A core outcome set for the treatment of pregnant women with pregestational diabetes: an international consensus study.

OBJECTIVE: To develop a core outcome set (COS) for randomised controlled trials (RCTs) evaluating the effectiveness of interventions for the treatment of pregnant women with pregestational diabetes mellitus (PGDM). DESIGN: A consensus developmental study. SETTING: International. POPULATION: Two hundred and five stakeholders completed the first round. METHODS: The study consisted of three components. (1) A systematic review of the literature to produce a list of outcomes reported in RCTs assessing the effectiveness of interventions for the treatment of pregnant women with PGDM. (2) A three-round, online eDelphi survey to prioritise these outcomes by international stakeholders (including healthcare professionals, researchers and women with PGDM). (3) A consensus meeting where stakeholders from each group decided on the final COS. MAIN OUTCOME MEASURES: All outcomes were extracted from the literature. RESULTS: We extracted 131 unique outcomes from 67 records meeting the full inclusion criteria. Of the 205 stakeholders who completed the first round, 174/205 (85%) and 165/174 (95%) completed rounds 2 and 3, respectively. Participants at the subsequent consensus meeting chose 19 outcomes for inclusion into the COS: trimester-specific haemoglobin A1c, maternal weight gain during pregnancy, severe maternal hypoglycaemia, diabetic ketoacidosis, miscarriage, pregnancy-induced hypertension, pre-eclampsia, maternal death, birthweight, large for gestational age, small for gestational age, gestational age at birth, preterm birth, mode of birth, shoulder dystocia, neonatal hypoglycaemia, congenital malformations, stillbirth and neonatal death. CONCLUSIONS: This COS will enable better comparison between RCTs to produce robust evidence synthesis, improve trial reporting and optimise research efficiency in studies assessing treatment of pregnant women with PGDM. TWEETABLE ABSTRACT: 165 key stakeholders have developed #Treatment #CoreOutcomes in pregnant women with #diabetes existing before pregnancy.

AMP-activated protein kinase mediates effects of oxidative stress on embryo gene expression in a mouse model of diabetic embryopathy.

AIMS/HYPOTHESIS: Neural tube defects (NTDs) are a common malformation associated with diabetic embryopathy. Maternal hyperglycaemia-induced oxidative stress inhibits the expression of Pax3, a gene that is essential for neural tube closure, and increases the incidence of NTDs. Because oxidative stress can stimulate AMP-activated kinase (AMPK) activity, and AMPK can regulate gene transcription, we hypothesised that increased AMPK activity would mediate the adverse effects of maternal hyperglycaemia-induced oxidative stress on Pax3 expression and NTDs. METHODS: Pregnant mice were made transiently hyperglycaemic by glucose injection, or hypoxic by housing in a hypoxic chamber, or were treated with antimycin A to induce oxidative stress, and AMPK activity in the embryos was assayed. The effects of stimulating AMPK activity with 5-aminoimidazole-4-carboxamide-1-beta-4-ribofuranoside (AICAR) on Pax3 expression and NTDs were determined. Vitamin E or glutathione ethyl ester was used to reduce oxidative stress, and compound C was used to inhibit AMPK activation. Murine embryonic stem cells were employed as an in vitro model to study the effects of oxidative stress on AMPK activity and the effects of AMPK stimulation on Pax3 expression. RESULTS: Maternal hyperglycaemia stimulated AMPK activity, and stimulation of AMPK with AICAR inhibited Pax3 expression (in vivo and in vitro) and increased NTDs (in vivo). Stimulation of AMPK by hyperglycaemia, hypoxia or antimycin A was inhibited by antioxidants. The AMPK inhibitor compound C blocked the effects of hyperglycaemia or AA on Pax3 expression and NTDs. CONCLUSIONS/INTERPRETATION: Stimulation of AMPK in embryos during a diabetic pregnancy mediates the effects of hyperglycaemia-induced oxidative stress to disturb the expression of the critical Pax3 gene, thereby causing NTDs.

