PE) is a complication affecting 3 to 5% of pregnancies. It is characterized by symptoms such as hyperten- sion and proteinuria that usually appear after 20 weeks of gestation (wg). Currently, the diagnosis is mainly based on clinical symptoms and recently on the measurement of placental angiogenic and anti-angiogenic factors that are released by the placenta in response to the maintenance of a hypoxic environment beyond the physiological period of 10/12 wg. Unfortunately, these biomarkers are not deregulated early enough to predict PE occurrence by the end of first trimester. Our team conducted a clinical study called "EGEVE, NCT01490489" that included 148 pregnant women between 14-18 wg. Out of this cohort, 4 women developed PE later. Proteomic analysis of their plasma identified a glycoprotein called Pregnancy Zone Protein (PZP), whose circulating levels were significantly lower in PE patients. The objective of this study was to characterize the role of PZP in placentas collected from the first trimester of pregnancy and to determine whether PZP expression is also deregulated in the placentas of PE women, during the third trimester of pregnancy. We used, human placentas (6 to13wg) that were collected from elective termination of pregnancy at Grenoble hospital (NCT05188066) to characterize the physiological expression profile of PZP. Placentas were also cultured in 3D system as placenta explants to determine the ontology of PZP expression and secretion throughout the first trimester of gestation and to determine the effect of hypoxia on its expression and secretion. Finally, 3rd trimester CTL and PE human placentas were used to compare PZP expression when the PE is established. We demonstrated, using immunohistochemistry and western blot analysis, that PZP is expressed in different cell types of the human placenta, with specific expression in the most differentiated trophoblasts: syncytiotrophoblasts and extravillous trophoblasts. We also demonstrated a trend to an upregulation of the PZP protein under hypoxic conditions in the 3D culture system. These findings strongly suggest a positive regulation of PZP expression and or secretion by hypoxia. Finally, we demonstrated that PZP placental expression was higher in PE specimens compared to CTL. This finding strongly suggests a potential compensatory mechanism that occurs in the placenta to overcome the initial decrease that occurs during the first trimester in PE placenta. Ongoing experiments are in progress to validate these results. To conclude on a final role of PZP in PE, a larger cohort is needed, as it will allow better stratification of PE women. In fact, PZP levels are influenced by other factors such as, contraception or the age of the women. In conclusion, we demonstrated for the first time that PZP protein may be considered as a biomarker of PE occurrence early-on during pregnancy and that this protein may contribute to placental development during early pregnancy. Background: Nitric oxide (NO), produced by endothelial NO synthase (eNOS), has a key role in reducing vascular tone in the fetoplacental circulation. Shear stress is the most effective stimulator of NO production in this vascular bed. Pregnancies complicated by fetal growth restriction (FGR) have raised vascular resistance and a different eNOS profile in this circulation, with higher expression and activity compared to normal pregnancy. In umbilical arteries from healthy pregnancies, arginase-2 competes with eNOS for L-arginine and prevents the NOS-dependent relaxation. Here we test the hypothesis that therapeutic intervention aimed at arginase inhibition by BEC, alone and with tetrahydrobiopterin (BH4), an eNOS co-factor with anti-oxidant properties, would augment NO production by the placenta, with potential future therapeutic benefit in FGR.

Methods Human placental arterial endothelial cells (HPAECs) from normal pregnancy (n=6) were grown to confluency in 0.4 Luer T/C treated micro-chambers (Ibidi BioDiagnostics). Cells were subjected to unidirectional laminar shear stress (20 dynes/cm2) for 48 hours. Four cell flow systems operated in parallel and each cell line was exposed to (i) PBS control, (ii) 25 μM BEC; (iii) 20 μM BH4 or (iv) BEC + BH4. At 48 hours, the conditioned medium was tested for nitrite levels (NO stable breakdown product) using the Griess reaction, following nitrate to nitrite conversion. The concentration dependency of BEC (0125-1250 µM) and BH4 (0.2-2000µM) on cell growth was assessed via a metabolism (MTT assay, N=6); and a cell stress protein array was performed on static cultured HPAECs (12.5 µM BEC; 20 µM BH4 n=3).

