The role of microRNAs in pregnancies complicated by maternal diabetes.

With the global prevalence of diabetes increasing, more people of reproductive age are experiencing hyperglycaemic pregnancies. Maternal Type 1 (T1DM) or Type 2 (T2DM) diabetes mellitus, and gestational diabetes mellitus (GDM) are associated with maternal cardiovascular and metabolic complications. Pregnancies complicated by maternal diabetes also increase the risk of short- and long-term health complications for the offspring, including altered fetal growth and the onset of T2DM and cardiometabolic diseases throughout life. Despite advanced methods for improving maternal glucose control, the prevalence of adverse maternal and offspring outcomes associated with maternal diabetes remains high. The placenta is a key organ at the maternal-fetal interface that regulates fetal growth and development. In pregnancies complicated by maternal diabetes, altered placental development and function has been linked to adverse outcomes in both mother and fetus. Emerging evidence suggests that microRNAs (miRNAs) are key molecules involved in mediating these changes. In this review, we describe the role of miRNAs in normal pregnancy and discuss how miRNA dysregulation in the placenta and maternal circulation is associated with suboptimal placental development and pregnancy outcomes in individuals with maternal diabetes. We also discuss evidence demonstrating that miRNA dysregulation may affect the long-term health of mothers and their offspring. As such, miRNAs are potential candidates as biomarkers and therapeutic targets in diabetic pregnancies at risk of adverse outcomes.

MicroRNA-322 overexpression reduces neural tube defects in diabetic pregnancies.

BACKGROUND: Hyperglycemia from pregestational diabetes mellitus induces neural tube defects in the developing fetus. Folate supplementation is the only effective way to prevent neural tube defects; however, some cases of neural tube defects are resistant to folate. Excess folate has been linked to higher maternal cancer risk and infant allergy. Therefore, additional interventions are needed. Understanding the mechanisms underlying maternal diabetes mellitus-induced neural tube defects can identify potential targets for preventing such defects. Despite not yet being in clinical use, growing evidence suggests that microRNAs are important intermediates in embryonic development and can serve as both biomarkers and drug targets for disease intervention. Our previous studies showed that maternal diabetes mellitus in vivo activates the inositol-requiring transmembrane kinase/endoribonuclease 1α (IRE1α) in the developing embryo and that a high glucose condition in vitro reduces microRNA-322 (miR-322) levels. IRE1α is an RNA endonuclease; however, it is unknown whether IRE1α targets and degrades miR-322 specifically or whether miR-322 degradation leads to neural tube defects via apoptosis. We hypothesize that IRE1α can inhibit miR-322 in maternal diabetes mellitus-induced neural tube defects and that restoring miR-322 expression in developing neuroepithelium ameliorates neural tube defects. OBJECTIVE: This study aimed to identify potential targets for preventing maternal diabetes mellitus-induced neural tube defects and to investigate the roles and relationship of a microRNA and an RNA endonuclease in mouse embryos exposed to maternal diabetes mellitus. STUDY DESIGN: To determine whether miR-322 reduction is necessary for neural tube defect formation in pregnancies complicated by diabetes mellitus, male mice carrying a transgene expressing miR-322 were mated with nondiabetic or diabetic wide-type female mice to generate embryos with or without miR-322 overexpression. At embryonic day 8.5 when the neural tube is not yet closed, embryos were harvested for the assessment of 3 miR-322 transcripts (primary, precursor, and mature miR-322), tumor necrosis factor receptor-associated factor 3 (TRAF3), and neuroepithelium cell survival. Neural tube defect incidences were determined in embryonic day 10.5 embryos when the neural tube should be closed if there is no neural tube defect formation. To identify which miR-322 transcript is affected by maternal diabetes mellitus and high glucose conditions, 3 miR-322 transcripts were assessed in embryos from dams with or without diabetes mellitus and in C17.2 mouse neural stem cells treated with different concentrations of glucose and at different time points. To determine whether the endonuclease IRE1α targets miR-322, small interfering RNA knockdown of IRE1α or overexpression of inositol-requiring transmembrane kinase/endoribonuclease 1α by DNA plasmid transfection was used to determine the effect of IRE1α deficiency or overexpression on miR-322 expression. RNA immunoprecipitation was performed to reveal the direct targets of inositol-requiring transmembrane kinase/endoribonuclease 1α. RESULTS: Maternal diabetes mellitus suppressed miR-322 expression in the developing neuroepithelium. Restoring miR-322 expression in the neuroepithelium blocked maternal diabetes mellitus-induced caspase-3 and caspase-8 cleavage and cell apoptosis, leading to a neural tube defect reduction. Reversal of maternal diabetes mellitus-inhibited miR-322 via transgenic overexpression prevented TRAF3 up-regulation in embryos exposed to maternal diabetes mellitus. Activated IRE1α acted as an endonuclease and degraded precursor miR-322, resulting in mature miR-322 reduction. CONCLUSION: This study supports the crucial role of the IRE1α-microRNA-TRAF3 circuit in the induction of neuroepithelial cell apoptosis and neural tube defect formation in pregnancies complicated by diabetes mellitus and identifies IRE1α and miR-322 as potential targets for preventing maternal diabetes mellitus-induced neural tube defects.

