Trophoblasts, invasion, and microRNA.

International audienceMicroRNAs (miRNAs) have recently become essential actors in various fields of physiology and medicine, especially as easily accessible circulating biomarkers, or as modulators of cell differentiation. To this respect, terminal differentiation of trophoblasts (the characteristic cells of the placenta in Therian mammals) into syncytiotrophoblast, villous trophoblast, or extravillous trophoblast constitutes a good example of such a choice, where miRNAs have recently been shown to play an important role. The aim of this review is to provide a snapshot of what is known today in placentation mechanisms that are mediated by miRNA, under the angles of materno-fetal immune dialog regulation, trophoblast differentiation, and angiogenesis at the materno-fetal interface. Also, two aspects of regulation of these issues will be highlighted: the part played by oxygen concentration and the specific function of imprinted genes in the developing placenta

In Vitro Fertilization and Embryo Culture Strongly Impact the Placental Transcriptome in the Mouse Model

BACKGROUND: Assisted Reproductive Technologies (ART) are increasingly used in humans; however, their impact is now questioned. At blastocyst stage, the trophectoderm is directly in contact with an artificial medium environment, which can impact placental development. This study was designed to carry out an in-depth analysis of the placental transcriptome after ART in mice. METHODOLOGY/PRINCIPAL FINDINGS: Blastocysts were transferred either (1) after in vivo fertilization and development (control group) or (2) after in vitro fertilization and embryo culture. Placentas were then analyzed at E10.5. Six percent of transcripts were altered at the two-fold threshold in placentas of manipulated embryos, 2/3 of transcripts being down-regulated. Strikingly, the X-chromosome harbors 11% of altered genes, 2/3 being induced. Imprinted genes were modified similarly to the X. Promoter composition analysis indicates that FOXA transcription factors may be involved in the transcriptional deregulations. CONCLUSIONS: For the first time, our study shows that in vitro fertilization associated with embryo culture strongly modify the placental expression profile, long after embryo manipulations, meaning that the stress of artificial environment is memorized after implantation. Expression of X and imprinted genes is also greatly modulated probably to adapt to adverse conditions. Our results highlight the importance of studying human placentas from ART

FOXD1 mutations are related to repeated implantation failure, intra-uterine growth restriction and preeclampsia

Background: Human reproductive disorders consist of frequently occurring dysfunctions including a broad range of phenotypes affecting fertility and women's health during pregnancy. Several female-related diseases have been associated with hypofertility/infertility phenotypes, such as recurrent pregnancy loss (RPL). Other occurring diseases may be life-threatening for the mother and foetus, such as preeclampsia (PE) and intra-uterine growth restriction (IUGR). FOXD1 was defined as a major molecule involved in embryo implantation in mice and humans by regulating endometrial/placental genes. FOXD1 mutations in human species have been functionally linked to RPL's origin. Methods: FOXD1 gene mutation screening, in 158 patients affected by PE, IUGR, RPL and repeated implantation failure (RIF), by direct sequencing and bioinformatics analysis. Plasmid constructs including FOXD1 mutations were used to perform in vitro gene reporter assays. Results: Nine non-synonymous sequence variants were identified. Functional experiments revealed that p.His267Tyr and p.Arg57del led to disturbances of promoter transcriptional activity (C3 and PlGF genes). The FOXD1 p.Ala356Gly and p.Ile364Met deleterious mutations (previously found in RPL patients) have been identified in the present work in women suffering PE and IUGR. Conclusions: Our results argue in favour of FOXD1 mutations' central role in RPL, RIF, IUGR and PE pathogenesis via C3 and PlGF regulation and they describe, for the first time, a functional link between FOXD1 and implantation/placental diseases. FOXD1 could therefore be used in clinical environments as a molecular biomarker for these diseases in the near future. Recurrent pregnancy loss, Preeclampsia, Intra-uterine growth restriction, FOXD1. © 2019 The Author(s)

The Intensity of IUGR-Induced Transcriptome Deregulations Is Inversely Correlated with the Onset of Organ Function in a Rat Model

