Study of Offspring of Women With Type 1 Diabetes
Condition: Type 1 Diabetes
MeSH terms: Diabetes Mellitus; Diabetes Mellitus, Type 1
Study type: Observational

Summary: 
      There is an increasing trend in the society for developing obesity, type 2 diabetes and
      cardiovascular diseases. It is therefore important to identify the risk factors behind this
      tendency. Recent studies have shown that exposure to high blood sugar levels in pregnancy (as
      in mothers with type 1 diabetes) may play a role in the development of obesity, type 2
      diabetes and cardiovascular diseases later in life for the children. Some studies suggest
      that high blood sugar levels during pregnancy can also affects cognitive function as well as
      growth and development of puberty. The mechanisms behind this are not sufficiently clarified
      yet.

      In the period 1993-1999, pregnant women with type 1 diabetes in Denmark were followed and
      information about the course of pregnancy was collected, including the long-term blood sugar
      level during pregnancy and the children's' condition at birth. The unique opportunities in
      Denmark to identify and follow the children of these mothers and the possibility to select a
      comparison group of children of non-diabetic mothers gives a unique opportunity to examine
      the children of mothers with type 1 diabetes and accurately investigate the effect of blood
      sugar levels in pregnancy on conditions later in life.

      There has not previously been made any study of this size and it is the investigators hope to
      be able to quantify the effect of blood sugar level during pregnancy on outcomes later in
      life. This will potentially give the possibility to detect individuals at risk for
      cardiovascular diseases earlier and to improve prevention targets in children of mothers with
      diabetes.
    

Detailed description: 
      Introduction

      Health conditions result from a combination of genetic susceptibility and environmental
      influences. While the genotype is determined at conception the phenotype is modulated and
      influenced by environmental and epigenetic factors throughout life, not only postnatally but
      already in uteri and possibly even at preimplantation stages.

      Recent studies have highlighted the possible role of intrauterine exposure to maternal
      diabetes in the pathogenesis of overweight, type 2 diabetes and cardiovascular disease. A
      hyperglycaemic intrauterine environment may also affect cognitive function, childhood growth
      and pubertal development in the offspring. During 1993-99 all pregnancies in women with
      pre-gestational type 1 diabetes were prospectively reported to a national registry in the
      Danish Diabetes Association. This nationwide registry contain detailed information of
      maternal demography, pregnancy outcome and diabetes status including glycated haemoglobin
      (HbA1c) in a prospective cohort of 900 women and their newborn offspring. These data are
      therefore ideal for a large-scale study on long-term effects of a diabetic intrauterine
      environment.

      The study consists of 3 substudies.

      Aims

      Study I

      To study long-term effects of a diabetic intrauterine environment in offspring of women with
      type 1 diabetes compared to matched controls from the background population with respect to:

      A)

        -  Cognitive function

        -  Pubertal development

        -  Diabetes/pre-diabetes

        -  Overweight

      B) To study if HbA1c-level in pregnancy is an independent predictor of these outcomes within
      the study group.

      Study II

      To study differences in offspring of women with type 1 diabetes compared to offspring from
      the background population with respect to:

        -  morbidity and congenital malformations

        -  medical treatment

        -  mortality

      Study III

      To study differences in offspring of women with type 1 diabetes compared to compared to
      matched controls from the background population without a family history of diabetes with
      respect to:

      A) State-of-the-art metabolic characterization using euglycemic-hyperinsulinemic clamp

      B) DNA methylation, RNA transcription and protein quantification in muscle and adipose-tissue

      Design

      The study is a prospective follow-up study. The study group includes the offspring of women
      with type 1 diabetes from the national diabetes birth registry (1993-99, n=900) with
      information of HbA1c prior to conception and/or 1st trimester HbA1c and a control group
      including offspring of women without diabetes who delivered during the same period matched
      with respect to gender and age of offspring and the family's postcode as an indirect marker
      of the socioeconomic background.