Role of EBAF/Nodal/p27 signaling pathway in development of placenta in normal and diabetic rats.

Placentas control the maternal-fetal transport of nutrients and gases. Placental reactions to adverse intrauterine conditions affect fetal development. Such adverse conditions occur in pregnancies complicated by diabetes, leading to alterations in placental anatomy and physiology. In this study, streptozocin (STZ) injection produced sustained hyperglycemia during pregnancy in rats. Hyperglycemic pregnant rats had gained significantly less weight than normal pregnant rats on embryonic day 15.5. We investigated the influence of diabetes on placental anatomy and physiology. Compared with controls, the diabetic group had a markedly thicker junctional zone at embryonic day 15.5. To explore a mechanism for this abnormality, we examined Nodal expression in the junctional zone of control and diabetic groups. We found lower expression of Nodal in the diabetic group. We then investigated the expression of its target gene p27Kip1 (p27), which is related to cell proliferation. In vitro, Nodal overexpression up-regulated p27 protein levels while interfered EBAF up-regulated p27. In vivo, the expression of p27 was lower in diabetic compared with normal rats, and localization was similar between the two groups. In contrast, a higher expression of PCNA was found in diabetic versus normal placenta. Endometrial bleeding associated factor (EBAF), an up-stream molecular regulator of Nodal, was expressed at higher levels in placenta from diabetic versus normal rats. Based on these results, we speculate that the EBAF/Nodal/p27 signaling pathway plays a role in morphological change of diabetic placenta.

Fetal growth regulation via insulin-like growth factor axis in normal and diabetic pregnancy.

OBJECTIVES: Diabetes mellitus (DM) in pregnancy and gestational diabetes remain a considerable cause of pregnancy complications, and fetal macrosomia is among them. Insulin, insulin-like growth factors (IGFs), and components of their signal-transduction axes belong to the predominant growth regulators and are implicated in glucose homeostasis. This study aimed to evaluate the available evidence on the association between the IGF axis and fetal anthropometric parameters in human diabetic pregnancy. METHODS: PubMed, Medline, Web of Science, and CNKI databases (1981-2021) were searched. RESULTS: Maternal and cord serum IGF-I levels are suggested to be positively associated with weight and length of neonates born to mothers with type 1 DM. The results concerning IGF-II and IGFBPs in type 1 DM or any of the IGF axis components in type 2 DM remain controversial. The alterations of maternal serum IGFs concentrations throughout diabetic and non-diabetic pregnancy do not appear to be the same. Maternal 1st trimester IGF-I level is positively associated with fetal birth weight in DM. CONCLUSIONS: Research on the IGF axis should take gestational age of sampling, presence of DM, and insulin administration into account. Maternal 1st trimester IGF-I level might become a predictor for macrosomia development in diabetic pregnancy.

Placental Transcriptome Profiling in Subtypes of Diabetic Pregnancies Is Strongly Confounded by Fetal Sex.

The placenta is a temporary organ with a unique structure and function to ensure healthy fetal development. Placental dysfunction is involved in pre-eclampsia (PE), fetal growth restriction, preterm birth, and gestational diabetes mellitus (GDM). A diabetic state affects maternal and fetal health and may lead to functional alterations of placental metabolism, inflammation, hypoxia, and weight, amplifying the fetal stress. The placental molecular adaptations to the diabetic environment and the adaptive spatio-temporal consequences to elevated glucose or insulin are largely unknown (2). We aimed to identify gene expression signatures related to the diabetic placental pathology of placentas from women with diabetes mellitus. Human placenta samples (n = 77) consisting of healthy controls, women with either gestational diabetes mellitus (GDM), type 1 or type 2 diabetes, and women with GDM, type 1 or type 2 diabetes and superimposed PE were collected. Interestingly, gene expression differences quantified by total RNA sequencing were mainly driven by fetal sex rather than clinical diagnosis. Association of the principal components with a full set of clinical patient data identified fetal sex as the single main explanatory variable. Accordingly, placentas complicated by type 1 and type 2 diabetes showed only few differentially expressed genes, while possible effects of GDM and diabetic pregnancy complicated by PE were not identifiable in this cohort. We conclude that fetal sex has a prominent effect on the placental transcriptome, dominating and confounding gene expression signatures resulting from diabetes mellitus in settings of well-controlled diabetic disease. Our results support the notion of placenta as a sexual dimorphic organ.

Impact of maternal hyperglycemia on cardiac development: Insights from animal models.

Congenital heart disease (CHD) is the leading cause of birth defect-related death in infants and is a global pediatric health concern. While the genetic causes of CHD have become increasingly recognized with advances in genome sequencing technologies, the etiology for the majority of cases of CHD is unknown. The maternal environment during embryogenesis has a profound impact on cardiac development, and numerous environmental factors are associated with an elevated risk of CHD. Maternal diabetes mellitus (matDM) is associated with up to a fivefold increased risk of having an infant with CHD. The rising prevalence of diabetes mellitus has led to a growing interest in the use of experimental diabetic models to elucidate mechanisms underlying this associated risk for CHD. The purpose of this review is to provide a comprehensive summary of rodent models that are being used to investigate alterations in cardiac developmental pathways when exposed to a maternal diabetic setting and to summarize the key findings from these models. The majority of studies in the field have utilized the chemically induced model of matDM, but recent advances have also been made using diet based and genetic models. Each model provides an opportunity to investigate unique aspects of matDM and is invaluable for a comprehensive understanding of the molecular and cellular mechanisms underlying matDM-associated CHD.