Results BEC did not affect MTT at any concentration used and BH4 was without effect at 200µM (Wilcoxon vs control 100%: NS). BEC (25µM) did not increase nitrite levels in conditioned medium from normal pregnancy compared to the PBS control (14.5±7.6µM and 17.1±4.7 µM, mean±SE, respectively), but BH4 (20µM) neared significance in increasing nitrite levels (31.3±5.7 µM; P=0.06, Wilcoxon). Experiments with BEC+BH4 had reduced numbers (n=3) due to technical problems and data were not statistically tested. BEC reduced 17 of 26 human cell stress related proteins in normal HPAECs; and notably included a 61% reduction in expression of the antioxidant SOD2 and a 28% reduction in HIF-1alpha, which dysregulates NOS expression.

Conclusion

The arginase inhibitor BEC did not enhance NO production by the HPAECs in normal pregnancy. This result might arise if increasing L-arginine availability alone is not enough to promote NO production. Alternatively, BEC could have off target actions to elevate oxidative stress and it might be possible to counteract this by concomitant treatment with the eNOS co-factor BH4. Further work is required to assess the potential of BEC, and BEC+BH4, as therapeutic strategy to enhance the capacity of placental endothelial cells to generate NO in FGR.

Methods: Flow culture experiments were performed using differentiated BeWo cells and placental villi (first trimester and term) subjected to flow chambers. They were cultured under physiological shear stress values, ranging from 0,3-1dyne/cm 2 up to 6dyne/cm 2 for pathological conditions, and low oxygen concentration. The expression of RNA was investigated with RNA Sequencing and was confirmed with qPCR. Protein levels were analyzed using Immunofluorescence and Western Blot. All results from cells exposed to shear stress were compared to static conditions to identify important metabolic players under physiological flow conditions. Mitochondrial activity was measured by IF. Glucose consumption of the cells was investigated after silencing the main glucose transporter (SLC2A1) GLUT1 and under different glucose concentrations. To gain insights about the glucose transport under (patho)-physiological conditions, we silenced the main glucose transporter GLUT1 in an artificial placental barrier and observed GOT1 and GOT2 (Glutamic-oxaloacetic transaminase) expression as well as the glucose uptake in different cell layers.

Results: With RNA Sequencing we were able to observe lower expression of genes involved in the glycolytic pathway, beginning with the main glucose transporter itself, GLUT1. Genes, which encode for key enzymes in this pathway are downregulated (HK2, PFKP). The main product of the glycolytic pathway, pyruvate and further lactate, were increased on cellular level as well as in the supernatant, under flow conditions. Mitochondria in cells exposed to shear stress show lower glucose dependency to hold their membrane potential. Upregulation of genes involved in the energy metabolism could be observed for amino acid pathways. GOT1 and GOT2 were significantly upregulated in cells exposed to shear stress. These two enzymes are also altered in IUGR, as well as in glucose uptake studies, by silencing the glucose transporter GLUT1. Conclusion: We suggest, that there is a shift of the energy metabolism from the glycolytic to the amino acid pathway in placenta with physiological shear rates. The use of amino acids to gain energy in the placenta can be important, to support the glycolysis and to force glucose net transport to the fetus.

Methods: Fresh term placental explants were obtained from healthy pregnancies. An in vitro model of inflammation was established using LPS (0.1µg/ml). Placental explants (n=6) were incubated with vehicle controls or LPS+/-ASA (50, 100µM), FA (10, 50µM) or MA (10, 50µM) for 24 or 48 hours. To assess the effects of pre-treatment, placental explants (n=7) were incubated with ASA, FA or MA at 2 concentrations for 24 hours, then incubated with LPS for another 48 hours. Culture medium was collected to assess the release of pro-and anti-inflammatory cytokines (IL-1β, IL-6, IL-10, IL-1ra) downstream of NLRP3 activation. HCG release was measured to assess placental endocrine function. Explants were also collected for immunohistochemistry to detect cleaved caspase 3 (CC3) to assess induction of apoptosis.

