A. Vambergue

Maternal Diets Trigger Sex-Specific Divergent Trajectories of Gene Expression and Epigenetic Systems in Mouse Placenta

Males and females responses to gestational overnutrition set the stage for subsequent sex-specific differences in adult onset non communicable diseases. Placenta, as a widely recognized programming agent, contibutes to the underlying processes. According to our previous findings, a high-fat diet during gestation triggers sex-specific epigenetic alterations within CpG and throughout the genome, together with the deregulation of clusters of imprinted genes. We further investigated the impact of diet and sex on placental histology, transcriptomic and epigenetic signatures in mice. Both basal gene expression and response to maternal high-fat diet were sexually dimorphic in whole placentas. Numerous genes showed sexually dimorphic expression, but only 11 genes regardless of the diet. In line with the key role of genes belonging to the sex chromosomes, 3 of these genes were Y-specific and 3 were X-specific. Amongst all the genes that were differentially expressed under a high-fat diet, only 16 genes were consistently affected in both males and females. The differences were not only quantitative but remarkably qualitative. The biological functions and networks of genes dysregulated differed markedly between the sexes. Seven genes of the epigenetic machinery were dysregulated, due to effects of diet, sex or both, including the Y- and X-linked histone demethylase paralogues Kdm5c and Kdm5d, which could mark differently male and female epigenomes. The DNA methyltransferase cofactor Dnmt3l gene expression was affected, reminiscent of our previous observation of changes in global DNA methylation. Overall, this striking sexual dimorphism of programming trajectories impose a considerable revision of the current dietary interventions protocols.

Whole-Exome Sequencing and High Throughput Genotyping Identified KCNJ11 as the Thirteenth MODY Gene

Background Maturity-onset of the young (MODY) is a clinically heterogeneous form of diabetes characterized by an autosomal-dominant mode of inheritance, an onset before the age of 25 years, and a primary defect in the pancreatic beta-cell function. Approximately 30% of MODY families remain genetically unexplained (MODY-X). Here, we aimed to use whole-exome sequencing (WES) in a four-generation MODY-X family to identify a new susceptibility gene for MODY. Methodology WES (Agilent-SureSelect capture/Illumina-GAIIx sequencing) was performed in three affected and one non-affected relatives in the MODY-X family. We then performed a high-throughput multiplex genotyping (Illumina-GoldenGate assay) of the putative causal mutations in the whole family and in 406 controls. A linkage analysis was also carried out. Principal Findings By focusing on variants of interest (i.e. gains of stop codon, frameshift, non-synonymous and splice-site variants not reported in dbSNP130) present in the three affected relatives and not present in the control, we found 69 mutations. However, as WES was not uniform between samples, a total of 324 mutations had to be assessed in the whole family and in controls. Only one mutation (p.Glu227Lys in KCNJ11) co-segregated with diabetes in the family (with a LOD-score of 3.68). No KCNJ11 mutation was found in 25 other MODY-X unrelated subjects. Conclusions/Significance Beyond neonatal diabetes mellitus (NDM), KCNJ11 is also a MODY gene (‘MODY13’), confirming the wide spectrum of diabetes related phenotypes due to mutations in NDM genes (i.e. KCNJ11, ABCC8 and INS). Therefore, the molecular diagnosis of MODY should include KCNJ11 as affected carriers can be ideally treated with oral sulfonylureas.

Consequences of gestational and pregestational diabetes on placental function and birth weight

Maternal diabetes constitutes an unfavorable environment for embryonic and fetoplacental development. Despite current treatments, pregnant women with pregestational diabetes are at increased risk for congenital malformations, materno-fetal complications, placental abnormalities and intrauterine malprogramming. The complications during pregnancy concern the mother (gravidic hypertension and/or preeclampsia, cesarean section) and the fetus (macrosomia or intrauterine growth restriction, shoulder dystocia, hypoglycemia and respiratory distress). The fetoplacental impairment and intrauterine programming of diseases in the offsprings later life induced by gestational diabetes are similar to those induced by type 1 and type 2 diabetes mellitus. Despite the existence of several developmental and morphological differences in the placenta from rodents and women, there are similarities in the alterations induced by maternal diabetes in the placenta from diabetic patients and diabetic experimental models. From both human and rodent diabetic experimental models, it has been suggested that the placenta is a compromised target that largely suffers the impact of maternal diabetes. Depending on the maternal metabolic and proinflammatory derangements, macrosomia is explained by an excessive availability of nutrients and an increase in fetal insulin release, a phenotype related to the programming of glucose intolerance. The degree of fetal damage and placental dysfunction and the availability and utilisation of fetal substrates can lead to the induction of macrosomia or intrauterine growth restriction. In maternal diabetes, both the maternal environment and the genetic background are important in the complex and multifactorial processes that induce damage to the embryo, the placenta, the fetus and the offspring. Nevertheless, further research is needed to better understand the mechanisms that govern the early embryo development, the induction of congenital anomalies and fetal overgrowth in maternal diabetes.