Is the erythropoietin-erythroferrone-hepcidin axis intact in human neonates?

In a two-part process, we assessed elements of the principal hormonal pathway regulating iron homeostasis in human neonates. Part 1: Quantifying erythropoietin (Epo), erythroferrone (ERFE), hepcidin, and relevant serum and erythrocytic iron-related metrics in umbilical cord blood from term (n = 13) and preterm (n = 10) neonates, and from neonates born to mothers with diabetes and obesity (n = 13); Part 2: Quantifying serum Epo, ERFE, and hepcidin before and following darbepoetin administration. Part 1: We measured Epo, ERFE and hepcidin in all cord blood samples. Epo and ERFE levels did not differ between the three groups. Preterm neonates had the lowest hepcidin levels, while neonates born to diabetic women with a very high BMI had the lowest ferritin and RET-He levels. Part 2: Following darbepoetin dosing, ERFE levels generally increased (p < 0.05) and hepcidin levels generally fell (p < 0.05). Our observations suggest that the Epo/ERFE/hepcidin axis is intact in the newborn period.

Interaction between Metformin, Folate and Vitamin B12 and the Potential Impact on Fetal Growth and Long-Term Metabolic Health in Diabetic Pregnancies.

Metformin is the first-line treatment for many people with type 2 diabetes mellitus (T2DM) and gestational diabetes mellitus (GDM) to maintain glycaemic control. Recent evidence suggests metformin can cross the placenta during pregnancy, thereby exposing the fetus to high concentrations of metformin and potentially restricting placental and fetal growth. Offspring exposed to metformin during gestation are at increased risk of being born small for gestational age (SGA) and show signs of 'catch up' growth and obesity during childhood which increases their risk of future cardiometabolic diseases. The mechanisms by which metformin impacts on the fetal growth and long-term health of the offspring remain to be established. Metformin is associated with maternal vitamin B12 deficiency and antifolate like activity. Vitamin B12 and folate balance is vital for one carbon metabolism, which is essential for DNA methylation and purine/pyrimidine synthesis of nucleic acids. Folate:vitamin B12 imbalance induced by metformin may lead to genomic instability and aberrant gene expression, thus promoting fetal programming. Mitochondrial aerobic respiration may also be affected, thereby inhibiting placental and fetal growth, and suppressing mammalian target of rapamycin (mTOR) activity for cellular nutrient transport. Vitamin supplementation, before or during metformin treatment in pregnancy, could be a promising strategy to improve maternal vitamin B12 and folate levels and reduce the incidence of SGA births and childhood obesity. Heterogeneous diagnostic and screening criteria for GDM and the transient nature of nutrient biomarkers have led to inconsistencies in clinical study designs to investigate the effects of metformin on folate:vitamin B12 balance and child development. As rates of diabetes in pregnancy continue to escalate, more women are likely to be prescribed metformin; thus, it is of paramount importance to improve our understanding of metformin's transgenerational effects to develop prophylactic strategies for the prevention of adverse fetal outcomes.

Cord blood metabolic markers are strong mediators of the effect of maternal adiposity on fetal growth in pregnancies across the glucose tolerance spectrum: the PANDORA study.

AIMS/HYPOTHESIS: We aimed to assess associations between cord blood metabolic markers and fetal overgrowth, and whether cord markers mediated the impact of maternal adiposity on neonatal anthropometric outcomes among children born to Indigenous and Non-Indigenous Australian women with normal glucose tolerance (NGT), gestational diabetes mellitus (GDM) and pregestational type 2 diabetes mellitus. METHODS: From the Pregnancy and Neonatal Outcomes in Remote Australia (PANDORA) study, an observational cohort of 1135 mother-baby pairs, venous cord blood was available for 645 singleton babies (49% Indigenous Australian) of women with NGT (n = 129), GDM (n = 419) and type 2 diabetes (n = 97). Cord glucose, triacylglycerol, HDL-cholesterol, C-reactive protein (CRP) and C-peptide were measured. Multivariable logistic and linear regression were used to assess the associations between cord blood metabolic markers and the outcomes of birthweight z score, sum of skinfold thickness (SSF), being large for gestational age (LGA) and percentage of body fat. Pathway analysis assessed whether cord markers mediated the associations between maternal and neonatal adiposity. RESULTS: Elevated cord C-peptide was significantly associated with increasing birthweight z score (ß 0.57 [95% CI 0.42, 0.71]), SSF (ß 0.83 [95% CI 0.41, 1.25]), percentage of body fat (ß 1.20 [95% CI 0.69, 1.71]) and risk for LGA [OR 3.14 [95% CI 2.11, 4.68]), after adjusting for age, ethnicity and diabetes type. Cord triacylglycerol was negatively associated with birthweight z score for Indigenous Australian women only. No associations between cord glucose, HDL-cholesterol and CRP >0.3 mg/l (2.9 nmol/l) with neonatal outcomes were observed. C-peptide mediated 18% (95% CI 13, 36) of the association of maternal BMI with LGA and 11% (95% CI 8, 17) of the association with per cent neonatal fat. CONCLUSIONS/INTERPRETATION: Cord blood C-peptide is an important mediator of the association between maternal and infant adiposity, across the spectrum of maternal glucose tolerance.