A low-protein diet applied during pregnancy in the rat results in intrauterine growth restricted (IUGR) fetuses. In humans, IUGR is associated with increased perinatal morbidity, higher incidence of neuro-developmental defects and increased risk of adult metabolic anomalies, such as diabetes and cardiovascular disease. Development and function of many organs are affected by environmental conditions such as those inducing fetal and early postnatal growth restriction. This phenomenon, termed “fetal programming” has been studied unconnectedly in some organs, but very few studies (if any) have investigated at the same time several organs, on a more comparative basis. However, it is quite probable that IUGR affects differentially most organ systems, with possible persistent changes in gene expression. In this study we address transcriptional alterations induced by IUGR in a multi-organ perspective, by systematic analysis of 20-days rat fetuses. We show that (1) expressional alterations are apparently stronger in organs functioning late in foetal or postnatal life than in organs that are functioning early (2) hierarchical classification of the deregulations put together kidney and placenta in one cluster, liver, lungs and heart in another; (3) the epigenetic machinery is set up especially in the placenta, while its alterations are rather mild in other organs; (4) the genes appear deregulated in chromosome clusters; (5) the altered expression cascades varies from organ to organ, with noticeably a very significant modification of the complement and coagulation cascades in the kidney; (6) we found a significant increase in TF binding site for HNF4 proteins specifically for liver genes that are down-regulated in IUGR, suggesting that this decrease is achieved through the action of HNF transcription factors, that are themselves transcriptionnally induced in the liver by IUGR (x 1.84 fold). Altogether, our study suggests that a combination of tissue-specific mechanisms contributes to bring about tissue-driven modifications of gene cascades. The question of these cascades being activated to adapt the organ to harsh environmental condition, or as an endpoint consequence is still raised

Vers l'identification de marqueurs biologiques dans le retard de croissance intra-utérin humain

Le retard de croissance intra-utérin (RCIU) est une complication fréquente de la grossesse qui se traduit à la naissance par un poids et/ou une taille inférieurs au dixième percentile par rapport à l âge gestationnel. Il représente un problème majeur de santé publique avec une augmentation de la morbi-mortalité néonatale et un risque accru de développer des maladies cardiovasculaires et un diabète à l âge adulte. Le RCIU humain est une pathologie complexe et multifactorielle avec une physiopathologie incomplètement élucidée dans près de 30 à 40% des cas. L objectif de ce travail a été de progresser sur la physiopathologie du RCIU humain et de proposer des gènes candidats impliqués. J ai analysé les perturbations d expression génique dans des placentas issus de grossesses avec et sans RCIU, participé à la caractérisation d un modèle animal de rate gestante soumise à un régime hypoprotidique, mis au point et validé un modèle cellulaire de cellules JEG-3 transformées par surexpression du gène PDX1 et/ou déplétées en acides aminés constituant un modèle important des effets du RCIU. J ai étudié le rôle et l implication de plusieurs gènes candidats : IMP3, les Cullines et PDX1. J ai enfin participé à l identification de nouveaux gènes soumis à empreinte dans le placenta. Ce travail de thèse a confirmé le rôle essentiel de l apoptose et des régulations épigénétiques, plus particulièrement les modifications de profil de méthylation de l ADN, dans le RCIU. L analyse transcriptomique des placentas humains de grossesse avec RCIU a confirmé le rôle essentiel de 3 gènes : la leptine, IGFBP1 et RBP4. Enfin l utilisation de notre modèle cellulaire a permis de confirmer l implication de PDX1 dans le RCIU, PDX1 contrebalance les effets transcriptomiques de la déplétion en acides aminés sur les cellules JEG en culture. En extrapolant ces résultats à la pathologie humaine, on peut émettre l hypothèse que l induction placentaire de PDX1 en situation de RCIU permettrait de contrebalancer ou de limiter les effets délétères du RCIU pour la survie du fœtus. La leptine, IGFBP, RBP4 et PDX1 sont de bons candidats à la fois pour l origine du RCIU et également pour la prédiction de l évolution possible vers un syndrome métabolique chez les adultes anciens RCIU. Des études complémentaires sur le rôle et l implication de ces 4 gènes dans la physiopathologie du RCIU ainsi que dans la programmation fœtale des pathologies cardiovasculaires, de l obésité et de l intolérance glucidique à l âge adulte restent nécessaires.Intra-uterine growth restriction (IUGR) is a frequent complication of pregnancy that leads to a baby with a birth weight and/or size below the 10th percentile for a given gestational age. IUGR represents a major public health problem associated with neonatal increased morbidity and mortality and an increased risk to develop cardiovascular pathologies and diabetes in adulthood. Human IUGR is a complex and multi-factorial pathology with an incompletely characterised physiopathology in up to 30 to 40% of cases. The goal of this work was to improve the knowledge on the physiopathology of IUGR and to propose implicated candidate genes.I analysed modulations of gene expression in placentas from pregnancies with and without IUGR, participated to the characterisation of an IUGR animal model of female rat fed with a hypoprotidic diet during gestation, set up and validated a cellular model of JEG-3 cells transformed by over-expression of the PDX1 gene and/or depletion of amino acids to provide a handy model of IUGR effects. I studied the role and implication of candidate genes: IMP3, the Cullin family and PDX1. Finally I participated to the identification of new imprinted genes in the human placenta. This thesis work confirmed the essential role of apoptosis and epigenetic regulations, in particular modifications of DNA methylation profiles, in IUGR. Transcriptomic analysis of human placentas from IUGR and control pregnancies confirmed the crucial role of 3 genes: leptin, IGFBP1 and RBP4. Finally, the use of our cellular model strengthened the role of PDX1 in IUGR, where it counterbalances the transcriptomic effects of amino acid depletion in cultured JEG-3 cells. By extrapolating these results to the human pathology, we can suggest the hypothesis that the placental induction of PDX1 in IUGR could counterbalance or limit the deleterious effects of IUGR on the foetus survival. Leptin, IGFBP1, RBP4 and PDX1 are good candidates to explain the origin of IUGR as well as to predict the potential evolution towards a metabolic syndrome in adults that suffered from IUGR. Complementary studies on the role and function of these 4 genes in IUGR physiopathology and also in foetal programming of cardiovascular pathologies, obesity and glucidic intolerance in adulthood remain necessary.PARIS11-SCD-Bib. électronique (914719901) / SudocSudocFranceF