Results: LPS treatment significantly increased IL-10 secretion at 24 and 48 hours (p=0.02, p=0.01) and IL-1ra secretion at 24 hours (p=0.05), compared to controls. Co-treatment with ASA (100µM) significantly reduced IL-10 secretion compared to the LPS group after 24 and 48 hours (p=0.006, p=0.005). Co-treatment with FA acid increased median pro-inflammatory IL-6 levels at 24 and 48 hours. Pre-treatment with MA (10, 50µM) significantly decreased LPS-induced release of IL-1β (p=0.003, p<0.0001), IL-10 (p=0.0073, p=0.0068) and IL-1ra (p<0.0001, p<0.0001). HCG secretion was unchanged by all treatments, indicating intact placental endocrine function. A low number of CC3-positive cells was observed in all explants, suggesting that none of the treatments induced cell death at the concentrations tested.

Conclusion:

ASA and MA reduced LPS-induced placental inflammation in healthy placental explants, without altering endocrine function or causing cytotoxicity. FA may increase inflammation in some placentas. This study suggests that drugs known to target the NLRP3 inflammasome can alter cytokine release from placental tissue, thus merit further investigation to determine whether this approach could reduce sterile inflammation. Mefenamic acid is licenced in the UK for dysmenorrhoea and menorrhagia (0.5g three times a day), which may facilitate further studies on placental inflammation.

Introduction

Pro-and anticoagulant mechanisms of the placenta play an important role during pregnancy. One of these mechanisms involves the hydrolysis of extracellular adenosine triphosphate (ATP) via adenosine di-and monophosphate (ADP, AMP) to adenosine by the ectonucleotidases CD39 and CD73. ATP can be released from cells into the extracellular space in response to metabolic disturbances and tissue insults. ATP and ADP are also released by platelets into their environment upon activation, which in turn can lead to inflammatory processes and cell damage at elevated concentrations. Dysregulations in this process can thus lead to the development of pregnancy pathologies, such as preeclampsia. In this study, we aimed to investigate the role of the ectonucleotidases CD39 and CD73 in the human placenta.

Methods

The expression of CD39 and CD73 was investigated in placental tissue from the first and third trimester of healthy and preeclamptic pregnancies as well as in trophoblast cell lines at protein and gene expression levels. Placental explant cultures were incubated in presence of either platelet releasate or isolated activated platelets from autologous donors for 24h. Total RNA and protein were subjected to qPCRs and Western blots, whereas FFPE placental tissue was analysed by immunohistochemistry.

Results

Analysis of placental tissue from first and third trimester revealed an increase of CD39 towards term on protein as well as on gene expression level, whereas the expression of CD73 decreases towards the end of pregnancy. Furthermore, CD39 expression was shown to be decreased in preeclamptic tissue compared to healthy controls.

Conclusion

Decreased placental expression of CD39 could result in reduced hydrolysis of extracellular ATP, which may contribute to a pro-inflammatory microenvironment in the intervillous space in preeclampsia.

18 th EP G m ee tin g | Am ph ith ea te r of Bi ot ec hn ol og ie s, Bu ild in g 44 0 IN RA E Île -d e-Fr an ce -Jo uy -e n-Jo sa s SESSION 9

2 DNA

 2 18 th EP G m ee tin g | Am ph ith ea te r of Bi ot ec hn ol og ie s, Bu ild in g 44 0 IN RA E Île -d e-Fr an ce -Jo uy -e n-Jo sa s SESSION

  18 th EP G m ee tin g | Am ph ith ea te r of Bi ot ec hn ol og ie s, Bu ild in g 44 0 IN RA E Île -d e-Fr an ce -Jo uy -e n-Jo sa s SESSION 6 TOWARDS A HUMAN-BASED PLACENTA-EMBRYO CHIP FOR DEVELOPMENTAL TOXICITY AS-SESSMENT OF NANOPARTICLES Murdeu, M. 1* , Boos, J. 2 , Di Renzo, E. 1 , Aengenheister, L. 1 , Furer, L.A. 1 , Hierlemann, A. 2 , Buerki-Thurnherr, T.

  Our data demonstrating decreased fetal weight and fetal:placental weight ratio suggest that increased protein O-GlcNAcylation during implantation compromises placental function, though identification of the underlying mechanism(s) requires further investigation. 18 th EP G m ee tin g | Am ph ith ea te r of Bi ot ec hn ol og ie s, Bu ild in g 44 0 IN RA E Île -d e-Fr an ce -Jo uy -e n-Jo sa s SESSION 7 PERINATAL, METABOLIC AND REPRODUCTIVE FEATURES IN PPARG-RELATED LIPODYSTROPHY Fournier T. *1# , Gosseaume C. 1,2# , Jéru I. 2,3 , Missotte I. 4 , Archambeaud F. 5 , Debussche X. 6 , Fève B.