Embryonic fatty acid metabolism in diabetic pregnancy: the difference between embryoblasts and trophoblasts.

During the first days of development the preimplantation embryo is supplied with nutrients from the surrounding milieu. Maternal diabetes mellitus affects the uterine microenvironment, leading to a metabolic adaptation processes in the embryo. We analysed embryonic fatty acid (FA) profiles and expression of processing genes in rabbit blastocysts, separately in embryoblasts (EBs) and trophoblasts (TBs), to determine the potential consequences of maternal diabetes mellitus on intracellular FA metabolism. Insulin-dependent diabetes was induced by alloxan in female rabbits. On Day 6 post coitum, FA profiles in blastocysts (EB, TB and blastocoel fluid) and maternal blood were analysed by gas chromatography. The expression levels of molecules involved in FA elongation (fatty acid elongases, ELOVLs) and desaturation (fatty acid desaturases, FADSs) were measured in EB and TB. Maternal diabetes mellitus influenced the FA profile in maternal plasma and blastocysts. Independent from metabolic changes, rabbit blastocysts contained a higher level of saturated fatty acids (SFAs) and a lower level of polyunsaturated fatty acids (PUFAs) compared to the FA profile of the maternal plasma. Furthermore, the FA profile was altered in the EB and TB, differently. While SFAs (palmitic and stearic acid) were elevated in EB of diabetic rabbits, PUFAs, such as docosahexaenoic acid, were decreased. In contrast, in the TB, lower levels of SFAs and higher levels of oleic acid were observed. EB and TB specific alterations in gene expression were found for ELOVLs and FADSs, key enzymes for FA elongation and desaturation. In conclusion, maternal diabetes mellitus alters embryonic FA metabolism differently in EB and TB, indicating a lineage-specific metabolic adaptive response.

Need for less tight glucose control in early pregnancy after embryogenesis due to high risk of maternal hypoglycaemia in women with pre-existing diabetes can be compensated by good control in late pregnancy.

AIM: Poor glucose control is associated with adverse outcomes in pregnancies with pre-existing diabetes. However, strict glucose control increases the risk of severe hypoglycaemia, particularly in the first trimester. Therefore, we aimed to investigate whether less tight glucose control in the first trimester determines adverse outcomes or can be compensated for by good control in late pregnancy. METHODS: Retrospective data were collected from 517 singleton pregnancies complicated by pre-existing diabetes delivering between 2010 and 2017. Three hundred and thirty-six pregnancies fulfilled the inclusion criteria of having available HbA1c values either pre-conception or in the first trimester (65% type 1 diabetes, 35% type 2 diabetes). RESULTS: Higher HbA1c values in the first trimester were associated with increasing rates of large for gestational age (LGA) neonates, preterm delivery or neonatal intensive care unit admissions. Multiple regression analysis demonstrated third trimester HbA1c , type 1 diabetes, multiparity and excess weight gain, but not first trimester HbA1c , to be independently predictive for LGA. Pre-eclampsia and third trimester HbA1c increased the risk for preterm delivery. If HbA1c was ≤ 42 mmol/mol (6.0%) in the third trimester, rates of adverse outcomes were not significantly higher even if HbA1c targets of ≤ 48 mmol/mol (6.5%) had not been met in the first trimester. Good first trimester glucose control did not modify the rates of adverse outcomes if HbA1c was > 42 mmol/mol (6.0%) in the third trimester. CONCLUSIONS: Less tight glycaemic control, for example due to high frequency of severe hypoglycaemia in the first trimester, does not lead to increased adverse neonatal events if followed by tight control in the third trimester. Besides glycaemic control, excess weight gain is a modifiable predictor of adverse outcome.

Deficiency of the oxidative stress-responsive kinase p70S6K1 restores autophagy and ameliorates neural tube defects in diabetic embryopathy.