Increasing incidence of abnormal glucose tolerance in women with prior abnormal glucose tolerance during pregnancy: DIAGEST 2 study.

Aims  Mild blood glucose abnormalities during pregnancy may be linked to later glucose tolerance abnormalities or diabetes mellitus. Our aim was to determine the prevalence of diabetes mellitus (DM), impaired glucose tolerance (IGT) or impaired fasting glucose (IFG) 6.75 years after delivery in women with differential blood glucose status during pregnancy.

Is mild gestational hyperglycaemia associated with maternal and neonatal complications? The Diagest Study

Aims To evaluate the maternal and neonatal complications rates of mild gestational hyperglycaemia (MGH) compared to a control group in France.

Pregnancy induced hypertension in women with gestational carbohydrate intolerance: the diagest study

OBJECTIVE To determine the relationship between pregnancy induced hypertension (PIH) and gestational glucose intolerance. METHODS A 50g, 1h glucose loading test was offered to all pregnant women between 24 and 28 weeks of gestation in 15 centres in northern France during 8 months in 1992. If the test was positive (> or =7.2 mmol/l), the woman underwent a 3h oral glucose tolerance test (OGTT) as soon as possible. Using the criteria of Carpenter and Coustan, gestational diabetes mellitus (GDM) was defined by two abnormal values (n=218) and gestational mild hyperglycemia (GMH) by one abnormal value (n=130). Each control group was defined by a 50g, 1h loading test result of <7.2 mmol/l (n=108).PIH included gestational hypertension (GH) and preeclampsia (PE). GH was defined as a diastolic pressure of more than 85 mmHg on at least two occasions arising during pregnancy. PE was defined as GH with proteinuria > or =500 mg/24h. RESULTS The rate of PIH in the three groups (GDM; GMH and control group, C) was, respectively 17.0, 10.8, and 4.6%. All the six PE occurred in the GDM group. Univariate analysis showed significantly higher rate of hypertension in women with a history of PE, increasing body mass index before pregnancy (BMI) and glucose intolerance. In multivariate analysis with adjustment for primiparity, independent risk factors for PIH were a history of PE, BMI>27 and GDM, contrary to GMH and maternal age. CONCLUSIONS PIH appears to be linked to the level of glucose intolerance during pregnancy, independently of other known factors of hypertension.

Placental BDNF/TrkB signaling system is modulated by fetal growth disturbances in rat and human.

The brain-derived neurotrophic factor (BDNF) has been shown to exert an important role during implantation, placental development, and fetal growth control in mice. Its expression is closely related to the nutritional status in several tissues such as in the nervous system. In a previous study, we demonstrated that maternal undernutrition (MU), during the perinatal life, modified both the BDNF and its functional receptor, the tyrosine kinase receptor B (TrkB) gene expression in the brain of growth-restricted rat offspring during sensitive developmental windows, suggesting that these early modifications may have long-lasting consequences. In the present study, we measured BDNF/TrkB mRNA and protein levels in rat placentas from mothers submitted to a 50% food restriction during gestation, and in human placentas from pregnancies with fetal growth restriction or fetal macrosomia. In the rat, two subtypes of placental TrkB receptors have been identified: the TrkB-FL and TrkB-T1 receptors. We found that MU induced intrauterine growth restriction (IUGR) of fetuses at term and decreased the placental BDNF mRNA and protein levels. Placentae from undernourished mothers exhibited an increased mRNA expression of TrkB-FL whereas both TrkB-FL and TrkB-T1 receptors proteins levels were not modified. In human IUGR placentas, both BDNF and TrkB receptor mRNA expressions were up-regulated. Finally, although neither BDNF nor TrkB mRNA levels were altered by fetal macrosomia alone, BDNF mRNA levels were decreased when macrosomia was associated with maternal type 1 diabetes. These results show that the placental BDNF/TrkB system is modulated in rats and humans during pregnancies with fetal growth perturbations and is affected by the maternal energetic status. These data suggest that this system may exert an important role for the feto-placental unit development and that it may also be implicated in the etiology of pathologies related to placental and fetal growth disturbances.

Higher carbohydrate intake is associated with decreased incidence of newborn macrosomia in women with gestational diabetes.