Oocyte ERM and EWI Proteins Are Involved in Mouse Fertilization

International audienceIn mammalian fertilization, the link between the oocyte plasma membrane and underneath cytoskeleton has often been associated to key elements of successful gamete fusion, like microvilli shaping or CD9 function, but its effective role has poorly been studied. EWI-2 and EWI-F as cis partners of CD9, and ERM proteins (Ezrin, Radixin and Moesin) that both attach to the actin cytoskeleton and to the EWI are part of the molecules that make the link between the oocyte membrane and its cytoskeleton. This study aims to assay through siRNA inhibition, the involvement of these ERM and EWI molecules in mouse fertilization, their role in the microvilli morphology of the egg but also their possible contribution to the cortical tension, a parameter that reflects the mechanical behavior of the oocyte cortex. Whereas inhibiting separately the expression of each protein had no effect on fertilization, the combined inhibition of either EWI-2/EWI-F or the three ERM triggered a significant decrease of the fertilization index. This inhibition seems to correlate with an increase in the radius of curvature of the oocyte microvilli. It also causes a decrease of the oocyte cortical tension. These results show the importance of EWI-2 and EWI–F and ERM proteins in the smooth running of a fertilization event and support their involvement in the microvilli architecture of the oocyte and in its mechanical properties

Maternal Genetic Effects, Exerted by Genes Involved in Homocysteine Remethylation, Influence the Risk of Spina Bifida

There is currently considerable interest in the relationship between variation in genes that are involved in the folate-homocysteine metabolic axis and the risk of spina bifida. The evaluation of this relationship is, however, complicated by the potential involvement of both the maternal and the embryonic genotype in determination of disease risk. The present study was designed to address questions regarding both maternal and embryonic genetic risk factors for spina bifida by use of the two-step transmission/disequilibrium test. Analysis of data on variants of two genes involved in homocysteine remethylation/methionine biosynthesis—methionine synthase (MTR) A2756G and methionine synthase reductase (MTRR) A66G—provided evidence that both variants influence the risk of spina bifida via the maternal rather than the embryonic genotype. For both variants, the risk of having a child with spina bifida appears to increase with the number of high-risk alleles in the maternal genotype: MTR (R(1)=2.16, 95% CI 0.92–5.06; R(2)=6.58, 95% CI 0.87–49.67) and MTRR (R(1)=2.05, 95% CI 1.05–3.99; R(2)=3.15, 95% CI 0.92–10.85). These findings highlight the importance of considering both the maternal and embryonic genotype when evaluating putative spina bifida susceptibility loci