BACKGROUND: Autophagy is highly active in neuroepithelial cells of the developing neuroepithelium, and impairment of autophagy leads to neural tube defects. In this study, we have found that maternal diabetes suppresses autophagy that leads to neural tube defects and consequent cellular imbalance in the endoplasmic reticulum where critical events occur, leading to the induction of diabetic embryopathy. Because the mammalian target of rapamycin pathway suppresses autophagy, we hypothesized that 70 kDa ribosomal protein S6 kinase 1 (p70S6K1), a major downstream effector of mammalian target of rapamycin, mediates the inhibitory effect of maternal diabetes on autophagy in the developing neuroepithelium. OBJECTIVE: We investigated whether p70S6K1 mediates the inhibitory effect of maternal diabetes on autophagy during neurulation. We also examined whether p70S6K1 deficiency restores autophagy and therefore relieves endoplasmic reticulum stress and inhibits maternal diabetes-induced apoptosis, which leads to reduction in neural tube defect incidence in diabetic embryopathy. STUDY DESIGN: Female p70S6K1 heterogeneous knockout (p70S6K1+/-) mice were bred with male p70S6K1 heterogeneous knockout (p70S6K1+/-) mice to generate wild-type (WT), p70S6K1+/- and p70S6K1 knockout (p70S6K1-/-) embryos. Embryos at embryonic day 8.5 were harvested for the assessment of indices of autophagy, endoplasmic reticulum stress, and apoptosis. Neural tube defect incidence in embryos was determined at embryonic day 10.5. For in vitro studies, small interfering RNA knockdown of p70S6K1 in C17.2 mouse neural stem cells was used to determine the effect of p70S6K1 deficiency on autophagy impairment and endoplasmic reticulum stress under high glucose conditions. RESULTS: Knockout of the Rps6kb1 gene, which encodes for p70S6K1, ameliorated maternal diabetes-induced NTDs and restored autophagosome formation in neuroepithelial cells suppressed by maternal diabetes. Maternal diabetes-suppressed conversion of LC3-I (microtubule-associated protein 1A/1B-light chain 3) to LC3-II, an index of autophagic activity, in neurulation stage embryos was abrogated in the absence of p70S6K1. p70S6K1 knockdown in neural stem cells also restored autophagosome formation and the conversion of LC3-I to LC3-II. The activation of the major unfolded protein response, indicated by phosphorylation of inositol-requiring enzyme 1 alpha, and protein kinase R-like endoplasmic reticulum kinase, and eukaryotic translation initiation factor 2α, and the increase of the endoplasmic reticulum stress marker, C/EBP homologous protein, were induced by maternal diabetes in vivo and high glucose in vitro. Unfolded protein response and endoplasmic reticulum stress induced by maternal diabetes or high glucose were reduced by Rps6kb1 deletion or p70S6K1 knockdown, respectively. Rps6kb1 knockout blocked maternal diabetes-induced caspase cleavage and neuroepithelial cell apoptosis. The superoxide dismutase mimetic Tempol abolished high glucose-induced p70S6K1 activation. CONCLUSION: The study revealed the critical involvement of p70S6K1 in the pathogenesis of diabetic embryopathy.

Influences of Maternal Prepregnancy Obesity and Gestational Diabetes Mellitus on the Infant Gut Microbiome in Full-Term Infants.

This review examines the current evidence of how prepregnancy obesity (PPO) and gestational diabetes mellitus (GDM) influence the newborn gut microbiome. Scientific gaps in the literature are described to guide future research in this area. The prevalence of PPO and GDM increased to 64% in the United States over the past decade. Prepregnancy obesity and GDM influence newborn gut microbiome and contribute to adverse short- and long-term outcomes in full-term infants. This review aims to discuss current research findings related to the associations between PPO and GDM, separately, and together, on infant gut microbiome outcomes, provide an overview of short-term and long-term outcomes, describe clinical relevance, and identify avenues for future scientific inquiry. This review found that PPO and GDM influence infant gut microbiomes. Infants born to women with PPO and GDM were found to have lower levels of diversity in gut microbiota than infants born to normal prepregnancy weight women and those born to women without GDM.

Continuous glucose monitoring targets in type 1 diabetes pregnancy: every 5% time in range matters.

With randomised trial data confirming that continuous glucose monitoring (CGM) is associated with improvements in maternal glucose control and neonatal health outcomes, CGM is increasingly used in antenatal care. Across pregnancy, the ambition is to increase the CGM time in range (TIR), while reducing time above range (TAR), time below range (TBR) and glycaemic variability measures. Pregnant women with type 1 diabetes currently spend, on average, 50% (12 h), 55% (13 h) and 60% (14 h) in the target range of 3.5-7.8 mmol/l (63-140 mg/dl) during the first, second and third trimesters, respectively. Hyperglycaemia, as measured by TAR, reduces from 40% (10 h) to 33% (8 h) during the first to third trimester. A TIR of >70% (16 h, 48 min) and a TAR of <25% (6 h) is achieved only in the final weeks of pregnancy. CGM TBR data are particularly sensor dependent, but regardless of the threshold used for individual patients, spending ≥4% of time (1 h) below 3.5 mmol/l or ≥1% of time (15 min) below 3.0 mmol/l is not recommended. While maternal hyperglycaemia is a well-established risk factor for obstetric and neonatal complications, CGM-based risk factors are emerging. A 5% lower TIR and 5% higher TAR during the second and third trimesters is associated with increased risk of large for gestational age infants, neonatal hypoglycaemia and neonatal intensive care unit admissions. For optimal neonatal outcomes, women and clinicians should aim for a TIR of >70% (16 h, 48 min) and a TAR of <25% (6 h), from as early as possible during pregnancy.

Association between maternal diabetes, being large for gestational age and breast-feeding on being overweight or obese in childhood.