Stimulation of the adenovirus E2 promoter by simian virus 40 T antigen or E1A occurs by different mechanisms.

We have examined the ability of simian virus 40 T antigen to stimulate transcription from the adenovirus E2 promoter. T antigen, produced from a cotransfected plasmid, stimulated chloramphenicol acetyltransferase enzyme and mRNA production from an E2 promoter-chloramphenicol acetyltransferase fusion plasmid (pEC113) in monkey kidney CV-1 cells. The level of stimulation of E2 transcription by simian virus 40 T antigen was equal to that observed in cotransfections of pEC113 and the adenovirus E1A gene product. Deletion mutations from the 5' end of the E2 promoter were examined for their ability to express basal, T-antigen, or E1A trans-activated promoter activity. In each case, deletion of upstream promoter sequences to -70 base pairs reduced chloramphenicol acetyltransferase expression to approximately 30% of the level observed with the intact E2 promoter. Deletion to -59 base pairs resulted in chloramphenicol acetyltransferase expression that was 3 to 5% of that observed with the intact E2 promoter. At saturating levels of the stimulatory proteins, the chloramphenicol acetyltransferase levels obtained in response to T antigen and adenovirus E1A were additive. COS-1 cells, which are derived from CV-1 cells and constitutively express simian virus 40 T antigen, do not support E2 promoter trans activation by T antigen. E1A trans activation of the E2 promoter is efficient in COS-1 cells. These results suggest that although promoter sequence requirements are similar, T antigen and E1A trans activate the E2 promoter by different mechanisms.

Neural tube defects in embryos of diabetic mice: role of the Pax-3 gene and apoptosis.

Neural tube defects are among the most common of the malformations associated with diabetic embryopathy. To study the molecular mechanisms by which neural tube defects occur during diabetic pregnancy, we have developed a new experimental system using pregnant diabetic mice. In this system, the rate of neural tube defects is about three times higher in embryos of diabetic mice than in embryos of nondiabetic mice. Most of the defects affected presumptive midbrain and hindbrain structures and included open defects (i.e., exencephaly) and gross maldevelopment. By semiquantitative reverse transcription-polymerase chain reaction and in situ hybridization, we found that expression of Pax-3, a gene required for neural tube closure in the area of the midbrain and hindbrain, is significantly reduced in the embryos of diabetic mice. The same regions of the neural tube where Pax-3 had been underexpressed were found subsequently to contain high concentrations of cells undergoing apoptosis. Reduced expression of Pax-3 appears to be responsible for this apoptosis because apoptotic cells were also found at sites of neural tube defects in embryos carrying null mutation of the Pax-3 gene. Finally, mouse strains that carry null mutations in Pax-3 develop neural tube defects that resemble the malformations that occur in embryos of diabetic mice. These results suggest that Pax-3 is an important developmental control gene, expression of which is disturbed in embryos of diabetic mice, and that as a consequence, apoptosis of the neural tube occurs. This pathway may be responsible for many of the neural tube defects resulting from diabetic pregnancy.

Evidence that elevated glucose causes altered gene expression, apoptosis, and neural tube defects in a mouse model of diabetic pregnancy.