      Material and Methods

      Clinical characteristics at the time of exposure:

      Study group (diabetes-exposed):

      The following data were reported to the national diabetes birth registry:

      Mothers:

        -  Demographics (age, parity, height, pre-gestational weight)

        -  Diabetes status (pregestational /1st trimester HbA1c, 3rd trimester HbA1c,
           pre-gestational /1st trimester Urine Albumin Excretion Rate, hypertension, proliferative
           retinopathy, diabetes duration, pregestational insulin requirements (IE/d), occurrence
           of severe hypoglycemic events)

        -  Pregnancy complications (Preeclampsia, preterm delivery)

      Offspring:

        -  Demographics (gestational age, birth weight)

        -  Neonatal morbidity (congenital malformations, hypoglycaemia, respiratory distress
           syndrome, jaundice, systemic infection)

      Control group Maternal age, parity, height, pre-gestational weight, information on
      preeclampsia, gestational age, birth weight and neonatal morbidity will be collected from
      medical records.

      Examination program at follow-up:

      Study I: Clinical study (450 diabetes-exposed and 450 controls aged 13-19 years) Clinical
      examination to determine the effect of intrauterine hyperglycaemia on growth, pubertal
      development, metabolism and cognitive function in childhood.

      Examinations

      The participants meet fasting in the morning. The following examinations will be performed:

      Diabetes/prediabetes:

        -  Oral glucose tolerance test (OGTT) with glucose, insulin, C-peptide and proinsulin at 0,
           30, 120 minutes

        -  Blood pressure

        -  HbA1c, cytokines (CRP, Interleukin-6), adiponectin, leptin, incretins

        -  GAD-antibodies

        -  Blood sample for DNA analysis

      Obesity:

        -  Body mass index (BMI), waist/hip circumference

        -  Lipids (total, HDL-C, LDL-C, triglycerides)

        -  Dual energy X-ray absorptiometry (DEXA) scan to determine body composition

      Growth and pubertal development:

        -  Height, sitting height, weight, head circumference

        -  Pubertal stage (Breast development/testicular size and pubic hair)

        -  Hirsutism in girls?

        -  Insulin-like Growth Factor-1 (IGF-1), free EGF-1, Insulin-like growth factor-binding
           protein 3 (IGFBP-3), Insulin-like growth factor-binding protein 1 (IGFBP-1)

        -  Testosterone, estradiol, Sex hormone-binding globulin (SHBG), Follicle- stimulating
           hormone (FSH), Luteinizing hormone (LH), inhibin A, inhibin B, Anti-Müllerian hormone
           (AMH)

        -  Adrenal androgens: dehydroepiandrosterone sulphate (DHEA-S), and Δ4 androgen
           androstenedione (ADION)

        -  Thyroid hormones: Thyroid-stimulating hormone (TSH), Thyroxine (T4), free Thyroxine
           (fT4)

      Cognitive examination including:

        -  Assessment of global intelligence: Reynolds Intellectual Assessment Scales (RIAS)

        -  Assessment of specific cognitive functions Attention Learning and Memory Psychomotor
           speed and reaction time

      The children and their parents will be asked to fill in a questionnaire addressing
      psychosocial aspects, demographics (height, weight) and history of menstrual cycle.
      Furthermore, the children will be asked to fill in a questionnaire on Self-reported physical
      activity (IPAQ-questionnaire).

      Primary endpoints

        -  Cognitive function

        -  Pubertal development

        -  Diabetes/pre-diabetes

        -  Overweight (> 85-percentile for age and gender)

      Secondary endpoints

        -  BMI

        -  Body composition (% body fat)

        -  Blood pressure

        -  Dyslipidaemia

        -  Insulin levels

        -  Markers of endothelial function

        -  Markers of autoimmunity (GAD-antibodies)

        -  Polycystic ovary syndrome (PCOS)

      Study II: Register-based study (n=900 diabetes-exposed; for every case we will sex and age
      match 100 controls, resulting in n=90,000 controls aged 12-18 years) A register-based study
      will be performed to determine the effect of intrauterine hyperglycaemia on congenital
      malformations, morbidity and mortality in childhood.