AIMS/HYPOTHESIS: This study aimed to examine the association of maternal diabetes, being large for gestational age (LGA) and breast-feeding with being overweight or obese in pre-school-aged children. METHODS: Data on height and weight at the time of their pre-school (age 4-6 years) immunisation visit between January 2009 and August 2017, as well as breast-feeding status in the first 5 months of life, for 81,226 children born between January 2005 and August 2013 were linked with maternal hospitalisation and outpatient records and birth registry data. Children were grouped into six categories based on maternal diabetes status during pregnancy (no diabetes, gestational diabetes or pre-existing diabetes) and birthweight (appropriate for gestational age [AGA] or LGA). WHO criteria were used to identify children who were overweight or obese. RESULTS: There were 69,506 children in the no diabetes/AGA group (control), 5926 in the no diabetes/LGA group, 4563 in the gestational diabetes/AGA group, 573 in the gestational diabetes/LGA group, 480 in the pre-existing diabetes/AGA group and 178 in the pre-existing diabetes/LGA group. The rate of being overweight/obese at pre-school age ranged from 20.5% in the control group to 42.9% in the gestational diabetes/LGA group. The adjusted attributable risk per cent for LGA alone (39.4%) was significantly higher than that for maternal gestational diabetes (16.0%) or pre-existing diabetes alone (15.1%); the risk for the combinations of gestational diabetes/LGA and pre-existing diabetes/LGA were 50.1% and 39.1%, respectively. Further stratification of the pre-existing diabetes groups found the prevalence of being overweight/obese was 21.2% in the type 1/AGA group, 31.4% in the type 1/LGA group (similar to those in the no diabetes groups), 26.7% in the type 2/AGA group and 42.5% in the type 2/LGA group. Breast-feeding was associated with a lower likelihood of being overweight/obese in childhood in all groups except gestational diabetes/LGA and pre-existing diabetes/LGA (both type 1 and type 2). CONCLUSION/INTERPRETATION: LGA is a stronger marker for risk of being overweight/obese in early childhood, compared with maternal diabetes during pregnancy. Rates of being overweight/obese in childhood were highest in LGA children born to mothers with gestational diabetes or pre-existing type 2 diabetes. Breast-feeding was associated with a lower risk of being overweight/obese in childhood in the majority of children; however, this association was not maintained in LGA children of mothers with diabetes.

Disturbed intracellular calcium homeostasis in neural tube defects in diabetic embryopathy.

Pregnancies complicated by preexisting maternal diabetes mellitus are associated with a higher risk of birth defects in infants, known as diabetic embryopathy. The common defects seen in the central nervous system result from failure of neural tube closure. The formation of neural tube defects (NTDs) is associated with excessive programmed cell death (apoptosis) in the neuroepithelium under hyperglycemia-induced intracellular stress conditions. The early cellular response to hyperglycemia remains to be identified. We hypothesize that hyperglycemia may disturb intracellular calcium (Ca2+) homeostasis, which perturbs organelle function and apoptotic regulation, resulting in increased apoptosis and embryonic NTDs. In an animal model of diabetic embryopathy, we performed Ca2+ imaging and observed significant increases in intracellular Ca2+ ([Ca2+]i) in the embryonic neural epithelium. Blocking T-type Ca2+ channels with mibefradil, but not L-type with verapamil, significantly blunted the increases in [Ca2+]i, implicating an involvement of channel type-dependent Ca2+ influx in hyperglycemia-perturbed Ca2+ homeostasis. Treatment of diabetic pregnant mice with mibefradil during neurulation significantly reduced NTD rates in the embryos. This effect was associated with decreases in apoptosis, alleviation of endoplasmic reticulum stress, and increases of anti-apoptotic factors. Taken together, our data suggest an important role of Ca2+ influx in hyperglycemia-induced NTDs and of T-type Ca2+ channels as a potential target to prevent birth defects in diabetic pregnancies.

Hyperglycemia induces inflammatory mediators in the human chorionic villous.

This study was based on the hypothesis that IL-1ß and its central regulator, the inflammasome, may play a role in the inflammatory condition exhibited by placental tissues from mothers with different gestational hyperglycemia levels. Pregnant women were classified according to the glycemic reference as non-diabetic (n = 15), mild gestational hyperglycemia (n = 15), gestational diabetes mellitus (n = 15) and type 2 diabetes mellitus (n = 15). We investigated levels of pro-inflammatory factors in maternal plasma and placental tissues (by ELISA or immunohistochemistry) and, NFKB activity (by electrophoretic mobility shift assay) and inflammasome protein expression (by Western blot) in chorionic villous. Maternal plasma and placental levels of inflammatory factors (IL-1ß, IL-6, and MCP-1) were increased during all hyperglycemic conditions. Villous stroma cells showed strong immunoreactivity to CD68. In addition, with syncytiotrophoblast, the villous stroma cells were also stained to detect iNOS, MCP-1, TLR2, and TLR4. Although the levels of protein had fluctuated in the groups, NLRP1, NLRP3, ASC, and Caspase 1 were up-regulated in all hyperglycemic groups suggesting the inflammasome may be assembled in these pregnant women. The NFKB activity also exhibited higher levels in hyperglycemic groups, which might imply in pro-inflammatory cytokines production. In summary, increased maternal glucose levels during pregnancy changed systemic and placental inflammatory patterns, which occurred in parallel with the expression of inflammasome factors and processing and secretion of the pro-inflammatory cytokine IL-1ß. These results suggest an inflammatory condition in all gestational hyperglycemic conditions, even in hyperglycemia that is less severe than gestational or overt diabetes, likely associated with inflammasome activation and inflammatory cytokine secretion. Inflammasome activation as a possible source of inflammatory factors may be an important target to be considered while managing hyperglycemia and preventing adverse pregnancy outcomes.