Congenital malformations, including neural tube defects (NTDs), are significantly increased in the offspring of diabetic mothers. We previously reported that in the embryos of a mouse model of diabetic pregnancy, NTDs are associated with reduced expression of the gene Pax-3, which encodes a transcription factor that regulates neural tube development, and that reduced expression of Pax-3 leads to neuroepithelial apoptosis. In this study, we used three approaches to test whether glucose alone could be responsible for these adverse effects of diabetes on embryonic development. First, primary culture of embryo tissue in medium containing 15 mmol/l glucose inhibited Pax-3 expression compared with culture in medium containing 5 mmol/l glucose. Second, inducing hyperglycemia in pregnant mice by subcutaneous glucose administration significantly inhibited Pax-3 expression (P < 0.05), as demonstrated by quantitative reverse transcription-polymerase chain reaction assay of Pax-3 mRNA, and also increased neural tube apoptosis (P < 0.05). NTDs were significantly increased in glucose-injected pregnancies when blood glucose levels were >250 mg/dl (P < 0.002) but not in moderately hyperglycemic pregnancies (150-250 mg/dl, P = 0.37). Third, phlorizin administration to pregnant diabetic mice reduced blood glucose levels and the rate of NTDs. As seen with glucose-injected pregnancies, the rate of NTDs in phlorizin-treated diabetic pregnancies was related to the severity of hyperglycemia, since NTDs were significantly increased in severely hyperglycemic (>250 mg/dl) diabetic pregnancies (P < 0.001) but not in moderately hyperglycemic pregnancies (150-250 mg/dl, P = 0.35). These two findings, that elevated glucose alone can cause the changes in Pax-3 expression observed during diabetic pregnancy and that the NTD rate rises with significant increases in blood glucose levels, suggest that congenital malformations associated with diabetic pregnancy are caused by disruption of regulatory gene expression in the embryo in response to elevated glucose.

Heterogeneity of expression and secretion of native and mutant [AspB10]insulin in AtT20 cells.

AtT20 (pituitary corticotroph) cells were transfected with either the native or a mutant [AspB10]rat insulin II gene, using a plasmid containing the insulin gene and a neomycin resistance gene under the control of independent constitutive promoters. The cellular immunoreactive insulin (IRI) content ranged from 0.8-440 ng/10(6) cells, with the highest value similar to that found for a rat insulinoma cell line (RIN) and corresponding to approximately 1% that of native pancreatic B-cells. There was a direct correlation between insulin mRNA levels and IRI content and no correlation between mRNA levels and rat insulin II gene copy number. Furthermore, in some lines the insulin II transgene was lost even though the gene encoding neomycin resistance was retained. IRI release was stimulated up to 4-fold by isobutylmethylxanthine in all lines transfected with the native rat insulin II gene, and HPLC analysis showed most IRI as fully processed insulin, with less than 5% as proinsulin. These cells, thus, directed most proinsulin to secretory granules for conversion and regulated release regardless of the absolute amount of IRI expressed. One of the lines transfected with the AspB10 mutant gene (line AA9) released nearly 50% of IRI as proinsulin under basal conditions, with stimulation of insulin, but not proinsulin, release by isobutylmethylxanthine. This confirmed our previous finding of partial diversion of this mutant proinsulin from the regulated to the constitutive pathway. A second line (IC6) expressing the same mutant gene at much higher levels appeared to direct all mutant proinsulin to the regulated pathway, suggesting that for this particular mutant proinsulin, the secretory pathway employed by the transfected cells can be affected by the amount of proinsulin synthesized.

Stimulatory effect of luteinizing hormone upon relaxin secretion by cultured porcine preovulatory granulosa cells.

Relaxin, a peptide hormone capable of causing connective tissue alterations, is produced by the corpus luteum and traditionally considered a hormone of pregnancy. We have cultured granulosa cells from preovulatory porcine follicles and have found that these non-pregnancy associated cells secrete relaxin, and that luteinizing hormone, which stimulates ovulation, enhances relaxin secretion by cells from large preovulatory follicles. These results suggest that relaxin secreted prior to ovulation may have a local ovarian effect, perhaps facilitating ovulation.

Effects of mutation of the CREB binding site of the somatostatin promoter on cyclic AMP responsiveness in CV-1 cells.

The transcription factors CREB (cAMP response element binding protein) and ATF (activating transcription factor) recognize DNA containing the consensus sequence TGACGTCA. We compared the neuropeptide somatostatin promoter, which binds CREB and is activated by cAMP, to the adenovirus E2A promoter, which binds ATF but is not activated by cAMP, to determine which specific nucleotides within a CREB/ATF recognition sequence confer cAMP responsiveness. Several mutant somatostatin promoters were generated containing part of all of the E2A ATF binding site. Some of the hybrid CREB/ATF binding sites competed for factor binding to a wild-type somatostatin promoter probe. However, only the wild-type CREB binding site promoter could confer cAMP activation on a linked CAT plasmid. Furthermore, this wild-type CREB binding site could confer cAMP activation on the CAT plasmid only if it was adjacent to a wild-type somatostatin TATA box and cap site. These results suggest that slight deviation from a wild-type CREB recognition sequence might be tolerated by factor(s) binding to cAMP response element-like sequences. However, transcription activation may require a particular CREB recognition sequence, as well as additional promoter elements that bind proteins that interact with CREB.