  EP G m ee tin g | Am ph ith ea te r of Bi ot ec hn ol og ie s, Bu ild in g 44 0 IN RA E Île -d e-Fr an ce -Jo uy -e n-Jo sa s SESSION 8 CHARACTERIZATION OF THE ROLE OF PREGNANCY ZONE PROTEIN (PZP) IN PREECLAMPSIA Desseux, M. 1,2* , T. Barjat, V. Kieu, A. Adrait, N. Lemaitre, C. Collet, M. Digonnet, F. Sergent, M, Benharouga, Alfaidy, A. 1,2,3

  18 th EP G m ee tin g | Am ph ith ea te r of Bi ot ec hn ol og ie s, Bu ild in g 44 0 IN RA E Île -d e-Fr an ce -Jo uy -e n-Jo sa s SESSION 8 THE POTENTIAL OF S-(2-BORONOETHYL)-L-CYSTEINE (BEC) AS A THERAPY TO ENHANCE NITRIC OXIDE PRODUCTION BY THE FETOPLACENTAL ENDOTHELIUM Almohammadi L.

1 1

 1 Particles-Biology Interactions, Empa, Swiss Federal Laboratories for Materials Science and Technology, Ler-chenfeldstrasse 5, 9014 St.Gallen, Switzerland 2 Bioengineering Laboratory, Department of Biosystems Science and Engineering, ETH Zürich, Mattenstrasse 26, Basel 4058, Switzerland Abstract

* Presenting author; early stage investigator -Yes

2,7 , Guerci B. 8 , Horsmans Y. 9 , Nobecourt E. 10 , Pienkowski C. 11 , Poitou C. 12 , Thissen J. P. 9 , Lascols O. 2,3 , Vignaud M. L. 1 , Degrelle S. 1,13 , Tsatsaris V. 1, 3 , Vigouroux C. 2,3,7# , Vatier C. 2,7#

  Methods: The PE model was established in cytotrophoblast cells (CTB) isolated from healthy placentae by trypsin/DNAse digestions. CTB were placed either under normoxic (5% pCO2/21%pO2/24h), hypoxic (5% pCO2/1.5% pO2/24h) or hypoxia/reoxygenation (H/R, 6h interval each of normoxia and hypoxia until 24h) conditions. Total, esterified, and free intracellular cholesterol levels were assayed enzymatically. The mRNA expression of genes involved in cholesterol homeostasis (SREBP-1a, SREBP-2, LDLR, HMGCR, ACAT-1) and cholesterol transport (ABCA1, ABCG1 and SR-BI) was analysed by qPCR; protein expression of ABCA1, ABCG1 and SR-BI was determined by western blot-

ting. Additionally, we determined the efflux of cholesterol to maternal and fetal serum (5% serum/6h/37ºC) under normoxic, hypoxic and H/R conditions. Finally, we evaluated the protein secretion of apolipoproteins (ApoA-I and ApoE) and progesterone by ELISA.

1,2 Greenwood S. 1,2 , Johnstone E.D. 1,2 , Brownbill P. 1,2 1

  Maternal & Fetal Health Research Centre, Division of Developmental Biology & Medicine, School of Medical Sciences, Faculty of Biology, Medicine and Health, University of Manchester, Manchester, UK; 2 St Mary's Hospital, Manchester University Hospital NHS Foundation Trust, Manchester Academic Health Science Centre, Manchester, UK

	Abstract	*Early stage investigator

Bioinformatics Programme, Babraham Institute, Cambridge, UK;

Centro de Investigación Príncipe Felipe, Valencia, Spain;

Department of Biochemistry and Molecular Biology, University of Calgary, Calgary, Canada;

Department of Medical Genetics, University of Calgary. Calgary, Canada;

Université Paris Cité, Inserm, 3PHM, Paris, F-75006, France;

