Metformin use in pregnancy: promises and uncertainties

Metformin has been prescribed in pregnancy for over 40 years; for much of this time, use has been limited both in numbers and geographically, and the evidence base has been confined to observational studies. In early years, perceived safety concerns and lack of availability of the drug in many countries acted as a barrier to use. More recently, RCTs have begun to examine the role of metformin in pregnancy in much-needed detail. However, this evidence base has been interpreted differently in different countries, leading to very wide variation in its current application in pregnancy. In this short review, we will discuss the history of metformin in pregnancy and highlight some of the key clinical trials. We will then consider some of the remaining controversies associated with metformin use in pregnancy, most important of these being the potential for long-term ‘programming’ effects on the fetus as a result of metformin being able to cross the placenta. We will also consider clinical situations where metformin might be avoided. Finally, we will discuss some future directions for this drug as it reaches its sixtieth anniversary

Identification of a new binding motif for the paired domain of Pax-3 and unusual characteristics of spacing of bipartite recognition elements on binding and transcription activation

Pax-3, a transcription factor that is required for development of the embryonic neural tube, neural crest, and somitic derivatives, contains two DNA-binding domains, a paired domain, and a paired-type homeodomain. Although Pax-3 binds to sequences related to the e5 element of the Drosophila even-skipped gene, the sequence requirements of an optimal Pax-3 response element have not been well characterized. Using both DNA-binding domains and a pool of random oligonucleotides, we identified a new paired box consensus motif, “GTTAT,” which was located 1, 4, 5, 8, or 13 base pairs downstream of the homeobox binding motif, “ATTA.” Binding analysis of these sequences demonstrated that the distance between recognition elements for the homeodomain and the paired domain affects affinity. Specifically, spacing elements 1 or 13 base pairs apart from each other conferred low affinity Pax-3 binding, whereas intermediate spacing (5 or 8 base pairs) conferred high affinity binding. Contrary to previous reports, oligonucleotides deleted for either the ATTA or the GTTAT could also be bound by Pax-3, although both sites were necessary for maximal affinity. Finally, transient transfections demonstrated that Pax-3 trans-activation correlated with binding affinity. Because the Pax-3-responsive genes identified to date contain almost exclusively low affinity binding sequences, our analysis indicates that they may be responsive to Pax-3 only when cellular levels are high

Pax3 Stimulates p53 Ubiquitination and Degradation Independent of Transcription

Background: Pax3 is a developmental transcription factor that is required for neural tube and neural crest development. We previously showed that inactivating the p53 tumor suppressor protein prevents neural tube and cardiac neural crest defects in Pax3-mutant mouse embryos. This demonstrates that Pax3 regulates these processes by blocking p53 function. Here we investigated the mechanism by which Pax3 blocks p53 function. Methodology/Principal Findings We employed murine embryonic stem cell (ESC)-derived neuronal precursors as a cell culture model of embryonic neuroepithelium or neural crest. Pax3 reduced p53 protein stability, but had no effect on p53 mRNA levels or the rate of p53 synthesis. Full length Pax3 as well as fragments that contained either the DNA-binding paired box or the homeodomain, expressed as GST or FLAG fusion proteins, physically associated with p53 and Mdm2 both in vitro and in vivo. In contrast, Splotch Pax3, which causes neural tube and neural crest defects in homozygous embryos, bound weakly, or not at all, to p53 or Mdm2. The paired domain and homeodomain each stimulated Mdm2-mediated ubiquitination of p53 and p53 degradation in the absence of the Pax3 transcription regulatory domains, whereas Splotch Pax3 did not stimulate p53 ubiquitination or degradation. Conclusions/Significance: Pax3 inactivates p53 function by stimulating its ubiquitination and degradation. This process utilizes the Pax3 paired domain and homeodomain but is independent of DNA-binding and transcription regulation. Because inactivating p53 is the only required Pax3 function during neural tube closure and cardiac neural crest development, and inactivating p53 does not require Pax3-dependent transcription regulation, this indicates that Pax3 is not required to function as a transcription factor during neural tube closure and cardiac neural crest development. These findings further suggest novel explanations for PAX3 functions in human diseases, such as in neural crest-derived cancers and Waardenburg syndrome types 1 and 3

A core outcome set for studies of gestational diabetes mellitus prevention and treatment