Free radicals and birth defects.

Maternal diabetes significantly increases the risk for birth defects. Studies using animal models indicate that oxidative stress may play a causative role. Oxidative stress can result from exposure to certain drugs, ionizing radiation and folic acid deficiency. Therefore, study of the mechanisms by which maternal diabetes affects embryogenesis may provide insight into general processes by which birth defects occur. Study of embryonic gene expression has demonstrated that maternal diabetes causes birth defects by disturbing expression of genes that control essential developmental processes, and that oxidative stress is involved. A model in which oxidative stress-induced deficient gene expression leads to congenital defects involving p53-dependent apoptosis is discussed.

Expression of the gene encoding the high-Km glucose transporter 2 by the early postimplantation mouse embryo is essential for neural tube defects associated with diabetic embryopathy.

AIMS/HYPOTHESIS: Excess glucose transport to embryos during diabetic pregnancy causes congenital malformations. The early postimplantation embryo expresses the gene encoding the high-Km GLUT2 (also known as SLC2A2) glucose transporter. The hypothesis tested here is that high-Km glucose transport by GLUT2 causes malformations resulting from maternal hyperglycaemia during diabetic pregnancy. MATERIALS AND METHODS: Glut2 mRNA was assayed by RT-PCR. The Km of embryo glucose transport was determined by measuring 0.5-20 mmol/l 2-deoxy[3H]glucose transport. To test whether the GLUT2 transporter is required for neural tube defects resulting from maternal hyperglycaemia, Glut2+/- mice were crossed and transient hyperglycaemia was induced by glucose injection on day 7.5 of pregnancy. Embryos were recovered on day 10.5, and the incidence of neural tube defects in wild-type, Glut2+/- and Glut2-/- embryos was scored. RESULTS: Early postimplantation embryos expressed Glut2, and expression was unaffected by maternal diabetes. Moreover, glucose transport by these embryos showed Michaelis-Menten kinetics of 16.19 mmol/l, consistent with transport mediated by GLUT2. In pregnancies made hyperglycaemic on day 7.5, neural tube defects were significantly increased in wild-type embryos, but Glut2+/- embryos were partially protected from neural tube defects, and Glut2-/- embryos were completely protected from these defects. The frequency of occurrence of wild-type, Glut2+/- and Glut2-/- embryos suggests that the presence of Glut2 alleles confers a survival advantage in embryos before day 10.5. CONCLUSIONS/INTERPRETATIONS: High-Km glucose transport by the GLUT2 glucose transporter during organogenesis is responsible for the embryopathic effects of maternal diabetes.

Simian virus 40 small t antigen trans activates the adenovirus E2A promoter by using mechanisms distinct from those used by adenovirus E1A.

As reported previously for simian virus 40 small t antigen, polyomavirus small t antigen stimulates transcription directed by the adenovirus E2A and VA-I promoters during transient transfection assays. To determine whether papovaviral small t antigens might employ biochemical mechanisms during transcription activation that are either similar to or distinct from other viral trans activators, I compared the abilities of simian virus 40 small t antigen and adenovirus E1A to regulate the E2A promoter during transient transfection assays. I determined that, whereas activation of the E2A promoter by E1A involves the transcription factors ATF and EIIF, activation by small t antigen involves only EIIF. The effects of cotransfecting maximal concentrations of plasmids encoding small t antigen with E1A suggested that they activate the E2A promoter by different mechanisms. To determine whether small t antigen employs a mechanism different from that encoded in E1A domain II, domain III, or both, I compared the effects of transfecting plasmids expressing small t antigen, the 12S product of E1A, or the 13S product with a mutation in domain II on trans activation of the E2A promoter in two cellular backgrounds. On the basis of these comparisons, it appears that small t antigen does not activate transcription by a mechanism similar to either of the activities encoded in E1A. This suggests that papovavirus small t antigens belong to a distinct class of trans-acting proteins.