      Study population In the cohort of 900 children from the national diabetes birth cohort, we
      will study all subjects utilizing register data concerning morbidity, mortality and use of
      prescription medicine. For every diabetes-exposed person Statistics Denmark will identify 100
      age, sex and calendar-time matched controls from the background population from the Central
      Person Register (Statistics Denmark).

      Morbidity and mortality - Operational strategy

        1. In the National Registry of Patients (NRP) and the National Cancer Registry all
           diagnoses from identified diabetes-exposed and controls will be identified.

        2. In the National Drug Prescription Database all information concerning prescription drugs
           on identified diabetes-exposed and controls will be identified.

        3. In the National Registry of Death all diagnoses from identified diabetes-exposed and
           controls will be identified.

      Endpoints Morbidity: diabetes-exposed vs. controls from the background population Medical
      treatment: diabetes-exposed vs. controls from the background population Mortality:
      diabetes-exposed vs. controls from the background population

      Statistics Incidence rates will be calculated as new cases per 100.000 per year and analyzed
      by Poisson regression. Via Statistics Denmark controls will be identified and matched
      appropriately.

      Morbidity and mortality will be analyzed by the Kaplan-Meier statistic, with log-rank test
      and Cox regression with relevant covariates.

      Study III: Sub-study (n=50 diabetes-exposed and n=50 un-exposed controls aged 18-19 years)
      Offspring of mothers with type 1 diabetes, born before 1 January 1994 (over 18 years of age
      at the date of invitation), and who participated in the study A will be invited to
      participate in study B. Study B is anticipated to involve approximately 50 diabetes-exposed
      and approximately 50 un-exposed controls 18 - 19 years of age at time of examination. The
      study groups will be matched according to BMI, gender, date of birth and level of physical
      activity. All participants should be drug-naive and healthy with a BMI between 20 and 30, and
      controls should have no family history of diabetes. Since all participants will be over the
      age of 18, they will be able to provide informed written consent.

      Exclusion criteria:

        1. Any unknown disease or need for medication that occurs after inclusion,

        2. Abnormal ECG, screening blood tests and/or severe hypertension, and

        3. Impaired glucose tolerance in non-diabetic subjects.

      The selected subgroup of offspring of mothers with type 1 diabetes and matched controls will
      be investigated by state-of-the-art metabolic characterization using
      euglycemic-hyperinsulinemic clamp with tracer glucose combined with indirect calorimetry
      allowing reliable estimates of whole-body glucose disposal rates, endogenous glucose
      production and glucose and lipid oxidation. Moreover assessment of physical activity level
      using the IPAQ-questionnaire will be complemented by studies of maximal oxygen consumption
      (VO2max). During the clamp studies, tissue biopsies from subcutaneous abdominal fat and thigh
      muscle (m. vastus lateralis) will be obtained for studies of potential long-term molecular
      consequences of intrauterine exposure to hyperglycemia - metabolic memory of birth. This will
      include application of several discovery-mode (hypothesis free), global approaches such as
      transcriptional profiling using microarray-based technologies, quantitative proteomics,
      bioinformatics including pathway analysis and subsequent validation of observed abnormalities
      using qRT-PCR, immunoblotting and other more classical protein technologies.

      Examinations:

      Physical activity (Day 1)

      The children and their parents will be asked to fill in a questionnaire addressing
      psychosocial and demographic aspects and self-reported physical activity
      (IPAQ-questionnaire). VO2-max is determined by a graded maximal test (VO2-peak) on a cycle
      ergometer using indirect calorimetry.