AIMS/HYPOTHESIS: The aim of this systematic review was to develop core outcome sets (COSs) for trials evaluating interventions for the prevention or treatment of gestational diabetes mellitus (GDM). METHODS: We identified previously reported outcomes through a systematic review of the literature. These outcomes were presented to key stakeholders (including patient representatives, researchers and clinicians) for prioritisation using a three-round, e-Delphi study. A priori consensus criteria informed which outcomes were brought forward for discussion at a face-to-face consensus meeting where the COS was finalised. RESULTS: Our review identified 74 GDM prevention and 116 GDM treatment outcomes, which were presented to stakeholders in round 1 of the e-Delphi study. Round 1 was completed by 173 stakeholders, 70% (121/173) of whom went on to complete round 2; 84% (102/121) of round 2 responders completed round 3. Twenty-two GDM prevention outcomes and 30 GDM treatment outcomes were discussed at the consensus meeting. Owing to significant overlap between included prevention and treatment outcomes, consensus meeting stakeholders agreed to develop a single prevention/treatment COS. Fourteen outcomes were included in the final COS. These consisted of six maternal outcomes (GDM diagnosis, adherence to the intervention, hypertensive disorders of pregnancy, requirement and type of pharmacological therapy for hyperglycaemia, gestational weight gain and mode of birth) and eight neonatal outcomes (birthweight, large for gestational age, small for gestational age, gestational age at birth, preterm birth, neonatal hypoglycaemia, neonatal death and stillbirth). CONCLUSIONS/INTERPRETATION: This COS will enable future GDM prevention and treatment trials to measure similar outcomes that matter to stakeholders and facilitate comparison and combination of these studies. TRIAL REGISTRATION: This study was registered prospectively with the Core Outcome Measures in Effectiveness Trials (COMET) database: http://www.comet-initiative.org/studies/details/686/

Hypoxic stress in diabetic pregnancy contributes to impaired embryo gene expression and defective development by inducing oxidative stress

We have shown that neural tube defects (NTD) in a mouse model of diabetic embryopathy are associated with deficient expression of Pax3, a gene required for neural tube closure. Hyperglycemia-induced oxidative stress is responsible. Before organogenesis, the avascular embryo is physiologically hypoxic (2-5% O(2)). Here we hypothesized that, because O(2) delivery is limited at this stage of development, excess glucose metabolism could accelerate the rate of O(2) consumption, thereby exacerbating the hypoxic state. Because hypoxia can increase mitochondrial superoxide production, excessive hypoxia may contribute to oxidative stress. To test this, we assayed O(2) flux, an indicator of O(2) availability, in embryos of glucose-injected hyperglycemic or saline-injected mice. O(2) flux was reduced by 30% in embryos of hyperglycemic mice. To test whether hypoxia replicates, and hyperoxia suppresses, the effects of maternal hyperglycemia, pregnant mice were housed in controlled O(2) chambers on embryonic day 7.5. Housing pregnant mice in 12% O(2), or induction of maternal hyperglycemia (>250 mg/dl), decreased Pax3 expression fivefold, and increased NTD eightfold. Conversely, housing pregnant diabetic mice in 30% O(2) significantly suppressed the effect of maternal diabetes to increase NTD. These effects of hypoxia appear to be the result of increased production of mitochondrial superoxide, as indicated by assay of lipid peroxidation, reduced glutathione, and H(2)O(2). Further support of this interpretation was the effect of antioxidants, which blocked the effects of maternal hypoxia, as well as hyperglycemia, on Pax3 expression and NTD. These observations suggest that maternal hyperglycemia depletes O(2) in the embryo and that this contributes to oxidative stress and the adverse effects of maternal hyperglycemia on embryo developmen

Diabetic Embryopathy Susceptibility in Mice Is Associated with Differential Dependence on Glucosamine and Modulation of High Glucose-Induced Oxidative Stress

The high KM glucose transporter, GLUT2 (SLC2A2), is expressed by embryos and causes high rates of glucose transport during maternal hyperglycemic episodes in diabetic pregnancies and causes congenital malformations (diabetic embryopathy). GLUT2 is also a low KM transporter of the amino sugar, glucosamine (GlcN), which enters the hexosamine biosynthetic pathway (HBP) and provides substrate for glycosylation reactions. Exogenous GlcN also increases activity of the pentose phosphate pathway (PPP), which increases production of NADPH reducing equivalents. GLUT2-transported GlcN is inhibited by high glucose concentrations. Not all mouse strains are susceptible to diabetic embryopathy. The aim of this study was to test the hypothesis that susceptibility to diabetic embryopathy is related to differential dependence on exogenous GlcN for glycosylation or stimulation of the PPP. We tested this using murine embryonic stem cell (ESC) lines that were derived from embryopathy-susceptible FVB/NJ (FVB), and embryopathy-resistant C57Bl/6J (B6), embryos in the presence of low or high glucose, and in the presence or absence of GlcN. There were no significant differences in Glut2 expression, or of glucose or GlcN transport, between FVB and B6 ESC. GlcN effects on growth and incorporation into glycoproteins indicated that FVB ESC are more dependent on exogenous GlcN than are B6 ESC. GlcN stimulated PPP activity in FVB but not in B6 ESC. High glucose induced oxidative stress in FVB ESC but not in B6 ESC. These results indicate that FVB embryos are more dependent on exogenous GlcN for glycosylation, but also for stimulation of the PPP and NADPH production, than are B6 embryos, thereby rendering FVB embryos more susceptible to high glucose to induce oxidative stress