Sorbonne University, Inserm, Saint-Antoine Research Centre, Paris, F-75011, France;

Assistance Publique-Hôpitaux de Paris;

Department of Pediatrics, Nouméa, New Caledonia, France;

Department of Endocrinology, Limoges, France;

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The present study is supported by the Austrian Science Fund (grant P-31470-B30)

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SESSION 1

This study is supported by the Austrian Science Fund (grant P-34588). ph ith ea te r of Bi ot ec hn ol og ie s, Bu ild in g 44 0

IN RA E Île -d e-Fr an ce -Jo uy -e n-Jo sa s SESSION 1

SESSION 8

DIFFERENTIAL EXPRESSION OF GLUCOSE TRANSPORTER PROTEINS GLUT-1, GLUT-3, GLUT-8 AND GLUT-12 IN THE PLACENTA OF MACROSOMIC, SMALL-FOR-GESTATIONAL-AGE AND GROWTH-RESTRICTED FETUSES are dysregulated in pregnancy pathologies such as preeclampsia, which has been associated with aberrant trophoblast differentiation as well as turbulent blood flow in the intervillous space and increased wall shear stress at the villous syncytiotrophoblast. Here, we aimed to analyze expression dynamics of placental S1P receptors in human first trimester, pre-term and term cases, and characterize their expression pattern in different primary placental cell types and on differentially treated BeWo cells and first trimester placental explants. Methods: The expression of the five S1P receptors was analyzed by qPCR in first trimester placental samples, preterm and healthy third trimester placentas. In addition, the expression behavior of these receptors was measured in the BeWo cell line and placental explant cultures cultured under specific flow conditions. Immunohistochemistry and Western blot were used to characterize S1P receptors in specific primary cell types of placental tissue. Moreover, we investigated whether or not the placental S1PR subtype repertoire is deregulated in BeWo cells as well as first trimester placental explants, upon treatment with platelet-derived factors and different culture conditions including hypoxia and different flow rates. Results: qPCR analysis revealed that S1PR2 is the predominant placental S1P receptor in first trimester and vanishes towards term. All other S1P receptors increased from first trimester towards term. RNA and Protein data as well as immunohistochemical stainings confirmed that S1PR1 is predominantly found in the endothelium of placental villi, while S1PR2 and S1PR3 are mainly located in trophoblasts. Flow culture of the BeWo cell line showed no effect on the expression level of S1PR2, while platelet-derived factors decreased the expression of some receptors significantly.

In conclusion, our study suggests that the placental S1PR repertoire is differentially expressed across gestation. S1P receptor expression pattern in trophoblasts depends on its differentiation status and may be deregulated in response to hemodynamic forces and the presence of platelet-derived factors. Additional experiments are needed to further decipher the role of S1P receptors in the placenta over the course of human pregnancy and in pregnancy complications.

EFFECT OF PRAVASTATIN ON PLACENTAL EXPRESSION OF EGFL7 IN PE AND IUGR :

A NEW POTENTIAL THERAPEUTIC APPROACH Salvi S.