      State-of-the-art metabolic characterization (Day 2):

      Euglycemic-hyperinsulinemic clamp Subjects are admitted after a 12-h overnight fast to the
      centre at Odense University Hospital. They are instructed to consume a standardized diet, and
      to refrain from physical activity for 48-h before the experiments. The study subjects are
      examined by a euglycemic-hyperinsulinemic clamp (insulin 40mU/min/m2 for 4-h) using tracer
      technology as described55. The studies are combined with indirect calorimetry allowing
      estimates of glucose disposal rates, endogenous glucose production, glucose and lipid
      oxidation, and non-oxidative glucose metabolism as described. Blood samples are drawn every
      20 min during the clamp for assessment of plasma glucose, FFA, adipokines, and serum insulin
      and C-peptide. During the basal and insulin-stimulated states, tissue biopsies are taken from
      m. vastus lateralis and subcutaneous abdominal fat using a modified Bergstrom needle with
      suction under local anesthesia. Each biopsy is rapidly frozen in liquid nitrogen within 30 s
      and stored at -130°C for later analysis as described below. Body fat (%) is determined by the
      bioimpedance method.

      Transcriptomics and proteomic analysis of skeletal muscle and adipose tissue include:

      Microarray-based transcriptional profiling and biological pathway analysis:

      Discovery-mode quantitative proteomics of tissue biopsies:

      Targeted quantitative proteomics:

      Quantitative RT-PCR

      Immunoblotting

      Genetic analysis:

      Analysis of DNA methylation

      Whole genome methylation analysis

      Methylation-Sensitive High Resolution Melting analysis (MS-HRM)

      Gene Expression Analysis

      Sequencing

      Statistical Analysis Mann-Whitney U test will be used to assess for each CpG, whether two
      groups had the same distribution of methylation. To eliminate probes that did not work and
      other diverging values, beta values in each group will be trimmed for extremes prior to the
      calculation of the average beta value. Next, only genes with the highest 5% tail of the
      absolute average beta value differences between the two groups will be selected. Thus CpGs
      with a p-value below 0.005 and with the highest beta value differences between the two groups
      will be used for identification of genes with statistically significant diabetes-specific
      changes in methylation. Comparison of array methylation results with MS-HRM results will be
      performed with a Mann-Whitney U test and a Chi2 test for trend. Spearman coefficients will be
      calculated to assess the correlation between methylation and gene expression.

      Novelty and importance

      The rapidly increasing burden of overweight and cardiovascular disease is becoming a threat
      to both the individual and global economy. It is therefore essential to identify risk groups
      to target preventive strategies. Exposure to intrauterine hyperglycemia contributes to the
      epidemic through a vicious-cycle passing increased susceptibility on to the next generation
      via pathways that are only sparsely understood.

      This study includes a very large cohort of diabetes-exposed offspring of well-characterized
      prospectively studied women with type 1 diabetes. It includes information on maternal
      glycemia during pregnancy, which is imperative to assess possible associations with estimates
      of offspring metabolism and cognitive function. The register-based study is unique and
      enables collection of information on morbidity and mortality in the whole cohort. The
      specific molecular signature of intrauterine exposure to hyperglycemia in human tissues has
      to our knowledge not been examined before, and although the above-mentioned studies suggest
      the possible involvement of certain pathways in skeletal muscle and adipose tissue, it is
      important to investigate the metabolic memory of birth using hypothesis-free, discovery-mode
      global approaches to discover potential perturbations in both expected and unexpected
      pathways.
    

Observational model: Case-Control
Time perspective: Prospective
Sampling method: Probability Sample
Study population: 
        Offspring of mothers with type one diabetes.
      
Biospecimen description: 
      Whole blood, serum and plasma. Tissue biopsies from m. vastus lateralis and subcutaneous
      abdominal fat (only selected subgroup of participants)
    
Keywords: Offspring of mothers with type 1 diabetes; Pregnancy and programing; Intrauterine hyperglycaemia; Study focuses on offspring of mothers with type 1 diabetes