Short summary

Through analyses of the structural components of the vessel wall in the arteries and veins of the placenta and umbilical cord and umbilical cord blood, we aim to detect stuctural changes between females diagnosed with gestational diabetes mellitus (GDM), compared to females without the diagnosis. This is executed through a plethora of proteomic analyses, and with the establishment of a biobank containing donated tissues from participating pregnant couples delivering at OUH.

Rationale

Diabetes is a chronic metabolic disorder characterized by hyperglycemia affecting nearly half a billion people worldwide (1). Individuals living with diabetes face a profound increased risk of developing macrovascular complications, including coronary artery disease (CAD), stroke and peripheral vascular disease as well as microvascular complications, such as end-stage renal disease, retinopathy and neuropathy, along with lower-extremity amputations. Despite improvements in managing diabetes, this patient group continues to have a two- to fourfold higher risk of hospitalisation for major cardiovascular disease (CVD) events compared with those without diabetes (2). CAD results from decades of developing atherosclerotic plaque in the coronary bed and is accelerated in the presence of other modifiable risk factors including hypertension, smoking, obesity, and diabetes (3). Although the mechanisms behind increased CVD among patients with diabetes are largely unknown, they may involve direct effects of the diabetic milieu on vascular smooth muscle cells (VSMCs). Proteomic analysis revealed that the level of basement membrane (BM) proteins including collagen type IV (Col4) is increased in non-atherosclerotic arteries from patients with type 2 diabetes, whereas no difference in the level of interstitial collagens or other matrix molecules could be detected (4). The causal factors behind altered vascular molecular pathology is unknown, but putative associations between the diabetic milieu and vascular molecular changes may however be studied in placental vessels from patients with and without diabetes. In pregnancy the exposure to hyperglycemia has a profound impact where fetal overgrowth is an important neonatal complication. Accordingly, women with pre-gestational diabetes mellitus (PGDM) or gestational diabetes mellitus (GDM) have an increased risk of delivering infants who are large for gestational age (LGA) (5). The impact of hyperglycemia on vessels in placenta and the umbilical cord, however, is largely unknown and functional and molecular studies are scarce. One study investigated the maternal diabetic environment and provided evidence for altered umbilical cord gene expression of genes involved in the regulation of vascular development and function with simultaneous umbilical vessel muscle layer thickening (6). Several studies have shown that exposure to hyperglycemic conditions for prolonged time causes VSMCs to increase the expression of BM proteins (7-9), especially the expression of Col4 (10). The BM surrounds every VSMC and is known to regulate important cellular functions and to determine the development of at least some arterial diseases (3). These studies have investigated how fully developed arteries compensate for a milieu of high glucose levels over longer periods of time, where compensatory changes occur. There are, however, insufficient amount of conclusive research related to how a hyperglycemic environment affects the development of arteries in the gestational period and for the fetus, and whether there are detectable changes within the structure of arteries after exposure to hyperglycemia during a shorter timeframe as the gestational period. The changes within the VSMCs identified for adults with diabetes have concluded a reduction in the arteries' ability to remodel (7). By investigating the blood vessels of the placenta and the umbilical cord, there is an opportunity to identify whether the ability to remodel will be affected in the same way after a short time of exposure of a hyperglycemic environment during the vessels' development, and how this can affect the blood supply to the fetus. We envision that hyperglycemia in diabetic pregnancies leads to changes in molecular composition (i.e. protein and RNA expression) and morphology, in addition to altered structural and functional properties in blood vessels of the placenta and the umbilical cord at term, compared to non-diabetic controls. These changes may be associated with the level of dysglycemia. The overall aim of the study is to uncover possible association between hyperglycemia and alterations in blood vessels of the placenta and the umbilical cord to provide evidence for short-term influence of hyperglycemia in relation to vascular complications in diabetes. The present pregraduate study focuses on the following aims: 1. To perform a pilot study to develop a procedure for dissection and handling of umbilical cord and placenta arteries and veins in relation to downstream microscopic and molecular analyses. 2. To perform a case-control study of placenta and umbilical cords from women and their offspring. We will compare diet-treated GDM (n=10) and insulin-treated GDM (n=10) to normoglycemic healthy controls (n=10). The study will include microscopic, as well as molecular analyses as described below.

Description of the cohort

Non-diabetic controls with at negative oral glucose tolerance test (2-hour OGTT < 8,5 mM) at gestational week 24-28 have been included from the department of Clinical Biochemistry, OUH. Participants diagnosed with gestational diabetes mellitus (GDM) have been included either at the department of Clinical Biochemistry with a positive OGTT-result at gestational week 24-28, or during their follow up consultations at the Department of Gynecology and Obstetrics (Dep. D) at OUH at gestational week 32-37. All participants have delivered at dep. D, OUH.

