Study of Offspring of Women With Type 1 Diabetes
Study of Offspring of Women With Type 1 Diabetes
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.
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.
Inclusion Criteria:

 -  Children (diabetes exposed) from the national diabetes birth registry born between
 1993 and 1999 (approximately n = 450) The same number of controls from the Central
 Person Register matched according to gender, age and postcode.

 Exclusion Criteria:

 -  Offspring with major handicaps or chronic disease will be registered, but will not be
 examined.

 -  Multiple pregnancies and recurrent pregnancies will not be included in the study.