Multiple, distinct trans-activation functions are encoded by the simian virus 40 large T and small t antigens, only some of which require the 82-residue amino-terminal common domain.

Simian virus 40 (SV40) small t and large T antigens can each trans activate the adenovirus (Ad) E2A and the Ad VA-I promoters. The first 82 amino acids of large T and small t are identical. However, this large T-small t common domain between residues 1 and 82 does not trans activate, suggesting that large T and small t each encode separate trans-activation functions. To determine whether the large T or small t unique domains, which are required for trans activation of the E2A promoter, are sufficient for this activity, we have employed expression plasmids separately encoding the common and unique domains of large T and small t. Cotransfection of a large T unique domain expression plasmid efficiently trans activated the E2A promoter. Optimal trans activation by large T required the motif that binds cellular proteins such as the retinoblastoma gene product, which is located in the large T unique domain, and additional large T structures outside this motif. In contrast, the small t unique domain did not trans activate the E2A promoter. Experiments utilizing E2A promoter mutants containing only the ATF- or EIIF-binding sites demonstrated that trans activation by small t involves only the EIIF transcription factor and that this function requires both the common (residues 1 to 82) and the small t unique domains expressed as a colinear protein. trans activation by large T, in contrast, involves at least three mechanisms. There appear to be at least two mechanisms that involve the EIIF transcription factor, at least one of which does not require the common domain (residues 1 to 82) and one mechanism that involves the ATF factor and does require both the common and the large T unique domains.

Direct evidence for de-novo synthesis of LH receptors in cultured pig granulosa cells in response to FSH.

FSH plus insulin, cortisol, and thyroxine (IFT) stimulated incorporation of dense isotope-containing (2H, 13C, 15N) amino acids into soluble 125I-labelled hCG binding sites. Evidence of new synthesis of binding sites appeared as early as 3 h after the beginning of the pulse-labelling period. By 48 h the majority of detectable soluble 125I-labelled hCG binding sites appeared to be newly synthesized. Studies with FSH + IFT and puromycin indicated that FSH + IFT stimulated synthesis of new LH/hCG binding sites, and that internalization or degradation of LH/hCG binding sites may also require protein synthesis.

Analysis of maternal IgG subpopulations which are transported into the chicken oocyte.

Chicken serum and oocyte IgG were compared. Purified intact IgG and mercuripapain-produced Fc fragments of yolk and serum IgG were analysed by isoelectric focusing. All IgG bands were identical, indicating that all subpopulations of serum IgG were present in the yolk. Upon papain hydrolysis of both serum and yolk IgG, four identical Fc bands were produced from all serum and yolk samples. Sialic acid measurements showed that there was no significant difference in sialic acid content between serum and oocyte IgG. From these results we conclude that: (i) ovarian IgG receptor(s) selectively transports all subpopulations of maternal IgG; (ii) there is no selective destruction of IgG during transport; and (iii) yolk IgG has the same amount of sialic acid as the serum IgG.

Involvement of simian virus 40 (SV40) small t antigen in trans activation of SV40 early and late promoters.