OBJECTIVE To study the influence of energy and macronutrient intake on infant birthweight in women with gestational diabetes mellitus undergoing intensive management. DESIGN This prospective study evaluated the impact of intensive management of gestational diabetes on maternal and fetal morbidity, and addressed the relationship between food intake and infant birthweight. SETTING Fifteen maternity hospitals in northern France. SUBJECTS Ninety-nine women with gestational diabetes or gestational mild hyperglycemia diagnosed between 24 and 34 weeks of gestation were surveyed. After 1 was excluded because of a premature birth and 18 were excluded as underreporters, 80 women were included in the final analysis. Diet intake was assessed by a dietary history at the first interview, and by two 3-day diet records at the 3rd and 7th week after diagnosis. RESULTS In a forward-stepwise regression analysis (controlling for maternal age; smoking; parity; prepregnancy BMI; pregnancy weight gain; gestational duration; infant sex; fasting and 2-hour postprandial serum glucose; insulin therapy; and energy, fat, protein and carbohydrate intake during treatment) infant birthweight was positively associated with gestational duration (beta = +0.34, P<.002), and negatively with smoking (beta = -0.27, P<.02) and carbohydrate intake (beta = -0.24, P<.03). There were no large-for-gestational-age infants among women whose carbohydrate intake exceeded 210 g/day. CONCLUSION For women with gestational diabetes undergoing intensive management, higher carbohydrate intake is associated with decreased incidence of macrosomia. APPLICATION These findings suggest that nutrition counseling in gestational diabetes must be directed to maintain a sufficient carbohydrate intake (at least 250 g per day), which implies a low-fat diet to limit energy intake. A careful distribution of carbohydrate throughout the day and the use of low-glycemic index foods may help limit postprandial hyperglycemia.

Time course analysis of RNA stability in human placenta

BackgroundEvaluation of RNA quality is essential for gene expression analysis, as the presence of degraded samples may influence the interpretation of expression levels. Particularly, qRT-PCR data can be affected by RNA integrity and stability. To explore systematically how RNA quality affects qRT-PCR assay performance, a set of human placenta RNA samples was generated by two protocols handlings of fresh tissue over a progressive time course of 4 days. Protocol A consists of a direct transfer of tissue into RNA-stabilizing solution (RNAlater™) solution. Protocol B uses a dissection of placenta villosities before bio banking. We tested and compared RNA yields, total RNA integrity, mRNA integrity and stability in these two protocols according to the duration of storage.ResultsA long time tissue storage had little effect on the total RNA and mRNA integrity but induced changes in the transcript levels of stress-responsive genes as TNF-alpha or COX2 after 48 h. The loss of the RNA integrity was higher in the placental tissues that underwent a dissection before RNA processing by comparison with those transferred directly into RNA later™ solution. That loss is moderate, with average RIN (RNA Integration Numbers) range values of 4.5–6.05, in comparison with values of 6.44–7.22 in samples directly transferred to RNAlater™ (protocol A). Among the house keeping genes tested, the B2M is the most stable.ConclusionThis study shows that placental samples can be stored at + 4°C up to 48 h before RNA extraction without altering RNA quality. Rapid tissue handling without dissection and using RNA-stabilizing solution (RNAlater™) is a prerequisite to obtain suitable RNA integrity and stability.

Factor VII, Tissue Factor Pathway Inhibitor, and Monocyte Tissue Factor in Diabetes Mellitus: Influence of Type of Diabetes, Obesity Index, and Age

Changes of the tissue factor (TF) pathway of blood coagulation have been described in diabetes and could be involved in its vascular complications. In order to evaluate the influence of the type of diabetes and of the obesity index and age on these changes, factor VII coagulant activity, factor VII antigen, activated factor VII, monocyte TF expression, and plasma Tissue Factor Pathway Inhibitor (TFPI) were examined in 18 Type 1 and 16 Type 2 diabetic patients compared to non-diabetic control subjects matched for age, sex, and obesity index (Types 1 and 2 controls, respectively). Multicomplicated patients were excluded. FVIIc, FVIIAg, and FVIIa were higher in Type 2 diabetic patients and controls than in Type 1 diabetic patients and controls (P< .03). However, FVIIc and FVIIAg were lower in diabetic patients than in their matched controls (P< .03). Monocyte expression of TF was not different between Types 1 and 2 diabetic patients and their matched controls except for LPS-stimulated monocyte TF activity which was lower in Type 2 diabetic patients than in Type 2 controls (P< .05). Plasma TFPI was slightly but significantly higher in Type 1 diabetic patients than in Type 1 controls (P= .01) and was correlated to glycemia. However, both in Type 2 diabetic patients and controls, TFPI was higher than in Type 1 controls and was correlated with BMI (P< .0003). These results indicate that in not multicomplicated patients, the increase of FVII and TFPI was highly dependent on obesity index and age rather than on diabetes by itself.