Oxidative stress-induced miR-27a targets the redox gene nuclear factor erythroid 2-related factor 2 in diabetic embryopathy.

BACKGROUND: Maternal diabetes induces neural tube defects, and oxidative stress is a causal factor for maternal diabetes-induced neural tube defects. The redox gene nuclear factor erythroid 2-related factor 2 is the master regulator of the cellular antioxidant system. OBJECTIVE: In this study, we aimed to determine whether maternal diabetes inhibits nuclear factor erythroid 2-related factor 2 expression and nuclear factor erythroid 2-related factor 2-controlled antioxidant genes through the redox-sensitive miR-27a. STUDY DESIGN: We used a well-established type 1 diabetic embryopathy mouse model induced by streptozotocin for our in vivo studies. Embryos at embryonic day 8.5 were harvested for analysis of nuclear factor erythroid 2-related factor 2, nuclear factor erythroid 2-related factor 2-controlled antioxidant genes, and miR-27a expression. To determine if mitigating oxidative stress inhibits the increase of miR-27a and the decrease of nuclear factor erythroid 2-related factor 2 expression, we induced diabetic embryopathy in superoxide dismutase 2 (mitochondrial-associated antioxidant gene)-overexpressing mice. This model exhibits reduced mitochondria reactive oxygen species even in the presence of hyperglycemia. To investigate the causal relationship between miR-27a and nuclear factor erythroid 2-related factor 2 in vitro, we examined C17.2 neural stem cells under normal and high-glucose conditions. RESULTS: We observed that the messenger RNA and protein levels of nuclear factor erythroid 2-related factor 2 were significantly decreased in embryos on embryonic day 8.5 from diabetic dams compared to those from nondiabetic dams. High-glucose also significantly decreased nuclear factor erythroid 2-related factor 2 expression in a dose- and time-dependent manner in cultured neural stem cells. Our data revealed that miR-27a was up-regulated in embryos on embryonic day 8.5 exposed to diabetes, and that high glucose increased miR-27a levels in a dose- and time-dependent manner in cultured neural stem cells. In addition, we found that a miR-27a inhibitor abrogated the inhibitory effect of high glucose on nuclear factor erythroid 2-related factor 2 expression, and a miR-27a mimic suppressed nuclear factor erythroid 2-related factor 2 expression in cultured neural stem cells. Furthermore, our data indicated that the nuclear factor erythroid 2-related factor 2-controlled antioxidant enzymes glutamate-cysteine ligase catalytic subunit, glutamate-cysteine ligase modifier subunit, and glutathione S-transferase A1 were down-regulated by maternal diabetes in embryos on embryonic day 8.5 and high glucose in cultured neural stem cells. Inhibiting miR-27a restored expression of glutamate-cysteine ligase catalytic subunit, glutamate-cysteine ligase modifier subunit, and glutathione S-transferase A1. Overexpressing superoxide dismutase 2 reversed the maternal diabetes-induced increase of miR-27a and suppression of nuclear factor erythroid 2-related factor 2 and nuclear factor erythroid 2-related factor 2-controlled antioxidant enzymes. CONCLUSION: Our study demonstrates that maternal diabetes-induced oxidative stress increases miR-27a, which, in turn, suppresses nuclear factor erythroid 2-related factor 2 and its responsive antioxidant enzymes, resulting in diabetic embryopathy.

Software-guided insulin dosing improves intrapartum glycemic management in women with diabetes mellitus.