Altérations de l’expression des gènes dans les Retards de Croissance Intra-Utérin

Les retards de croissance intra-utérins constituent des complications fréquentes de la grossesse (3-5 %), caractérisées par une rupture de la courbe de croissance des foetus in utero. Dans les cas les plus graves le RCIU entraîne une souffrance fœtale chronique, qui en fait la troisième cause de létalité après la prématurité et les malformations. Dans de rares cas, le retard de croissance a été associé à des maladies impliquant l’empreinte génétique. De nombreux travaux se sont focalisés sur ce point probablement à la suite de l’identification des effets de l’invalidation du gène soumis à empreinte IGF2 au début des années 1990. Indépendamment de l’empreinte, le lien de cause à effet entre mutation d’un gène particulier et RCIU n’a été démontré que dans de très rares cas et en général dans le contexte de syndromes complexes. C’est le cas des mutations de la culline 7 dans le syndrome dit « 3M ». L’hétérogénéité évidente du RCIU provient de sa définition clinique extrêmement vague, et nécessitant des subdivisions. On peut par exemple distinguer des causes « endogènes » liées à des défauts de gènes impliqués dans le développement foeto-placentaire, et des causes vasculaires induites par un dysfonctionnement placentaire, comme dans le défaut trophique induit par la prééclampsie. Cette dernière catégorie de « RCIU vasculaires » est elle-même caractérisée par une très grande hétérogénéité. Un axe de recherche important part néanmoins de l’hypothèse que les conséquences physiologiques du RCIU pourraient présenter des tendances générales. Ces tendances peuvent être analysées sur le placenta, mais aussi sur des organes pérennes de l’individu, comme les reins, le foie, le cerveau, etc. La pérennisation des altérations nutritionnelles de la grossesse pourrait passer par des modifications « mémorisables » par les cellules, en particulier par exemple par l’intermédiaire d’un tissu commun à tous les organes : l’endothélium vasculaire. Les mécanismes qui permettent la transmission de l’information, la mémorisation d’un stress précoce sont matérialisés dans la chromatine par des mécanismes dits « épigénétiques ». Les modifications épigénétiques sont héritables mais ne modifient pas la séquence de l’ADN. On ne parle pas de mutations, mais d’épimutations. Elles sont réalisées par trois mécanismes complémentaires : des modifications chimiques de la molécule d’ADN (méthylation des cytosines dans les séquences CG), des modifications chimiques des histones (influant sur la structure de la chromatine, son état « ouvert », transcriptionnellement actif, ou « fermé », et des ARNs non codants capables de bloquer l’expression de gènes de façon ciblée). Le RCIU peut être induit expérimentalement par le régime alimentaire dans des modèles animaux (revue exhaustive dans Mc MILLEN et ROBINSON, Physiol Reviews, 2005). C’est ce que nous avons réalisé chez le rat par restriction protidique pendant la grossesse. Nous avons ensuite analysé les altérations trasncriptomiques dans cinq organes cibles : le placenta, le rein, le cœur, le foie et le poumon. L’analyse approfondie des altérations placentaires a suggéré l’implication du gène Pdx1 comme coordinateur majeur des altérations expressionnelles placentaires, en parallèle avec celles de nombreuses protéines à doigt de zinc. L’analyse comparée avec l’humain a révélé qu’une proportion élevée de facteurs coordonnant les altérations transcriptomiques est modifiée comme chez le rat, renforçant l’idée d’une unité dans les altérations placentaires (BUFFAT et al., 2007a). L’analyse des effets de PDX1 se poursuit dans un modèle cellulaire (GASCOIN-LACHAMBRE et al., en préparation). Parmi les acteurs révélés par des approches globales ou ciblées indépendantes, on retrouve une famille de 7 protéines appelées cullines. En particulier, la culline 4B et la culline 7 semblent altérées au niveau transcriptionnel par le RCIU, chez l’homme comme chez les rongeurs. Nous avons récemment montré que le RCIU induit des modifications subtiles de l’épissage de ces gènes, tout en altérant leur régulation expressionnelle, et leur marquage épigénétique au niveau de séquences CG spécifiques. Les cullines sont impliquées dans l’échafaudage de protéines cytoplasmiques structurant la cellule, ainsi que dans l’acheminement de protéines spécifiques vers le protéasome. Ce sont donc des acteurs importants de la physiologie et de l’homéostasie cellulaire, qui pourraient donc temporiser les effets du RCIU, et donc être systématiquement activées dans cette pathologie. En ce qui concerne les altérations des organes induites chez les fœtus par le RCIU, nous les avons analysées en détail dans le cas du rein. Nous avons pu montrer une augmentation massive de gènes impliqués dans la coagulation et l’activation du complément dans cet organe, ce qui est corrélé à la diminution du nombre de glomérules et donc globalement à un fonctionnement probablement moins efficace (BUFFAT et al., 2007b). L’analyse transcriptomique globale des cinq organes analysés dans le modèle rat conduit à l’idée que le placenta et le rein servent de « filtres » à deux niveaux entre la mère et le fœtus, car ces deux organes ont des altérations expressionnelles corrélées tandis que les trois autres organes fœtaux analysés (cœur, foie, poumon) sont corrélés entre eux. L’étude des effets à long terme sur les organes pérennes suite au RCIU est un sujet encore neuf, qui pose de très nombreuses questions en termes de régulation, d’adaptation des organes à un environnement agressif. La meilleure compréhension des effets du RCIU pourrait permettre par des apports nutritionnels ciblés, d’améliorer le pronostic à court et a long terme