Data and biological material

After consent from the participants, the following data and biological matter will be extracted: Biological material donated: Placenta Umbilical cord Umbilical cord blood for plasma and serum samples. Data: Baseline and outcome data will be obtained from patient files and medical records. This involves: Maternal pre-pregnanzy characteristics (age, height, weight, ethnicity, contact information, GDM risk factors, parity and information on earlier pregnancies (e.g. complications, miscarriages, etc), use of additional medicine prior to pregnancy, chronic disease), pregnancy outcomes (use of medicine during pregnancy, HbA1c in pregnancy, blood pressure, urinary ketones, daily insulin dose during pregnancy, insulin type(s), number of daily insulin injections, use of insulin pump, type of insulin pump as indicated, gestational hypertension, preeclampsia, fetal growth SD-score at scans at week 28, 32, and 36 weeks. Data from ASTRAIA (nationwide fetalmedicine database)), delivery outcomes (date of delivery, gestational age at delivery, mode of delivery, need for maternal corticosteroid treatment for fetal lung maturation, urinary tract infection, preterm labour rupture of the membranes, hydramnios, and placental weight), and neonatal outcomes (offspring gender, birthweight, LGA, apgar score) All data management will be handled according to the Danish "databeskyttelsesforordningen" and "databeskyttelsesloven".

Collaborating researchers and departments

Department of Clinical Biochemistry

• Martin Overgaard (PI), Associate Professor, PhD, Senior Specialist
• Lars Melholt Rasmussen, Professor, DMSci, Senior Consultant

Institute of Molecular Medicine, SDU

• Lasse Bach Steffensen, Associate Professor, MSc, PhD

Department of Gynaecology and Obstetrics, OUH

• Christina Anne Vinter, Associate Professor, PhD, Consultant

Steno Diabetes Center Odense and The Research Unit of Gynaecology and Obstetrics, OUH

• Dorte Møller Jensen, Professor, Senior Consultant, PhD

Perinatal Research Laboratory, Medical University of Graz, Austria

• Gernot Desoye, Professor, PhD, External cooperator

Publications associated with the project

Ogurtsova K, da Rocha Fernandes JD, Huang Y, et al. IDF Diabetes Atlas: Global estimates for the prevalence of diabetes for 2015 and 2040. Diabetes Res Clin Pract. 2017;128:40-50.

Einarson TR, Acs A, Ludwig C, Panton UH. Prevalence of cardiovascular disease in type 2 diabetes: a systematic literature review of scientific evidence from across the world in 2007-2017. Cardiovasc Diabetol. 2018;17(1):83.

Steffensen LB, Rasmussen LM. A role for collagen type IV in cardiovascular disease?. Am J Physiol Heart Circ Physiol. 2018;315(3):H610-H625.

Preil SA, Kristensen LP, Beck HC, et al. Quantitative Proteome Analysis Reveals Increased Content of Basement Membrane Proteins in Arteries From Patients With Type 2 Diabetes Mellitus and Lower Levels Among Metformin Users. Circ Cardiovasc Genet. 2015;8(5):727-735.

Ringholm L, Damm P, Mathiesen ER. Improving pregnancy outcomes in women with diabetes mellitus: modern management. Nat Rev Endocrinol. 2019;15(7):406-416.

Koskinen A, Lehtoranta L, Laiho A, Laine J, Kääpä P, Soukka H. Maternal diabetes induces changes in the umbilical cord gene expression. Placenta. 2015;36(7):767-774.

Steffensen LB, Iversen XES, Hansen RS, et al. Basement membrane proteins in various arterial beds from individuals with and without type 2 diabetes mellitus: a proteome study. Cardiovasc Diabetol. 2021;20(1):182.

Rasmussen LM, Ledet T. Serum from diabetic patients enhances synthesis of arterial basement membrane-like material in cultured smooth muscle cells. APMIS. 1988;96(1):77-83.

Moritani S, Negishi K, Watanabe T, et al. Glucose-induced production of type IV collagen and laminin P1 from cultured human umbilical vein endothelial cells. J Diabet Complications. 1991;5(2-3):201-203.

Thorsen AF, Riber LPS, Rasmussen LM, Overgaard M. A targeted multiplex mass spectrometry method for quantitation of abundant matrix and cellular proteins in formalin-fixed paraffin embedded arterial tissue. J Proteomics. 2023;272:104775