Abstract

* Early stage investigator -Yes | Corresponding author : Luisa Campagnolo 3 E-mail: campagnolo@med.uniroma2.it M aternal systemic manifestations of Preeclampsia (PE) are mainly due to the placental release of anti-angiogenic factors. Epidermal Growth Factor Like Domain 7 (Egfl7) is an angiogenic factor, highly expressed in proliferating endothelial cells during embryogenesis and in physiologic and pathologic angiogenesis discovered by our group simultaneously with other authors. Recently the trophoblast as a novel source of EGFL7 in the human placenta was also identified. In PE-complicated pregnancies, a significant EGFL7 downregulation in placental tissues is on the other hand accompanied by a significant increasein the maternal plasma. Moreover, circulating EGFL7 levels can discriminate between women with early-and late-onset PE (e-PE and l-PE) and isolated intrauterine growth restriction (IUGR). Pravastatin (PRA) is a lipid-lowering drug with hydrophilic activity widely taken to reduce the risk of cardiovascular events. In the last years, the potential of PRA in the prevention and treatment of PE was highlighted. Aim of our pilot case-control study is to evaluate the ability of PRA to modulate EGFL7 expression in human chorionic villous explant culture from uncomplicated pregnancies and from women with PE and IUGR. Methods: 19 women were enrolled for this study: 10 healthy controls, 4 e-PE, 3 l-PE and 2 IUGR. In all groups, placenta was collected after caesarean delivery with standard biopsy procedure. Explants of chorionic villi were prepared as described by Miller et al and chorionic villous explants were cultured for 24 hours with or without 10 µM PRA. Gene and protein expression of EGFL7 and other angiogenic factors was quantified by qRT-PCR and Western Blot analysis on RNA and protein extracts from chorionic villous cultures, respectively. Results: PRA significantly increased EGFL7 gene expression in villous explant cultures obtained from healthy, l-PE and IUGR pregnancies (p < 0.001, p = 0.006, and p = 0.014, respectively), while its levels decreased in e-PE villi after PRA treatment (p = 0.025). This trend was confirmed at protein level, although in a not-significant way. Differently from the other groups, in samples obtained from l-PE patients all NOTCH target genes were significantly upregulated by PRA treatment. NOTCH1 transmembrane subunit (NOTCH1 NTM) protein levels were higher in villous cultures obtained from treated compared to untreated l-and e-PE patients; no changes were observed for healthy controls. Conclusion: PRA is able to modulate the expression of EGFL7 in human placenta; the differences observed between groups and in particular between l-PE and e-PE underlines the well-known different nature of the two forms of PE and isolated IUGR; from a clinical point of view, the effect of PRA on villi from e-PE encourages the use of this molecule in prevention rather than as treatment when the disease is already established and the severe damaged placental villi may not be able anymore to correctly answer to PRA treatment. Background: The human placenta is a multifunctional organ essential for fetal development and growth, whose function can be disrupted by xenobiotics in maternal blood. Benzo-[a]-pyrene (B[a]P) is a carcinogenic, mutagenic and reprotoxic pollutant as well as an endocrine disruptor that can be internalized in the human body by respiratory and/or oral exposure. Cerium dioxide nanoparticles (CeO 2 NPs) have been introduced into our environment, mainly for their catalytic properties. These nanoparticles currently share the same emission sources as B[a]P (e.g. cigarette smoke, diesel engine exhaust). CeO 2 NPs are also being studied for their antioxidant and pro-oxidant properties for therapeutic applications. The study of these nanoparticles must take into account their ability to coat themselves with surrounding molecules such as B[a]P from common emission sources. In order to understand their impact on human health, in 2010 the OECD included CeO 2 NPs in the priority list of nanomaterials requiring urgent assessment. The aim of our study is to determine the impact of these two atmospheric pollutants on the human placental barrier in concomitant exposure, in order to get closer to the environmental reality.

Methods:

The chosen concentrations ratio used in this study was 10 μg/cm2 NPs CeO 2 for 1 μM B[a]P, which is the quantity of B[a]P necessary to cover the entire surface of the NPs when they are encountered together. Sections of chorionic villi from human placentas at term of pregnancy exposed in vitro to the pollutants were observed by confocal and Raman microscopy. Villous cytotrophoblasts (VCT) isolated from human placentas at term of pregnancy were cultured and exposed in vitro to these pollutants. Cytotoxicity was assessed using metabolic activity of mitochondrial dehydrogenase (WST-1 test) and extracellular lactate dehydrogenase assay. Placental endocrine activity was assessed by ELISA. The signaling pathways impacted by exposure to these pollutants have been studied by Western Blot and RT-qPCR. The production of reactive oxygen species (ROS) was assessed by the DCF probe.

Results: We demonstrated that CeO 2 NPs coated with B[a]P can be internalized within the chorionic villi. The two pollutants used at realistic exposure doses do not induce toxicity nor disturbance of hCG production on VCT in vitro. However, B[a]P alone or in co-exposure with CeO 2 NPs activate the metabolic pathway of the aryl hydrocarbon receptor (AhR), causing DNA damage. B[a]P also stabilizes the stress transcription factor p53 and its transcriptional target p21. At the studied doses and for short exposure times, CeO 2 NPs alone lead to an increase in ROS production, unlike B[a]P.

Conclusion:

Although these two pollutants do not cause major toxicity on term human trophoblasts after in vitro exposures, cellular stress markers are induced.