Gene polymorphisms of the renin-angiotensin system and early development of hypertension

BACKGROUND: A case-control association study was conducted to investigate a possible involvement of polymorphisms of three renin-angiotensin system genes: ACE (I/D and T-3892C), AGT (M235T and T174M), and AT1R (A1166C) in the early development of hypertension. ----- METHODS: One hundred nineteen hypertensive and 125 normotensive participants aged 18 to 40 years were selected from a broader sample representative of the general population of Croatia. The selection criteria for hypertensive cases were systolic blood pressure (BP) higher than 140 mm Hg or diastolic BP higher than 90 mm Hg and a history of hypertension according to patient interview. ----- RESULTS: Among the polymorphisms investigated, only those located on the ACE gene were associated with hypertension. For ACE I/D, the odds ratio for hypertension of DD versus II homozygote individuals was 2.50 (95% confidence interval [CI] 1.19-5.25) and for ACE T-3892C, the odds ratio of CC versus TT individuals was 2.32 (95% CI 1.05-5.10). Both polymorphisms of the ACE gene were in tight linkage disequilibrium. Of the investigated risk factors for hypertension, only body mass index (BMI) showed an influence on the early development of hypertension, acting independently of the ACE polymorphism. Their additive effect gives rise to 86% of hypertensives in subjects having both the DD genotype and BMI >or=30 kg/m(2). ----- CONCLUSIONS: The present study provides evidence of the association of the ACE gene polymorphisms and premature hypertension. In addition, BMI proved to be another important predictor of the disorder acting independently of the ACE gene

Partial Sperm beta1 Integrin Subunit Deletion Proves Its Involvement in Mouse Gamete Adhesion/Fusion

We have previously shown, using antibodies, that the sperm alpha6beta1 integrin is involved in mouse gamete fusion in vitro. Here we report the conditional knockdown of the sperm Itgb1 gene. It induced a drastic failure of sperm fusogenic ability with sperm accumulation in the perivitelline space of in vitro inseminated oocytes deleted or not for the Itgb1 gene. These data demonstrate that sperm, but not oocyte, beta1 integrin subunit is involved in gamete adhesion/fusion. Curiously, knockdown males were fertile in vivo probably because of the incomplete Cre-mediated deletion of the sperm Itgb1 floxed gene. Indeed, this was shown by Western blot analysis and confirmed by both the viability and litter size of pups obtained by mating partially sperm Itgb1 deleted males with females producing completely deleted Itgb1 oocytes. Because of the total peri-implantation lethality of Itgb1 deletion in mice, we assume that sperm that escaped the Itgb1 excision seemed to be preferentially used to fertilize in vivo. Here, we showed for the first time that the deletion, even partial, of the sperm Itgb1 gene makes the sperm unable to normally fertilize oocytes. However, to elucidate the question of the essentiality of its role during fertilization, further investigations using a mouse expressing a recombinase more effective in male germ cells are necessary