BACKGROUND: During labor, maintenance of maternal euglycemia is critical to decrease the risk of neonatal hypoglycemia and associated morbidities. When continuous intravenous insulin infusion is needed, standardized insulin dosing charts have been used for titration of insulin to maintain glucose in target range. The GlucoStabilizer software program (Indiana University Health Inc, Indianapolis, IN) is a software-guided insulin dosing system that calculates the dose of intravenous insulin that is needed based on metabolic parameters, target glucose concentration, and an individual's response to insulin. Although this tool has been validated and shown to reduce both hypoglycemia and errors in critical care settings, the utility of this software has not been examined in obstetrics. OBJECTIVE: The purpose of this study was to determine whether the use of intravenous insulin dosing software in women with pregestational or gestational diabetes mellitus that requires intrapartum insulin infusion can improve the rate of glucose concentration in target range (70-100 mg/dL; 3.9-5.5 mmol/L) at the time delivery. STUDY DESIGN: We performed a retrospective cohort study comparing laboring patients with diabetes mellitus that required insulin infusion who were dosed by standard insulin dosing chart vs the GlucoStabilizer software program from January 2012 to December 2017. The GlucoStabilizer software program, which was implemented in May 2016, replaced the standard intravenous insulin dosing chart. Inclusion criteria were women with pregestational or gestational diabetes mellitus who were treated with an intravenous insulin infusion intrapartum for at least 2 hours. Maternal characteristics, glucose values in labor, and neonatal outcomes were extracted from delivery and neonatal records. The primary outcome was the percentage of women who achieved the target glucose range (defined as a blood glucose between 70-100 mg/dL; 3.9-5.5 mmol/L) before delivery. Parametric and nonparametric statistics were used to compare both groups; a probability value of <.05 was considered statistically significant. RESULTS: We identified 22 patients who were dosed by a standard insulin dosing chart and 11 patients who were dosed by the GlucoStabilizer software program during intrapartum management. The GlucoStabilizer software program was superior in achieving glucose values in target range at delivery (81.8% vs 9.1%; P<.001) compared with standard insulin dosing without increasing maternal hypoglycemia (0% vs 4.3%; P=.99). Patients whose insulin dosing was managed by the GlucoStabilizer software program also had lower mean capillary blood glucose values compared with the standard insulin infusion (102.9±5.9 mg/dL [5.7±0.33 mmol/L] vs 121.7±5.9 mg/dL [6.8±0.33 mmol/L]; P=.02). Before the initiation of the infusion, both groups demonstrated mean capillary blood glucose values outside of target range (122.6±8.8 mg/dL [6.7±0.49 mmol/L] for the GlucoStabilizer software program vs 131.9±10.1 mg/dL [7.3±0.56 mmol/L] for standard insulin treatment group; P=not significant). There were no significant differences in baseline maternal characteristics between the groups or neonatal outcomes. CONCLUSION: This study is the first to demonstrate that the use of software-guided intravenous insulin dosing in obstetrics can improve intrapartum glycemic management without increasing hypoglycemia in women with both pregestational and gestational diabetes mellitus that is treated with an insulin infusion.

Modulation of nuclear factor-κB signaling and reduction of neural tube defects by quercetin-3-glucoside in embryos of diabetic mice.

BACKGROUND: Diabetes mellitus in early pregnancy increases the risk of birth defects in infants. Maternal hyperglycemia stimulates the expression of nitric oxide synthase 2, which can be regulated by transcription factors of the nuclear factor-κB family. Increases in reactive nitrogen species generate intracellular stress conditions, including nitrosative, oxidative, and endoplasmic reticulum stresses, and trigger programmed cell death (or apoptosis) in the neural folds, resulting in neural tube defects in the embryo. Inhibiting nitric oxide synthase 2 can reduce neural tube defects; however, the underlying mechanisms require further delineation. Targeting nitric oxide synthase 2 and associated nitrosative stress using naturally occurring phytochemicals is a potential approach to preventing birth defects in diabetic pregnancies. OBJECTIVE: This study aims to investigate the effect of quercetin-3-glucoside, a naturally occurring polyphenol flavonoid, in reducing maternal diabetes-induced neural tube defects in an animal model, and to delineate the molecular mechanisms underlying quercetin-3-glucoside action in regulating nitric oxide synthase 2 expression. STUDY DESIGN: Female mice (C57BL/6) were induced to develop diabetes using streptozotocin before pregnancy. Diabetic pregnant mice were administered quercetin-3-glucoside (100 mg/kg) daily via gavage feeding, introduction of drug to the stomach directly via a feeding needle, during neurulation from embryonic day 6.5-9.5. After treatment at embryonic day 10.5, embryos were collected and examined for the presence of neural tube defects and apoptosis in the neural tube. Expression of nitric oxide synthase 2 and superoxide dismutase 1 (an antioxidative enzyme) was quantified using Western blot assay. Nitrosative, oxidative, and endoplasmic reticulum stress conditions were assessed using specific biomarkers. Expression and posttranslational modification of factors in the nuclear factor-κB system were investigated. RESULTS: Treatment with quercetin-3-glucoside (suspended in water) significantly decreased neural tube defect rate and apoptosis in the embryos of diabetic mice, compared with those in the water-treated diabetic group (3.1% vs. 24.7%; P < .001). Quercetin-3-glucoside decreased the expression of nitric oxide synthase 2 and nitrosative stress (P < .05). It also increased the levels of superoxide dismutase 1 (P < .05), further increasing the antioxidative capacity of the cells. Quercetin-3-glucoside treatment also alleviated of endoplasmic reticulum stress in the embryos of diabetic mice (P < .05). Quercetin-3-glucoside reduced the levels of p65 (P < .05), a member of the nuclear factor-κB transcription factor family, but augmented the levels of the inhibitor of κBα (P < .05), which suppresses p65 nuclear translocation. In association with these changes, the levels of inhibitor of κB kinase-α and inhibitor of κBα phosphorylation were elevated (P < .05). CONCLUSION: Quercetin-3-glucoside reduces the neural tube defects rate in the embryos of diabetic dams. Quercetin-3-glucoside suppresses nitric oxide synthase 2 and increases superoxide dismutase 1 expression, leading to alleviation of nitrosative, oxidative, and endoplasmic reticulum stress conditions. Quercetin-3-glucoside may regulate the expression of nitric oxide synthase 2 via modulating the nuclear factor-κB transcription regulation system. Quercetin-3-glucoside, a naturally occurring polyphenol that has high bioavailability and low toxicity, is a promising candidate agent to prevent birth defects in diabetic pregnancies.