We have previously found that simian virus 40 (SV40) small t antigen (small t) can trans activate the E2A and VA-I genes of adenovirus in plasmid DNA-transfected cells (M. R. Loeken, I. Bikel, D. M. Livingston, and J. Brady, Cell 55:1171-1177, 1988). To determine whether trans activation by small t might be involved in the SV40 productive infection cycle, we examined the effects of cotransfecting plasmids encoding small t with plasmids containing the chloramphenicol acetyltransferase (CAT) gene linked to the SV40 early or late promoter. Small t increased three- to fivefold the expression of a CAT plasmid linked to the SV40 early promoter and enhancer. Small t expression had no effect by itself on CAT activity directed by the SV40 late promoter, but small t enhanced the effect of a suboptimal concentration of a plasmid expressing large T up to 10-fold. When the concentration of the plasmid expressing large T was increased to a level at which large T alone stimulated the late promoter ninefold, the enhancement by small t was only twofold. The effects of small t on both the SV40 early and late promoters depended on sequences within the small t-unique domain, since a plasmid expressing only the first 82 amino acids common to both large T and small t was inactive. The effects of small t on early- and late-promoter-directed CAT enzyme activity was reflected in increased CAT mRNA as measured by S1 analysis. These results suggest that SV40 small t may play a role in viral infection by increasing transcription from the early promoter and from the late promoter at times when large T levels are low.

The adenovirus EIIA enhancer. Analysis of regulatory sequences and changes in binding activity of ATF and EIIF following adenovirus infection.

Adenovirus EIIA upstream sequences which contain the binding sites for proteins ATF and EIIF act as an enhancer and can be trans-activated by both E1A and SV40 T/t-antigens. Specific mutation of either the ATF or EIIF binding site demonstrates that both act as positive regulators, decreasing transcription greater than 10-fold. Mutation of both the ATF and EIIF binding sites inhibited the EIIA enhancer 200-fold. Analysis of insertion mutations suggests that the spatial alignment of the upstream ATF and EIIF binding sites with respect to the downstream EIIF binding site on the DNA helix is important. Consistent with previous findings, using gel shift analysis we demonstrate that the binding activity of EIIF is increased following wild-type adenovirus infection. In contrast, using identical gel shift conditions, the binding activity of ATF is decreased by viral infection.

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.

Genotoxicity and diabetic embryopathy: impaired expression of developmental control genes as a cause of defective morphogenesis.

Since the advent of insulin therapy for diabetes mellitus, the survival of mothers with diabetes prior to pregnancy and their offspring has greatly improved. Nevertheless, the observation that the earliest stages of organogenesis can be impaired in the offspring of women with diabetes raises the question of how abnormal fuel metabolism disturbs embryogenesis. Research into this process has been made possible in recent years by advances in molecular biology which makes it possible to study gene expression in early embryos, and by the availability of genetically engineered mutant mouse strains. Using these approaches, a model is emerging in which elevated glucose, by disturbing expression of genes which regulate embryonic development and cell cycle progression, causes premature cell death of emerging organ structures, thereby causing defective morphogenesis. Investigation into the signaling mechanisms by which excess glucose metabolism exhibits toxic effects on embryo gene expression will explain how diabetic embryopathy occurs on a molecular and cellular level, as well as increase our understanding of the role of metabolic homeostasis in proper embryonic development.

Interaction between two transcriptional control sequences required for tumor-antigen-mediated simian virus 40 late gene expression.

Transcriptional control signals required for tumor (T)-antigen trans-activation of the simian virus 40 (SV40) late promoter include T-antigen binding sites I and II and the SV40 72-base-pair (bp) repeats. We have used in vivo competition studies to examine how these signals function in relationship to one another. In vivo competition with recombinant plasmids containing the entire SV40 late regulatory region and promoter sequences [map position (mp) 5171-272] results in quantitative removal of limiting trans-acting factor(s) required for late gene expression in COS-1 cells. Deletion of either the T-antigen binding sites (mp 5171-5243) or the 72-bp tandem repeat (mp 128-272) from the competitor plasmid results in markedly less efficient binding of the trans-acting factor, as judged by the loss of competition. Cotransfection of two separate plasmids, one containing the T-antigen binding sites I and II and the other containing the 72-bp repeats, fails to compete for the trans-acting factors. Insertion of increasing lengths of DNA sequences between the T-antigen binding sites and the enhancer sequences also dramatically reduces the efficiency of competition. These results suggest that efficient binding of trans-acting factors requires the presence, in cis, of at least two SV40 regulatory domains. Our studies further suggest that the distance separating these two transcriptional signals is important.